Source of infection Source of infection D-r Mitova MU-Sofia
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Source of infectionSource of infection
D-r MitovaMU-Sofia
D-r Mitova 2
Infectious disease epidemiology is a fundamental part of the whole of epidemiology In fact the subject of epidemiology originally developed from the study of epidemics of infectious diseaseInfectious disease epidemiology is a science of Public Health that studies distribution of infectious disease
D-r Mitova 3
Selected definitions
InfectionInfectionThe entry and development or
multiplication of an infectious agent in the body of man or animals It also implies that the body responds in some way to defend itself against the invader either in the form of an immune response or disease An infection does not always cause illness
D-r Mitova 4
There are several level of There are several level of infectioninfection
Colonization egSaureus in the skin and normal nasopharynxSubclinical or inapparent infection egpolioLatent infection egvirus of herpes symplexManifest or clinical infection
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Infectious disease A clinically manifest disease of man or animal
resulting from an infectionContagious disease
A disease that is transmitted through contactCommunicable disease
All illness due to specific infectious agent or its toxic products capable of being directly or indirectly transmitted from man to man animal to animal or environment (through air dust soil water food etc) to man or animal
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ContaminationContamination The presence of an infectious
agent on a body surface also in or on clothes bedding toys surgical instruments or dressings or other inanimate articles or substances including water milk and food (Lasts epidemiological definition)
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Epidemic processEpidemic process
The epidemic process defined as a chain of interrelated and interconnected infectious conditions in the populationCommunicable diseases are transmitted from the reservoir or source of infection to susceptible host This is the medical model of epidemic process
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Bacically there are 3 links in the chain of transmission-the reservoir mode of transmission and the susceptible host
Source Source reservoir reservoir of infectionof infection
ModesModesofof
transmissiontransmission
SusceptibleSusceptiblehosthost
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Courses types of the Courses types of the epidemic processepidemic process
A disease is said to be sporadicsporadic when only a few cases occur here and there in a given region
OutbreakOutbreak defines as a smallusually localised epidemic
D-r Mitova 10
Courses types of the Courses types of the epidemic processepidemic process
Epidemic (from Greek epi- upon + demos Epidemic (from Greek epi- upon + demos people)-people)- outbreak of disease that affects a much greater number of people than is usual for the locality or that spreads to regions where it is ordinarily not present A disease that tends to be restricted to a particular region (endemic disease) can become epidemic if nonimmune persons are present in large numbers (as in time of war or during pilgrimages) if the infectious agent is more virulent than usual or if distribution of the disease is more easily effected Cholera and plague endemic in parts of Asia can become epidemic under the above conditions as can dysentery and many other infections Epidemics may also be caused by new disease agents in the human population such as the Ebola virus
D-r Mitova 11
Courses types of the Courses types of the epidemic processepidemic process
A pandemicpandemic (from Greek pan all + demos people) is an epidemic (an outbreak of an infectious disease) that spreads worldwide or at least across a large regionA worldwide epidemic is known as a pandemicpandemic eg the influenza pandemic of 1918 or the AIDS pandemic beginning in the 1980s
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Examples Cholera Examples Cholera
first pandemic 1816ndash1826 Previously restricted to the Indian subcontinent the pandemic began in Bengal then spread across India by 1820 It extended as far as China and the Caspian Sea before recedingThe second pandemic (1829ndash1851) reached Europe London in 1832 Ontario Canada and New York in the same year and the Pacific coast of North America by 1834The third pandemic (1852ndash1860) mainly affected Russia with over a million deaths
D-r Mitova 13
Examples Cholera Examples Cholera
The fourth pandemic (1863ndash1875) spread mostly in Europe and AfricaIn 1866 there was an outbreak in North AmericaThe sixth pandemic (1899ndash1923) had little effect in Europe because of advances in public health but Russia was badly affected againThe seventh pandemic began in Indonesia in 1961 called El Tor after the strain and reached Bangladesh in 1963 India in 1964 and the USSR in 1966
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Examples InfluenzaExamples Influenza
The Spanish fluSpanish flu 1918ndash1919
An estimated 17 million died in India 500000 in the United States and 200000 in the UK The virus was recently reconstructed by scientists at the CDC studying remains preserved by the Alaskan permafrost They identified it as a type of H1N1 virus
D-r Mitova 15
Courses types of the Courses types of the epidemic processepidemic process
In epidemiology an infection is said to be endemic endemic (from Greek en- in or within + demos people) in a population when that infection is maintained in the population without the need for external inputs For example chickenpox is endemic in the UK but malaria is not Every year there are a few cases of malaria acquired in the UK but these do not lead to sustained transmission in the population due to the lack of a suitable vector (mosquitoes of the genus Anopheles)
D-r Mitova 16
SOURCE AND RESERVOIR
The source of infection is defined as the person animal object or substance from which an infectious agent passes or disseminated to the host
A reservoir is defined as ldquoany person animal arthropod plant soil or substance in which an infectious agent lives and multiplies on which it depends primarily for survival and where it reproduced itself in such manner that it can be transmitted to a susceptible hostrdquo In short the reservoir is the natural habitual in which the organism metabolized and replicates
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In Bulgaria the source of infection is defined only as the person or animal in which an infectious agent live and multiplies and from which an infectious agent passes to the host inanimate things and arthropods are defined as a vehicles of transmission
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HUMAN RESERVOIR
By far the most important source or reservoir of infection for humans is man himself He my be a case or carrier Man is often described as him own enemy because most of the communicable disease of which man is heir to a contacted from human sources
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HUMAN RESERVOIR - CASE
A case is defined as a person in the population or study group identified as having the particular disease health disorder or condition under investigation A variety of criteria ( clinical biochemical laboratory ) may be used to identify cases Broadly the presence of infection in the host may be clinical subclinical or latent
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A case is a risk factor hellip Infection in one person can be transmitted
to others
What is infectious disease epidemiology
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1The clinical illness
The clinical illness maybe mild or moderate typical or atypical severe or fatal
Epidemiologically mild cases may be more important sources of infection than severe cases because they are ambulant and spread the infection wherever they go whereas severe cases usually confined to bed
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2The subclinical cases are variously referred to as inapparent covert missed or abortive cases They are equally important sources of infection The disease agent may multiply in the host but does not manifest itself by sign and symptoms The disease agent is eliminated and contaminates the environment in the same way as clinical casesperson who are thus sick contribute more than symptomatic patient to the transmission of infection to others and what is more they do not appear in any of statistics
Subclinical cases play a dominant role in maintaining the chain of infection in the community The latent infection must be distinguished from subclinical infection In latent infection the host does not shed the infectious agent
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The subclinical cases
Subclinical infection can be detected only by laboratory tests
Barring a few(mesles varicella) subclinical infection occurs in most infectious disease In some disease(rubella mumps polio hepatitis A and B influenza diphteria) a great deal of subclinical infection occurs
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Latent infectionThe term latent infection must be
distinguished from subclinical infection In latent infection the host does not shed the infectious agent which lies dormant within the host without symptoms (and often without demonstrable presence in blood tissues or bodily secretions of the host) For examples latent infection occurs in herpes symplex Brill-Zincer disease
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CARRIERS
In some disease either due to inadequate treatment or immune response the disease agent is not completely eliminated leading to a carrier state A carrier is defined as an infected person or animal that harbors a specific infectious agent in the absence of
discernible clinical disease and serves as a potential source of infection for others
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CARRIERS
As a rule carriers are less infectious than cases but epidemiologically they are more dangerous than cases because they escape recognition and continuing as they do live a normal life among population or community they readily infect the susceptible individuals over a wider area and longer period of time under favorable conditionsthe ldquoTyphoid Maryrdquo is a classic example of a carrier-cooker Mary who gave rise to more than 1300 cases in her life time
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The elements in a carrier state are
The presence in the body of disease agent
The absence of recognizable symptoms and signs of disease
The shedding of disease agent in the discharges or excretion
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incubatory carriers are those who shed the infectious agent during the incubation period This usually occurs during the the last few days of incubation period As for example
Measles-the period of communicability is 4 days before the rush
Mumps-usually 4-6 days before onset of symptoms
Polio-7-10 days before onset of symptomsHepatitis B-for a month before
jaundiceabout 6 weeks after exposure
Carrier may be classified BY TYPE
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Carrier may be classified BY TYPE
convalescent carriers that is those who continue to shed the disease agent during the period of convalescence(typhoid fever cholera diphteria bacillary disentery) In the disease clinical recovery does not coincide with bacteriological recovery
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Carrier may be classified BY TYPE
healthy carriers they are victims of subclinical infection who have developed carrier state without suffering from overt disease but are nevertheless shedding the disease agent eg poliomyelitis cholera meningococcal meningitis salmonellosis diphtheria
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BY DURATION
Temporary carriers are those who shed the infectious agent for short period of timeChronic carriers are those who excretes the infectious agent for indefinite periods eg typhoid fever hepatitis B dysentery etcThey are known to reintroduce disease into areas which are otherwise free of infection
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Chronic carriers
Chronic carriers are far more important sources of infection than cases The longer the carrier state the greater the risk of community The duration of the carrier state varies with the disease In typhoid fever and hepatitis B the chronic carrier state may last for several yearsIn chronic dysentery it may last for year or longer In diphtheria the carrier state is associated with infected tonsils in typhoid fever with gall bladder disease
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BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
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EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
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Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
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Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
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Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
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S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
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Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
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Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
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Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
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MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
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DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
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After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
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The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
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Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
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Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
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LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
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LABORATORY DIAGNOSIS OF CHOLERA
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LABORATORY DIAGNOSIS
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Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
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Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
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MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
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At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
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N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
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Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
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A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
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Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
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Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
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D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 2
Infectious disease epidemiology is a fundamental part of the whole of epidemiology In fact the subject of epidemiology originally developed from the study of epidemics of infectious diseaseInfectious disease epidemiology is a science of Public Health that studies distribution of infectious disease
D-r Mitova 3
Selected definitions
InfectionInfectionThe entry and development or
multiplication of an infectious agent in the body of man or animals It also implies that the body responds in some way to defend itself against the invader either in the form of an immune response or disease An infection does not always cause illness
D-r Mitova 4
There are several level of There are several level of infectioninfection
Colonization egSaureus in the skin and normal nasopharynxSubclinical or inapparent infection egpolioLatent infection egvirus of herpes symplexManifest or clinical infection
D-r Mitova 5
Infectious disease A clinically manifest disease of man or animal
resulting from an infectionContagious disease
A disease that is transmitted through contactCommunicable disease
All illness due to specific infectious agent or its toxic products capable of being directly or indirectly transmitted from man to man animal to animal or environment (through air dust soil water food etc) to man or animal
D-r Mitova 6
ContaminationContamination The presence of an infectious
agent on a body surface also in or on clothes bedding toys surgical instruments or dressings or other inanimate articles or substances including water milk and food (Lasts epidemiological definition)
D-r Mitova 7
Epidemic processEpidemic process
The epidemic process defined as a chain of interrelated and interconnected infectious conditions in the populationCommunicable diseases are transmitted from the reservoir or source of infection to susceptible host This is the medical model of epidemic process
D-r Mitova 8
Bacically there are 3 links in the chain of transmission-the reservoir mode of transmission and the susceptible host
Source Source reservoir reservoir of infectionof infection
ModesModesofof
transmissiontransmission
SusceptibleSusceptiblehosthost
D-r Mitova 9
Courses types of the Courses types of the epidemic processepidemic process
A disease is said to be sporadicsporadic when only a few cases occur here and there in a given region
OutbreakOutbreak defines as a smallusually localised epidemic
D-r Mitova 10
Courses types of the Courses types of the epidemic processepidemic process
Epidemic (from Greek epi- upon + demos Epidemic (from Greek epi- upon + demos people)-people)- outbreak of disease that affects a much greater number of people than is usual for the locality or that spreads to regions where it is ordinarily not present A disease that tends to be restricted to a particular region (endemic disease) can become epidemic if nonimmune persons are present in large numbers (as in time of war or during pilgrimages) if the infectious agent is more virulent than usual or if distribution of the disease is more easily effected Cholera and plague endemic in parts of Asia can become epidemic under the above conditions as can dysentery and many other infections Epidemics may also be caused by new disease agents in the human population such as the Ebola virus
D-r Mitova 11
Courses types of the Courses types of the epidemic processepidemic process
A pandemicpandemic (from Greek pan all + demos people) is an epidemic (an outbreak of an infectious disease) that spreads worldwide or at least across a large regionA worldwide epidemic is known as a pandemicpandemic eg the influenza pandemic of 1918 or the AIDS pandemic beginning in the 1980s
D-r Mitova 12
Examples Cholera Examples Cholera
first pandemic 1816ndash1826 Previously restricted to the Indian subcontinent the pandemic began in Bengal then spread across India by 1820 It extended as far as China and the Caspian Sea before recedingThe second pandemic (1829ndash1851) reached Europe London in 1832 Ontario Canada and New York in the same year and the Pacific coast of North America by 1834The third pandemic (1852ndash1860) mainly affected Russia with over a million deaths
D-r Mitova 13
Examples Cholera Examples Cholera
The fourth pandemic (1863ndash1875) spread mostly in Europe and AfricaIn 1866 there was an outbreak in North AmericaThe sixth pandemic (1899ndash1923) had little effect in Europe because of advances in public health but Russia was badly affected againThe seventh pandemic began in Indonesia in 1961 called El Tor after the strain and reached Bangladesh in 1963 India in 1964 and the USSR in 1966
D-r Mitova 14
Examples InfluenzaExamples Influenza
The Spanish fluSpanish flu 1918ndash1919
An estimated 17 million died in India 500000 in the United States and 200000 in the UK The virus was recently reconstructed by scientists at the CDC studying remains preserved by the Alaskan permafrost They identified it as a type of H1N1 virus
D-r Mitova 15
Courses types of the Courses types of the epidemic processepidemic process
In epidemiology an infection is said to be endemic endemic (from Greek en- in or within + demos people) in a population when that infection is maintained in the population without the need for external inputs For example chickenpox is endemic in the UK but malaria is not Every year there are a few cases of malaria acquired in the UK but these do not lead to sustained transmission in the population due to the lack of a suitable vector (mosquitoes of the genus Anopheles)
D-r Mitova 16
SOURCE AND RESERVOIR
The source of infection is defined as the person animal object or substance from which an infectious agent passes or disseminated to the host
A reservoir is defined as ldquoany person animal arthropod plant soil or substance in which an infectious agent lives and multiplies on which it depends primarily for survival and where it reproduced itself in such manner that it can be transmitted to a susceptible hostrdquo In short the reservoir is the natural habitual in which the organism metabolized and replicates
D-r Mitova 17
In Bulgaria the source of infection is defined only as the person or animal in which an infectious agent live and multiplies and from which an infectious agent passes to the host inanimate things and arthropods are defined as a vehicles of transmission
D-r Mitova 18
HUMAN RESERVOIR
By far the most important source or reservoir of infection for humans is man himself He my be a case or carrier Man is often described as him own enemy because most of the communicable disease of which man is heir to a contacted from human sources
D-r Mitova 19
HUMAN RESERVOIR - CASE
A case is defined as a person in the population or study group identified as having the particular disease health disorder or condition under investigation A variety of criteria ( clinical biochemical laboratory ) may be used to identify cases Broadly the presence of infection in the host may be clinical subclinical or latent
D-r Mitova 20
A case is a risk factor hellip Infection in one person can be transmitted
to others
What is infectious disease epidemiology
D-r Mitova 21
1The clinical illness
The clinical illness maybe mild or moderate typical or atypical severe or fatal
Epidemiologically mild cases may be more important sources of infection than severe cases because they are ambulant and spread the infection wherever they go whereas severe cases usually confined to bed
D-r Mitova 22
2The subclinical cases are variously referred to as inapparent covert missed or abortive cases They are equally important sources of infection The disease agent may multiply in the host but does not manifest itself by sign and symptoms The disease agent is eliminated and contaminates the environment in the same way as clinical casesperson who are thus sick contribute more than symptomatic patient to the transmission of infection to others and what is more they do not appear in any of statistics
Subclinical cases play a dominant role in maintaining the chain of infection in the community The latent infection must be distinguished from subclinical infection In latent infection the host does not shed the infectious agent
D-r Mitova 23
The subclinical cases
Subclinical infection can be detected only by laboratory tests
Barring a few(mesles varicella) subclinical infection occurs in most infectious disease In some disease(rubella mumps polio hepatitis A and B influenza diphteria) a great deal of subclinical infection occurs
D-r Mitova 24
Latent infectionThe term latent infection must be
distinguished from subclinical infection In latent infection the host does not shed the infectious agent which lies dormant within the host without symptoms (and often without demonstrable presence in blood tissues or bodily secretions of the host) For examples latent infection occurs in herpes symplex Brill-Zincer disease
D-r Mitova 25
CARRIERS
In some disease either due to inadequate treatment or immune response the disease agent is not completely eliminated leading to a carrier state A carrier is defined as an infected person or animal that harbors a specific infectious agent in the absence of
discernible clinical disease and serves as a potential source of infection for others
D-r Mitova 26
CARRIERS
As a rule carriers are less infectious than cases but epidemiologically they are more dangerous than cases because they escape recognition and continuing as they do live a normal life among population or community they readily infect the susceptible individuals over a wider area and longer period of time under favorable conditionsthe ldquoTyphoid Maryrdquo is a classic example of a carrier-cooker Mary who gave rise to more than 1300 cases in her life time
D-r Mitova 27
The elements in a carrier state are
The presence in the body of disease agent
The absence of recognizable symptoms and signs of disease
The shedding of disease agent in the discharges or excretion
D-r Mitova 28
incubatory carriers are those who shed the infectious agent during the incubation period This usually occurs during the the last few days of incubation period As for example
Measles-the period of communicability is 4 days before the rush
Mumps-usually 4-6 days before onset of symptoms
Polio-7-10 days before onset of symptomsHepatitis B-for a month before
jaundiceabout 6 weeks after exposure
Carrier may be classified BY TYPE
D-r Mitova 29
Carrier may be classified BY TYPE
convalescent carriers that is those who continue to shed the disease agent during the period of convalescence(typhoid fever cholera diphteria bacillary disentery) In the disease clinical recovery does not coincide with bacteriological recovery
D-r Mitova 30
Carrier may be classified BY TYPE
healthy carriers they are victims of subclinical infection who have developed carrier state without suffering from overt disease but are nevertheless shedding the disease agent eg poliomyelitis cholera meningococcal meningitis salmonellosis diphtheria
D-r Mitova 31
BY DURATION
Temporary carriers are those who shed the infectious agent for short period of timeChronic carriers are those who excretes the infectious agent for indefinite periods eg typhoid fever hepatitis B dysentery etcThey are known to reintroduce disease into areas which are otherwise free of infection
D-r Mitova 32
Chronic carriers
Chronic carriers are far more important sources of infection than cases The longer the carrier state the greater the risk of community The duration of the carrier state varies with the disease In typhoid fever and hepatitis B the chronic carrier state may last for several yearsIn chronic dysentery it may last for year or longer In diphtheria the carrier state is associated with infected tonsils in typhoid fever with gall bladder disease
D-r Mitova 33
BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 3
Selected definitions
InfectionInfectionThe entry and development or
multiplication of an infectious agent in the body of man or animals It also implies that the body responds in some way to defend itself against the invader either in the form of an immune response or disease An infection does not always cause illness
D-r Mitova 4
There are several level of There are several level of infectioninfection
Colonization egSaureus in the skin and normal nasopharynxSubclinical or inapparent infection egpolioLatent infection egvirus of herpes symplexManifest or clinical infection
D-r Mitova 5
Infectious disease A clinically manifest disease of man or animal
resulting from an infectionContagious disease
A disease that is transmitted through contactCommunicable disease
All illness due to specific infectious agent or its toxic products capable of being directly or indirectly transmitted from man to man animal to animal or environment (through air dust soil water food etc) to man or animal
D-r Mitova 6
ContaminationContamination The presence of an infectious
agent on a body surface also in or on clothes bedding toys surgical instruments or dressings or other inanimate articles or substances including water milk and food (Lasts epidemiological definition)
D-r Mitova 7
Epidemic processEpidemic process
The epidemic process defined as a chain of interrelated and interconnected infectious conditions in the populationCommunicable diseases are transmitted from the reservoir or source of infection to susceptible host This is the medical model of epidemic process
D-r Mitova 8
Bacically there are 3 links in the chain of transmission-the reservoir mode of transmission and the susceptible host
Source Source reservoir reservoir of infectionof infection
ModesModesofof
transmissiontransmission
SusceptibleSusceptiblehosthost
D-r Mitova 9
Courses types of the Courses types of the epidemic processepidemic process
A disease is said to be sporadicsporadic when only a few cases occur here and there in a given region
OutbreakOutbreak defines as a smallusually localised epidemic
D-r Mitova 10
Courses types of the Courses types of the epidemic processepidemic process
Epidemic (from Greek epi- upon + demos Epidemic (from Greek epi- upon + demos people)-people)- outbreak of disease that affects a much greater number of people than is usual for the locality or that spreads to regions where it is ordinarily not present A disease that tends to be restricted to a particular region (endemic disease) can become epidemic if nonimmune persons are present in large numbers (as in time of war or during pilgrimages) if the infectious agent is more virulent than usual or if distribution of the disease is more easily effected Cholera and plague endemic in parts of Asia can become epidemic under the above conditions as can dysentery and many other infections Epidemics may also be caused by new disease agents in the human population such as the Ebola virus
D-r Mitova 11
Courses types of the Courses types of the epidemic processepidemic process
A pandemicpandemic (from Greek pan all + demos people) is an epidemic (an outbreak of an infectious disease) that spreads worldwide or at least across a large regionA worldwide epidemic is known as a pandemicpandemic eg the influenza pandemic of 1918 or the AIDS pandemic beginning in the 1980s
D-r Mitova 12
Examples Cholera Examples Cholera
first pandemic 1816ndash1826 Previously restricted to the Indian subcontinent the pandemic began in Bengal then spread across India by 1820 It extended as far as China and the Caspian Sea before recedingThe second pandemic (1829ndash1851) reached Europe London in 1832 Ontario Canada and New York in the same year and the Pacific coast of North America by 1834The third pandemic (1852ndash1860) mainly affected Russia with over a million deaths
D-r Mitova 13
Examples Cholera Examples Cholera
The fourth pandemic (1863ndash1875) spread mostly in Europe and AfricaIn 1866 there was an outbreak in North AmericaThe sixth pandemic (1899ndash1923) had little effect in Europe because of advances in public health but Russia was badly affected againThe seventh pandemic began in Indonesia in 1961 called El Tor after the strain and reached Bangladesh in 1963 India in 1964 and the USSR in 1966
D-r Mitova 14
Examples InfluenzaExamples Influenza
The Spanish fluSpanish flu 1918ndash1919
An estimated 17 million died in India 500000 in the United States and 200000 in the UK The virus was recently reconstructed by scientists at the CDC studying remains preserved by the Alaskan permafrost They identified it as a type of H1N1 virus
D-r Mitova 15
Courses types of the Courses types of the epidemic processepidemic process
In epidemiology an infection is said to be endemic endemic (from Greek en- in or within + demos people) in a population when that infection is maintained in the population without the need for external inputs For example chickenpox is endemic in the UK but malaria is not Every year there are a few cases of malaria acquired in the UK but these do not lead to sustained transmission in the population due to the lack of a suitable vector (mosquitoes of the genus Anopheles)
D-r Mitova 16
SOURCE AND RESERVOIR
The source of infection is defined as the person animal object or substance from which an infectious agent passes or disseminated to the host
A reservoir is defined as ldquoany person animal arthropod plant soil or substance in which an infectious agent lives and multiplies on which it depends primarily for survival and where it reproduced itself in such manner that it can be transmitted to a susceptible hostrdquo In short the reservoir is the natural habitual in which the organism metabolized and replicates
D-r Mitova 17
In Bulgaria the source of infection is defined only as the person or animal in which an infectious agent live and multiplies and from which an infectious agent passes to the host inanimate things and arthropods are defined as a vehicles of transmission
D-r Mitova 18
HUMAN RESERVOIR
By far the most important source or reservoir of infection for humans is man himself He my be a case or carrier Man is often described as him own enemy because most of the communicable disease of which man is heir to a contacted from human sources
D-r Mitova 19
HUMAN RESERVOIR - CASE
A case is defined as a person in the population or study group identified as having the particular disease health disorder or condition under investigation A variety of criteria ( clinical biochemical laboratory ) may be used to identify cases Broadly the presence of infection in the host may be clinical subclinical or latent
D-r Mitova 20
A case is a risk factor hellip Infection in one person can be transmitted
to others
What is infectious disease epidemiology
D-r Mitova 21
1The clinical illness
The clinical illness maybe mild or moderate typical or atypical severe or fatal
Epidemiologically mild cases may be more important sources of infection than severe cases because they are ambulant and spread the infection wherever they go whereas severe cases usually confined to bed
D-r Mitova 22
2The subclinical cases are variously referred to as inapparent covert missed or abortive cases They are equally important sources of infection The disease agent may multiply in the host but does not manifest itself by sign and symptoms The disease agent is eliminated and contaminates the environment in the same way as clinical casesperson who are thus sick contribute more than symptomatic patient to the transmission of infection to others and what is more they do not appear in any of statistics
Subclinical cases play a dominant role in maintaining the chain of infection in the community The latent infection must be distinguished from subclinical infection In latent infection the host does not shed the infectious agent
D-r Mitova 23
The subclinical cases
Subclinical infection can be detected only by laboratory tests
Barring a few(mesles varicella) subclinical infection occurs in most infectious disease In some disease(rubella mumps polio hepatitis A and B influenza diphteria) a great deal of subclinical infection occurs
D-r Mitova 24
Latent infectionThe term latent infection must be
distinguished from subclinical infection In latent infection the host does not shed the infectious agent which lies dormant within the host without symptoms (and often without demonstrable presence in blood tissues or bodily secretions of the host) For examples latent infection occurs in herpes symplex Brill-Zincer disease
D-r Mitova 25
CARRIERS
In some disease either due to inadequate treatment or immune response the disease agent is not completely eliminated leading to a carrier state A carrier is defined as an infected person or animal that harbors a specific infectious agent in the absence of
discernible clinical disease and serves as a potential source of infection for others
D-r Mitova 26
CARRIERS
As a rule carriers are less infectious than cases but epidemiologically they are more dangerous than cases because they escape recognition and continuing as they do live a normal life among population or community they readily infect the susceptible individuals over a wider area and longer period of time under favorable conditionsthe ldquoTyphoid Maryrdquo is a classic example of a carrier-cooker Mary who gave rise to more than 1300 cases in her life time
D-r Mitova 27
The elements in a carrier state are
The presence in the body of disease agent
The absence of recognizable symptoms and signs of disease
The shedding of disease agent in the discharges or excretion
D-r Mitova 28
incubatory carriers are those who shed the infectious agent during the incubation period This usually occurs during the the last few days of incubation period As for example
Measles-the period of communicability is 4 days before the rush
Mumps-usually 4-6 days before onset of symptoms
Polio-7-10 days before onset of symptomsHepatitis B-for a month before
jaundiceabout 6 weeks after exposure
Carrier may be classified BY TYPE
D-r Mitova 29
Carrier may be classified BY TYPE
convalescent carriers that is those who continue to shed the disease agent during the period of convalescence(typhoid fever cholera diphteria bacillary disentery) In the disease clinical recovery does not coincide with bacteriological recovery
D-r Mitova 30
Carrier may be classified BY TYPE
healthy carriers they are victims of subclinical infection who have developed carrier state without suffering from overt disease but are nevertheless shedding the disease agent eg poliomyelitis cholera meningococcal meningitis salmonellosis diphtheria
D-r Mitova 31
BY DURATION
Temporary carriers are those who shed the infectious agent for short period of timeChronic carriers are those who excretes the infectious agent for indefinite periods eg typhoid fever hepatitis B dysentery etcThey are known to reintroduce disease into areas which are otherwise free of infection
D-r Mitova 32
Chronic carriers
Chronic carriers are far more important sources of infection than cases The longer the carrier state the greater the risk of community The duration of the carrier state varies with the disease In typhoid fever and hepatitis B the chronic carrier state may last for several yearsIn chronic dysentery it may last for year or longer In diphtheria the carrier state is associated with infected tonsils in typhoid fever with gall bladder disease
D-r Mitova 33
BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 4
There are several level of There are several level of infectioninfection
Colonization egSaureus in the skin and normal nasopharynxSubclinical or inapparent infection egpolioLatent infection egvirus of herpes symplexManifest or clinical infection
D-r Mitova 5
Infectious disease A clinically manifest disease of man or animal
resulting from an infectionContagious disease
A disease that is transmitted through contactCommunicable disease
All illness due to specific infectious agent or its toxic products capable of being directly or indirectly transmitted from man to man animal to animal or environment (through air dust soil water food etc) to man or animal
D-r Mitova 6
ContaminationContamination The presence of an infectious
agent on a body surface also in or on clothes bedding toys surgical instruments or dressings or other inanimate articles or substances including water milk and food (Lasts epidemiological definition)
D-r Mitova 7
Epidemic processEpidemic process
The epidemic process defined as a chain of interrelated and interconnected infectious conditions in the populationCommunicable diseases are transmitted from the reservoir or source of infection to susceptible host This is the medical model of epidemic process
D-r Mitova 8
Bacically there are 3 links in the chain of transmission-the reservoir mode of transmission and the susceptible host
Source Source reservoir reservoir of infectionof infection
ModesModesofof
transmissiontransmission
SusceptibleSusceptiblehosthost
D-r Mitova 9
Courses types of the Courses types of the epidemic processepidemic process
A disease is said to be sporadicsporadic when only a few cases occur here and there in a given region
OutbreakOutbreak defines as a smallusually localised epidemic
D-r Mitova 10
Courses types of the Courses types of the epidemic processepidemic process
Epidemic (from Greek epi- upon + demos Epidemic (from Greek epi- upon + demos people)-people)- outbreak of disease that affects a much greater number of people than is usual for the locality or that spreads to regions where it is ordinarily not present A disease that tends to be restricted to a particular region (endemic disease) can become epidemic if nonimmune persons are present in large numbers (as in time of war or during pilgrimages) if the infectious agent is more virulent than usual or if distribution of the disease is more easily effected Cholera and plague endemic in parts of Asia can become epidemic under the above conditions as can dysentery and many other infections Epidemics may also be caused by new disease agents in the human population such as the Ebola virus
D-r Mitova 11
Courses types of the Courses types of the epidemic processepidemic process
A pandemicpandemic (from Greek pan all + demos people) is an epidemic (an outbreak of an infectious disease) that spreads worldwide or at least across a large regionA worldwide epidemic is known as a pandemicpandemic eg the influenza pandemic of 1918 or the AIDS pandemic beginning in the 1980s
D-r Mitova 12
Examples Cholera Examples Cholera
first pandemic 1816ndash1826 Previously restricted to the Indian subcontinent the pandemic began in Bengal then spread across India by 1820 It extended as far as China and the Caspian Sea before recedingThe second pandemic (1829ndash1851) reached Europe London in 1832 Ontario Canada and New York in the same year and the Pacific coast of North America by 1834The third pandemic (1852ndash1860) mainly affected Russia with over a million deaths
D-r Mitova 13
Examples Cholera Examples Cholera
The fourth pandemic (1863ndash1875) spread mostly in Europe and AfricaIn 1866 there was an outbreak in North AmericaThe sixth pandemic (1899ndash1923) had little effect in Europe because of advances in public health but Russia was badly affected againThe seventh pandemic began in Indonesia in 1961 called El Tor after the strain and reached Bangladesh in 1963 India in 1964 and the USSR in 1966
D-r Mitova 14
Examples InfluenzaExamples Influenza
The Spanish fluSpanish flu 1918ndash1919
An estimated 17 million died in India 500000 in the United States and 200000 in the UK The virus was recently reconstructed by scientists at the CDC studying remains preserved by the Alaskan permafrost They identified it as a type of H1N1 virus
D-r Mitova 15
Courses types of the Courses types of the epidemic processepidemic process
In epidemiology an infection is said to be endemic endemic (from Greek en- in or within + demos people) in a population when that infection is maintained in the population without the need for external inputs For example chickenpox is endemic in the UK but malaria is not Every year there are a few cases of malaria acquired in the UK but these do not lead to sustained transmission in the population due to the lack of a suitable vector (mosquitoes of the genus Anopheles)
D-r Mitova 16
SOURCE AND RESERVOIR
The source of infection is defined as the person animal object or substance from which an infectious agent passes or disseminated to the host
A reservoir is defined as ldquoany person animal arthropod plant soil or substance in which an infectious agent lives and multiplies on which it depends primarily for survival and where it reproduced itself in such manner that it can be transmitted to a susceptible hostrdquo In short the reservoir is the natural habitual in which the organism metabolized and replicates
D-r Mitova 17
In Bulgaria the source of infection is defined only as the person or animal in which an infectious agent live and multiplies and from which an infectious agent passes to the host inanimate things and arthropods are defined as a vehicles of transmission
D-r Mitova 18
HUMAN RESERVOIR
By far the most important source or reservoir of infection for humans is man himself He my be a case or carrier Man is often described as him own enemy because most of the communicable disease of which man is heir to a contacted from human sources
D-r Mitova 19
HUMAN RESERVOIR - CASE
A case is defined as a person in the population or study group identified as having the particular disease health disorder or condition under investigation A variety of criteria ( clinical biochemical laboratory ) may be used to identify cases Broadly the presence of infection in the host may be clinical subclinical or latent
D-r Mitova 20
A case is a risk factor hellip Infection in one person can be transmitted
to others
What is infectious disease epidemiology
D-r Mitova 21
1The clinical illness
The clinical illness maybe mild or moderate typical or atypical severe or fatal
Epidemiologically mild cases may be more important sources of infection than severe cases because they are ambulant and spread the infection wherever they go whereas severe cases usually confined to bed
D-r Mitova 22
2The subclinical cases are variously referred to as inapparent covert missed or abortive cases They are equally important sources of infection The disease agent may multiply in the host but does not manifest itself by sign and symptoms The disease agent is eliminated and contaminates the environment in the same way as clinical casesperson who are thus sick contribute more than symptomatic patient to the transmission of infection to others and what is more they do not appear in any of statistics
Subclinical cases play a dominant role in maintaining the chain of infection in the community The latent infection must be distinguished from subclinical infection In latent infection the host does not shed the infectious agent
D-r Mitova 23
The subclinical cases
Subclinical infection can be detected only by laboratory tests
Barring a few(mesles varicella) subclinical infection occurs in most infectious disease In some disease(rubella mumps polio hepatitis A and B influenza diphteria) a great deal of subclinical infection occurs
D-r Mitova 24
Latent infectionThe term latent infection must be
distinguished from subclinical infection In latent infection the host does not shed the infectious agent which lies dormant within the host without symptoms (and often without demonstrable presence in blood tissues or bodily secretions of the host) For examples latent infection occurs in herpes symplex Brill-Zincer disease
D-r Mitova 25
CARRIERS
In some disease either due to inadequate treatment or immune response the disease agent is not completely eliminated leading to a carrier state A carrier is defined as an infected person or animal that harbors a specific infectious agent in the absence of
discernible clinical disease and serves as a potential source of infection for others
D-r Mitova 26
CARRIERS
As a rule carriers are less infectious than cases but epidemiologically they are more dangerous than cases because they escape recognition and continuing as they do live a normal life among population or community they readily infect the susceptible individuals over a wider area and longer period of time under favorable conditionsthe ldquoTyphoid Maryrdquo is a classic example of a carrier-cooker Mary who gave rise to more than 1300 cases in her life time
D-r Mitova 27
The elements in a carrier state are
The presence in the body of disease agent
The absence of recognizable symptoms and signs of disease
The shedding of disease agent in the discharges or excretion
D-r Mitova 28
incubatory carriers are those who shed the infectious agent during the incubation period This usually occurs during the the last few days of incubation period As for example
Measles-the period of communicability is 4 days before the rush
Mumps-usually 4-6 days before onset of symptoms
Polio-7-10 days before onset of symptomsHepatitis B-for a month before
jaundiceabout 6 weeks after exposure
Carrier may be classified BY TYPE
D-r Mitova 29
Carrier may be classified BY TYPE
convalescent carriers that is those who continue to shed the disease agent during the period of convalescence(typhoid fever cholera diphteria bacillary disentery) In the disease clinical recovery does not coincide with bacteriological recovery
D-r Mitova 30
Carrier may be classified BY TYPE
healthy carriers they are victims of subclinical infection who have developed carrier state without suffering from overt disease but are nevertheless shedding the disease agent eg poliomyelitis cholera meningococcal meningitis salmonellosis diphtheria
D-r Mitova 31
BY DURATION
Temporary carriers are those who shed the infectious agent for short period of timeChronic carriers are those who excretes the infectious agent for indefinite periods eg typhoid fever hepatitis B dysentery etcThey are known to reintroduce disease into areas which are otherwise free of infection
D-r Mitova 32
Chronic carriers
Chronic carriers are far more important sources of infection than cases The longer the carrier state the greater the risk of community The duration of the carrier state varies with the disease In typhoid fever and hepatitis B the chronic carrier state may last for several yearsIn chronic dysentery it may last for year or longer In diphtheria the carrier state is associated with infected tonsils in typhoid fever with gall bladder disease
D-r Mitova 33
BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 5
Infectious disease A clinically manifest disease of man or animal
resulting from an infectionContagious disease
A disease that is transmitted through contactCommunicable disease
All illness due to specific infectious agent or its toxic products capable of being directly or indirectly transmitted from man to man animal to animal or environment (through air dust soil water food etc) to man or animal
D-r Mitova 6
ContaminationContamination The presence of an infectious
agent on a body surface also in or on clothes bedding toys surgical instruments or dressings or other inanimate articles or substances including water milk and food (Lasts epidemiological definition)
D-r Mitova 7
Epidemic processEpidemic process
The epidemic process defined as a chain of interrelated and interconnected infectious conditions in the populationCommunicable diseases are transmitted from the reservoir or source of infection to susceptible host This is the medical model of epidemic process
D-r Mitova 8
Bacically there are 3 links in the chain of transmission-the reservoir mode of transmission and the susceptible host
Source Source reservoir reservoir of infectionof infection
ModesModesofof
transmissiontransmission
SusceptibleSusceptiblehosthost
D-r Mitova 9
Courses types of the Courses types of the epidemic processepidemic process
A disease is said to be sporadicsporadic when only a few cases occur here and there in a given region
OutbreakOutbreak defines as a smallusually localised epidemic
D-r Mitova 10
Courses types of the Courses types of the epidemic processepidemic process
Epidemic (from Greek epi- upon + demos Epidemic (from Greek epi- upon + demos people)-people)- outbreak of disease that affects a much greater number of people than is usual for the locality or that spreads to regions where it is ordinarily not present A disease that tends to be restricted to a particular region (endemic disease) can become epidemic if nonimmune persons are present in large numbers (as in time of war or during pilgrimages) if the infectious agent is more virulent than usual or if distribution of the disease is more easily effected Cholera and plague endemic in parts of Asia can become epidemic under the above conditions as can dysentery and many other infections Epidemics may also be caused by new disease agents in the human population such as the Ebola virus
D-r Mitova 11
Courses types of the Courses types of the epidemic processepidemic process
A pandemicpandemic (from Greek pan all + demos people) is an epidemic (an outbreak of an infectious disease) that spreads worldwide or at least across a large regionA worldwide epidemic is known as a pandemicpandemic eg the influenza pandemic of 1918 or the AIDS pandemic beginning in the 1980s
D-r Mitova 12
Examples Cholera Examples Cholera
first pandemic 1816ndash1826 Previously restricted to the Indian subcontinent the pandemic began in Bengal then spread across India by 1820 It extended as far as China and the Caspian Sea before recedingThe second pandemic (1829ndash1851) reached Europe London in 1832 Ontario Canada and New York in the same year and the Pacific coast of North America by 1834The third pandemic (1852ndash1860) mainly affected Russia with over a million deaths
D-r Mitova 13
Examples Cholera Examples Cholera
The fourth pandemic (1863ndash1875) spread mostly in Europe and AfricaIn 1866 there was an outbreak in North AmericaThe sixth pandemic (1899ndash1923) had little effect in Europe because of advances in public health but Russia was badly affected againThe seventh pandemic began in Indonesia in 1961 called El Tor after the strain and reached Bangladesh in 1963 India in 1964 and the USSR in 1966
D-r Mitova 14
Examples InfluenzaExamples Influenza
The Spanish fluSpanish flu 1918ndash1919
An estimated 17 million died in India 500000 in the United States and 200000 in the UK The virus was recently reconstructed by scientists at the CDC studying remains preserved by the Alaskan permafrost They identified it as a type of H1N1 virus
D-r Mitova 15
Courses types of the Courses types of the epidemic processepidemic process
In epidemiology an infection is said to be endemic endemic (from Greek en- in or within + demos people) in a population when that infection is maintained in the population without the need for external inputs For example chickenpox is endemic in the UK but malaria is not Every year there are a few cases of malaria acquired in the UK but these do not lead to sustained transmission in the population due to the lack of a suitable vector (mosquitoes of the genus Anopheles)
D-r Mitova 16
SOURCE AND RESERVOIR
The source of infection is defined as the person animal object or substance from which an infectious agent passes or disseminated to the host
A reservoir is defined as ldquoany person animal arthropod plant soil or substance in which an infectious agent lives and multiplies on which it depends primarily for survival and where it reproduced itself in such manner that it can be transmitted to a susceptible hostrdquo In short the reservoir is the natural habitual in which the organism metabolized and replicates
D-r Mitova 17
In Bulgaria the source of infection is defined only as the person or animal in which an infectious agent live and multiplies and from which an infectious agent passes to the host inanimate things and arthropods are defined as a vehicles of transmission
D-r Mitova 18
HUMAN RESERVOIR
By far the most important source or reservoir of infection for humans is man himself He my be a case or carrier Man is often described as him own enemy because most of the communicable disease of which man is heir to a contacted from human sources
D-r Mitova 19
HUMAN RESERVOIR - CASE
A case is defined as a person in the population or study group identified as having the particular disease health disorder or condition under investigation A variety of criteria ( clinical biochemical laboratory ) may be used to identify cases Broadly the presence of infection in the host may be clinical subclinical or latent
D-r Mitova 20
A case is a risk factor hellip Infection in one person can be transmitted
to others
What is infectious disease epidemiology
D-r Mitova 21
1The clinical illness
The clinical illness maybe mild or moderate typical or atypical severe or fatal
Epidemiologically mild cases may be more important sources of infection than severe cases because they are ambulant and spread the infection wherever they go whereas severe cases usually confined to bed
D-r Mitova 22
2The subclinical cases are variously referred to as inapparent covert missed or abortive cases They are equally important sources of infection The disease agent may multiply in the host but does not manifest itself by sign and symptoms The disease agent is eliminated and contaminates the environment in the same way as clinical casesperson who are thus sick contribute more than symptomatic patient to the transmission of infection to others and what is more they do not appear in any of statistics
Subclinical cases play a dominant role in maintaining the chain of infection in the community The latent infection must be distinguished from subclinical infection In latent infection the host does not shed the infectious agent
D-r Mitova 23
The subclinical cases
Subclinical infection can be detected only by laboratory tests
Barring a few(mesles varicella) subclinical infection occurs in most infectious disease In some disease(rubella mumps polio hepatitis A and B influenza diphteria) a great deal of subclinical infection occurs
D-r Mitova 24
Latent infectionThe term latent infection must be
distinguished from subclinical infection In latent infection the host does not shed the infectious agent which lies dormant within the host without symptoms (and often without demonstrable presence in blood tissues or bodily secretions of the host) For examples latent infection occurs in herpes symplex Brill-Zincer disease
D-r Mitova 25
CARRIERS
In some disease either due to inadequate treatment or immune response the disease agent is not completely eliminated leading to a carrier state A carrier is defined as an infected person or animal that harbors a specific infectious agent in the absence of
discernible clinical disease and serves as a potential source of infection for others
D-r Mitova 26
CARRIERS
As a rule carriers are less infectious than cases but epidemiologically they are more dangerous than cases because they escape recognition and continuing as they do live a normal life among population or community they readily infect the susceptible individuals over a wider area and longer period of time under favorable conditionsthe ldquoTyphoid Maryrdquo is a classic example of a carrier-cooker Mary who gave rise to more than 1300 cases in her life time
D-r Mitova 27
The elements in a carrier state are
The presence in the body of disease agent
The absence of recognizable symptoms and signs of disease
The shedding of disease agent in the discharges or excretion
D-r Mitova 28
incubatory carriers are those who shed the infectious agent during the incubation period This usually occurs during the the last few days of incubation period As for example
Measles-the period of communicability is 4 days before the rush
Mumps-usually 4-6 days before onset of symptoms
Polio-7-10 days before onset of symptomsHepatitis B-for a month before
jaundiceabout 6 weeks after exposure
Carrier may be classified BY TYPE
D-r Mitova 29
Carrier may be classified BY TYPE
convalescent carriers that is those who continue to shed the disease agent during the period of convalescence(typhoid fever cholera diphteria bacillary disentery) In the disease clinical recovery does not coincide with bacteriological recovery
D-r Mitova 30
Carrier may be classified BY TYPE
healthy carriers they are victims of subclinical infection who have developed carrier state without suffering from overt disease but are nevertheless shedding the disease agent eg poliomyelitis cholera meningococcal meningitis salmonellosis diphtheria
D-r Mitova 31
BY DURATION
Temporary carriers are those who shed the infectious agent for short period of timeChronic carriers are those who excretes the infectious agent for indefinite periods eg typhoid fever hepatitis B dysentery etcThey are known to reintroduce disease into areas which are otherwise free of infection
D-r Mitova 32
Chronic carriers
Chronic carriers are far more important sources of infection than cases The longer the carrier state the greater the risk of community The duration of the carrier state varies with the disease In typhoid fever and hepatitis B the chronic carrier state may last for several yearsIn chronic dysentery it may last for year or longer In diphtheria the carrier state is associated with infected tonsils in typhoid fever with gall bladder disease
D-r Mitova 33
BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 6
ContaminationContamination The presence of an infectious
agent on a body surface also in or on clothes bedding toys surgical instruments or dressings or other inanimate articles or substances including water milk and food (Lasts epidemiological definition)
D-r Mitova 7
Epidemic processEpidemic process
The epidemic process defined as a chain of interrelated and interconnected infectious conditions in the populationCommunicable diseases are transmitted from the reservoir or source of infection to susceptible host This is the medical model of epidemic process
D-r Mitova 8
Bacically there are 3 links in the chain of transmission-the reservoir mode of transmission and the susceptible host
Source Source reservoir reservoir of infectionof infection
ModesModesofof
transmissiontransmission
SusceptibleSusceptiblehosthost
D-r Mitova 9
Courses types of the Courses types of the epidemic processepidemic process
A disease is said to be sporadicsporadic when only a few cases occur here and there in a given region
OutbreakOutbreak defines as a smallusually localised epidemic
D-r Mitova 10
Courses types of the Courses types of the epidemic processepidemic process
Epidemic (from Greek epi- upon + demos Epidemic (from Greek epi- upon + demos people)-people)- outbreak of disease that affects a much greater number of people than is usual for the locality or that spreads to regions where it is ordinarily not present A disease that tends to be restricted to a particular region (endemic disease) can become epidemic if nonimmune persons are present in large numbers (as in time of war or during pilgrimages) if the infectious agent is more virulent than usual or if distribution of the disease is more easily effected Cholera and plague endemic in parts of Asia can become epidemic under the above conditions as can dysentery and many other infections Epidemics may also be caused by new disease agents in the human population such as the Ebola virus
D-r Mitova 11
Courses types of the Courses types of the epidemic processepidemic process
A pandemicpandemic (from Greek pan all + demos people) is an epidemic (an outbreak of an infectious disease) that spreads worldwide or at least across a large regionA worldwide epidemic is known as a pandemicpandemic eg the influenza pandemic of 1918 or the AIDS pandemic beginning in the 1980s
D-r Mitova 12
Examples Cholera Examples Cholera
first pandemic 1816ndash1826 Previously restricted to the Indian subcontinent the pandemic began in Bengal then spread across India by 1820 It extended as far as China and the Caspian Sea before recedingThe second pandemic (1829ndash1851) reached Europe London in 1832 Ontario Canada and New York in the same year and the Pacific coast of North America by 1834The third pandemic (1852ndash1860) mainly affected Russia with over a million deaths
D-r Mitova 13
Examples Cholera Examples Cholera
The fourth pandemic (1863ndash1875) spread mostly in Europe and AfricaIn 1866 there was an outbreak in North AmericaThe sixth pandemic (1899ndash1923) had little effect in Europe because of advances in public health but Russia was badly affected againThe seventh pandemic began in Indonesia in 1961 called El Tor after the strain and reached Bangladesh in 1963 India in 1964 and the USSR in 1966
D-r Mitova 14
Examples InfluenzaExamples Influenza
The Spanish fluSpanish flu 1918ndash1919
An estimated 17 million died in India 500000 in the United States and 200000 in the UK The virus was recently reconstructed by scientists at the CDC studying remains preserved by the Alaskan permafrost They identified it as a type of H1N1 virus
D-r Mitova 15
Courses types of the Courses types of the epidemic processepidemic process
In epidemiology an infection is said to be endemic endemic (from Greek en- in or within + demos people) in a population when that infection is maintained in the population without the need for external inputs For example chickenpox is endemic in the UK but malaria is not Every year there are a few cases of malaria acquired in the UK but these do not lead to sustained transmission in the population due to the lack of a suitable vector (mosquitoes of the genus Anopheles)
D-r Mitova 16
SOURCE AND RESERVOIR
The source of infection is defined as the person animal object or substance from which an infectious agent passes or disseminated to the host
A reservoir is defined as ldquoany person animal arthropod plant soil or substance in which an infectious agent lives and multiplies on which it depends primarily for survival and where it reproduced itself in such manner that it can be transmitted to a susceptible hostrdquo In short the reservoir is the natural habitual in which the organism metabolized and replicates
D-r Mitova 17
In Bulgaria the source of infection is defined only as the person or animal in which an infectious agent live and multiplies and from which an infectious agent passes to the host inanimate things and arthropods are defined as a vehicles of transmission
D-r Mitova 18
HUMAN RESERVOIR
By far the most important source or reservoir of infection for humans is man himself He my be a case or carrier Man is often described as him own enemy because most of the communicable disease of which man is heir to a contacted from human sources
D-r Mitova 19
HUMAN RESERVOIR - CASE
A case is defined as a person in the population or study group identified as having the particular disease health disorder or condition under investigation A variety of criteria ( clinical biochemical laboratory ) may be used to identify cases Broadly the presence of infection in the host may be clinical subclinical or latent
D-r Mitova 20
A case is a risk factor hellip Infection in one person can be transmitted
to others
What is infectious disease epidemiology
D-r Mitova 21
1The clinical illness
The clinical illness maybe mild or moderate typical or atypical severe or fatal
Epidemiologically mild cases may be more important sources of infection than severe cases because they are ambulant and spread the infection wherever they go whereas severe cases usually confined to bed
D-r Mitova 22
2The subclinical cases are variously referred to as inapparent covert missed or abortive cases They are equally important sources of infection The disease agent may multiply in the host but does not manifest itself by sign and symptoms The disease agent is eliminated and contaminates the environment in the same way as clinical casesperson who are thus sick contribute more than symptomatic patient to the transmission of infection to others and what is more they do not appear in any of statistics
Subclinical cases play a dominant role in maintaining the chain of infection in the community The latent infection must be distinguished from subclinical infection In latent infection the host does not shed the infectious agent
D-r Mitova 23
The subclinical cases
Subclinical infection can be detected only by laboratory tests
Barring a few(mesles varicella) subclinical infection occurs in most infectious disease In some disease(rubella mumps polio hepatitis A and B influenza diphteria) a great deal of subclinical infection occurs
D-r Mitova 24
Latent infectionThe term latent infection must be
distinguished from subclinical infection In latent infection the host does not shed the infectious agent which lies dormant within the host without symptoms (and often without demonstrable presence in blood tissues or bodily secretions of the host) For examples latent infection occurs in herpes symplex Brill-Zincer disease
D-r Mitova 25
CARRIERS
In some disease either due to inadequate treatment or immune response the disease agent is not completely eliminated leading to a carrier state A carrier is defined as an infected person or animal that harbors a specific infectious agent in the absence of
discernible clinical disease and serves as a potential source of infection for others
D-r Mitova 26
CARRIERS
As a rule carriers are less infectious than cases but epidemiologically they are more dangerous than cases because they escape recognition and continuing as they do live a normal life among population or community they readily infect the susceptible individuals over a wider area and longer period of time under favorable conditionsthe ldquoTyphoid Maryrdquo is a classic example of a carrier-cooker Mary who gave rise to more than 1300 cases in her life time
D-r Mitova 27
The elements in a carrier state are
The presence in the body of disease agent
The absence of recognizable symptoms and signs of disease
The shedding of disease agent in the discharges or excretion
D-r Mitova 28
incubatory carriers are those who shed the infectious agent during the incubation period This usually occurs during the the last few days of incubation period As for example
Measles-the period of communicability is 4 days before the rush
Mumps-usually 4-6 days before onset of symptoms
Polio-7-10 days before onset of symptomsHepatitis B-for a month before
jaundiceabout 6 weeks after exposure
Carrier may be classified BY TYPE
D-r Mitova 29
Carrier may be classified BY TYPE
convalescent carriers that is those who continue to shed the disease agent during the period of convalescence(typhoid fever cholera diphteria bacillary disentery) In the disease clinical recovery does not coincide with bacteriological recovery
D-r Mitova 30
Carrier may be classified BY TYPE
healthy carriers they are victims of subclinical infection who have developed carrier state without suffering from overt disease but are nevertheless shedding the disease agent eg poliomyelitis cholera meningococcal meningitis salmonellosis diphtheria
D-r Mitova 31
BY DURATION
Temporary carriers are those who shed the infectious agent for short period of timeChronic carriers are those who excretes the infectious agent for indefinite periods eg typhoid fever hepatitis B dysentery etcThey are known to reintroduce disease into areas which are otherwise free of infection
D-r Mitova 32
Chronic carriers
Chronic carriers are far more important sources of infection than cases The longer the carrier state the greater the risk of community The duration of the carrier state varies with the disease In typhoid fever and hepatitis B the chronic carrier state may last for several yearsIn chronic dysentery it may last for year or longer In diphtheria the carrier state is associated with infected tonsils in typhoid fever with gall bladder disease
D-r Mitova 33
BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 7
Epidemic processEpidemic process
The epidemic process defined as a chain of interrelated and interconnected infectious conditions in the populationCommunicable diseases are transmitted from the reservoir or source of infection to susceptible host This is the medical model of epidemic process
D-r Mitova 8
Bacically there are 3 links in the chain of transmission-the reservoir mode of transmission and the susceptible host
Source Source reservoir reservoir of infectionof infection
ModesModesofof
transmissiontransmission
SusceptibleSusceptiblehosthost
D-r Mitova 9
Courses types of the Courses types of the epidemic processepidemic process
A disease is said to be sporadicsporadic when only a few cases occur here and there in a given region
OutbreakOutbreak defines as a smallusually localised epidemic
D-r Mitova 10
Courses types of the Courses types of the epidemic processepidemic process
Epidemic (from Greek epi- upon + demos Epidemic (from Greek epi- upon + demos people)-people)- outbreak of disease that affects a much greater number of people than is usual for the locality or that spreads to regions where it is ordinarily not present A disease that tends to be restricted to a particular region (endemic disease) can become epidemic if nonimmune persons are present in large numbers (as in time of war or during pilgrimages) if the infectious agent is more virulent than usual or if distribution of the disease is more easily effected Cholera and plague endemic in parts of Asia can become epidemic under the above conditions as can dysentery and many other infections Epidemics may also be caused by new disease agents in the human population such as the Ebola virus
D-r Mitova 11
Courses types of the Courses types of the epidemic processepidemic process
A pandemicpandemic (from Greek pan all + demos people) is an epidemic (an outbreak of an infectious disease) that spreads worldwide or at least across a large regionA worldwide epidemic is known as a pandemicpandemic eg the influenza pandemic of 1918 or the AIDS pandemic beginning in the 1980s
D-r Mitova 12
Examples Cholera Examples Cholera
first pandemic 1816ndash1826 Previously restricted to the Indian subcontinent the pandemic began in Bengal then spread across India by 1820 It extended as far as China and the Caspian Sea before recedingThe second pandemic (1829ndash1851) reached Europe London in 1832 Ontario Canada and New York in the same year and the Pacific coast of North America by 1834The third pandemic (1852ndash1860) mainly affected Russia with over a million deaths
D-r Mitova 13
Examples Cholera Examples Cholera
The fourth pandemic (1863ndash1875) spread mostly in Europe and AfricaIn 1866 there was an outbreak in North AmericaThe sixth pandemic (1899ndash1923) had little effect in Europe because of advances in public health but Russia was badly affected againThe seventh pandemic began in Indonesia in 1961 called El Tor after the strain and reached Bangladesh in 1963 India in 1964 and the USSR in 1966
D-r Mitova 14
Examples InfluenzaExamples Influenza
The Spanish fluSpanish flu 1918ndash1919
An estimated 17 million died in India 500000 in the United States and 200000 in the UK The virus was recently reconstructed by scientists at the CDC studying remains preserved by the Alaskan permafrost They identified it as a type of H1N1 virus
D-r Mitova 15
Courses types of the Courses types of the epidemic processepidemic process
In epidemiology an infection is said to be endemic endemic (from Greek en- in or within + demos people) in a population when that infection is maintained in the population without the need for external inputs For example chickenpox is endemic in the UK but malaria is not Every year there are a few cases of malaria acquired in the UK but these do not lead to sustained transmission in the population due to the lack of a suitable vector (mosquitoes of the genus Anopheles)
D-r Mitova 16
SOURCE AND RESERVOIR
The source of infection is defined as the person animal object or substance from which an infectious agent passes or disseminated to the host
A reservoir is defined as ldquoany person animal arthropod plant soil or substance in which an infectious agent lives and multiplies on which it depends primarily for survival and where it reproduced itself in such manner that it can be transmitted to a susceptible hostrdquo In short the reservoir is the natural habitual in which the organism metabolized and replicates
D-r Mitova 17
In Bulgaria the source of infection is defined only as the person or animal in which an infectious agent live and multiplies and from which an infectious agent passes to the host inanimate things and arthropods are defined as a vehicles of transmission
D-r Mitova 18
HUMAN RESERVOIR
By far the most important source or reservoir of infection for humans is man himself He my be a case or carrier Man is often described as him own enemy because most of the communicable disease of which man is heir to a contacted from human sources
D-r Mitova 19
HUMAN RESERVOIR - CASE
A case is defined as a person in the population or study group identified as having the particular disease health disorder or condition under investigation A variety of criteria ( clinical biochemical laboratory ) may be used to identify cases Broadly the presence of infection in the host may be clinical subclinical or latent
D-r Mitova 20
A case is a risk factor hellip Infection in one person can be transmitted
to others
What is infectious disease epidemiology
D-r Mitova 21
1The clinical illness
The clinical illness maybe mild or moderate typical or atypical severe or fatal
Epidemiologically mild cases may be more important sources of infection than severe cases because they are ambulant and spread the infection wherever they go whereas severe cases usually confined to bed
D-r Mitova 22
2The subclinical cases are variously referred to as inapparent covert missed or abortive cases They are equally important sources of infection The disease agent may multiply in the host but does not manifest itself by sign and symptoms The disease agent is eliminated and contaminates the environment in the same way as clinical casesperson who are thus sick contribute more than symptomatic patient to the transmission of infection to others and what is more they do not appear in any of statistics
Subclinical cases play a dominant role in maintaining the chain of infection in the community The latent infection must be distinguished from subclinical infection In latent infection the host does not shed the infectious agent
D-r Mitova 23
The subclinical cases
Subclinical infection can be detected only by laboratory tests
Barring a few(mesles varicella) subclinical infection occurs in most infectious disease In some disease(rubella mumps polio hepatitis A and B influenza diphteria) a great deal of subclinical infection occurs
D-r Mitova 24
Latent infectionThe term latent infection must be
distinguished from subclinical infection In latent infection the host does not shed the infectious agent which lies dormant within the host without symptoms (and often without demonstrable presence in blood tissues or bodily secretions of the host) For examples latent infection occurs in herpes symplex Brill-Zincer disease
D-r Mitova 25
CARRIERS
In some disease either due to inadequate treatment or immune response the disease agent is not completely eliminated leading to a carrier state A carrier is defined as an infected person or animal that harbors a specific infectious agent in the absence of
discernible clinical disease and serves as a potential source of infection for others
D-r Mitova 26
CARRIERS
As a rule carriers are less infectious than cases but epidemiologically they are more dangerous than cases because they escape recognition and continuing as they do live a normal life among population or community they readily infect the susceptible individuals over a wider area and longer period of time under favorable conditionsthe ldquoTyphoid Maryrdquo is a classic example of a carrier-cooker Mary who gave rise to more than 1300 cases in her life time
D-r Mitova 27
The elements in a carrier state are
The presence in the body of disease agent
The absence of recognizable symptoms and signs of disease
The shedding of disease agent in the discharges or excretion
D-r Mitova 28
incubatory carriers are those who shed the infectious agent during the incubation period This usually occurs during the the last few days of incubation period As for example
Measles-the period of communicability is 4 days before the rush
Mumps-usually 4-6 days before onset of symptoms
Polio-7-10 days before onset of symptomsHepatitis B-for a month before
jaundiceabout 6 weeks after exposure
Carrier may be classified BY TYPE
D-r Mitova 29
Carrier may be classified BY TYPE
convalescent carriers that is those who continue to shed the disease agent during the period of convalescence(typhoid fever cholera diphteria bacillary disentery) In the disease clinical recovery does not coincide with bacteriological recovery
D-r Mitova 30
Carrier may be classified BY TYPE
healthy carriers they are victims of subclinical infection who have developed carrier state without suffering from overt disease but are nevertheless shedding the disease agent eg poliomyelitis cholera meningococcal meningitis salmonellosis diphtheria
D-r Mitova 31
BY DURATION
Temporary carriers are those who shed the infectious agent for short period of timeChronic carriers are those who excretes the infectious agent for indefinite periods eg typhoid fever hepatitis B dysentery etcThey are known to reintroduce disease into areas which are otherwise free of infection
D-r Mitova 32
Chronic carriers
Chronic carriers are far more important sources of infection than cases The longer the carrier state the greater the risk of community The duration of the carrier state varies with the disease In typhoid fever and hepatitis B the chronic carrier state may last for several yearsIn chronic dysentery it may last for year or longer In diphtheria the carrier state is associated with infected tonsils in typhoid fever with gall bladder disease
D-r Mitova 33
BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 8
Bacically there are 3 links in the chain of transmission-the reservoir mode of transmission and the susceptible host
Source Source reservoir reservoir of infectionof infection
ModesModesofof
transmissiontransmission
SusceptibleSusceptiblehosthost
D-r Mitova 9
Courses types of the Courses types of the epidemic processepidemic process
A disease is said to be sporadicsporadic when only a few cases occur here and there in a given region
OutbreakOutbreak defines as a smallusually localised epidemic
D-r Mitova 10
Courses types of the Courses types of the epidemic processepidemic process
Epidemic (from Greek epi- upon + demos Epidemic (from Greek epi- upon + demos people)-people)- outbreak of disease that affects a much greater number of people than is usual for the locality or that spreads to regions where it is ordinarily not present A disease that tends to be restricted to a particular region (endemic disease) can become epidemic if nonimmune persons are present in large numbers (as in time of war or during pilgrimages) if the infectious agent is more virulent than usual or if distribution of the disease is more easily effected Cholera and plague endemic in parts of Asia can become epidemic under the above conditions as can dysentery and many other infections Epidemics may also be caused by new disease agents in the human population such as the Ebola virus
D-r Mitova 11
Courses types of the Courses types of the epidemic processepidemic process
A pandemicpandemic (from Greek pan all + demos people) is an epidemic (an outbreak of an infectious disease) that spreads worldwide or at least across a large regionA worldwide epidemic is known as a pandemicpandemic eg the influenza pandemic of 1918 or the AIDS pandemic beginning in the 1980s
D-r Mitova 12
Examples Cholera Examples Cholera
first pandemic 1816ndash1826 Previously restricted to the Indian subcontinent the pandemic began in Bengal then spread across India by 1820 It extended as far as China and the Caspian Sea before recedingThe second pandemic (1829ndash1851) reached Europe London in 1832 Ontario Canada and New York in the same year and the Pacific coast of North America by 1834The third pandemic (1852ndash1860) mainly affected Russia with over a million deaths
D-r Mitova 13
Examples Cholera Examples Cholera
The fourth pandemic (1863ndash1875) spread mostly in Europe and AfricaIn 1866 there was an outbreak in North AmericaThe sixth pandemic (1899ndash1923) had little effect in Europe because of advances in public health but Russia was badly affected againThe seventh pandemic began in Indonesia in 1961 called El Tor after the strain and reached Bangladesh in 1963 India in 1964 and the USSR in 1966
D-r Mitova 14
Examples InfluenzaExamples Influenza
The Spanish fluSpanish flu 1918ndash1919
An estimated 17 million died in India 500000 in the United States and 200000 in the UK The virus was recently reconstructed by scientists at the CDC studying remains preserved by the Alaskan permafrost They identified it as a type of H1N1 virus
D-r Mitova 15
Courses types of the Courses types of the epidemic processepidemic process
In epidemiology an infection is said to be endemic endemic (from Greek en- in or within + demos people) in a population when that infection is maintained in the population without the need for external inputs For example chickenpox is endemic in the UK but malaria is not Every year there are a few cases of malaria acquired in the UK but these do not lead to sustained transmission in the population due to the lack of a suitable vector (mosquitoes of the genus Anopheles)
D-r Mitova 16
SOURCE AND RESERVOIR
The source of infection is defined as the person animal object or substance from which an infectious agent passes or disseminated to the host
A reservoir is defined as ldquoany person animal arthropod plant soil or substance in which an infectious agent lives and multiplies on which it depends primarily for survival and where it reproduced itself in such manner that it can be transmitted to a susceptible hostrdquo In short the reservoir is the natural habitual in which the organism metabolized and replicates
D-r Mitova 17
In Bulgaria the source of infection is defined only as the person or animal in which an infectious agent live and multiplies and from which an infectious agent passes to the host inanimate things and arthropods are defined as a vehicles of transmission
D-r Mitova 18
HUMAN RESERVOIR
By far the most important source or reservoir of infection for humans is man himself He my be a case or carrier Man is often described as him own enemy because most of the communicable disease of which man is heir to a contacted from human sources
D-r Mitova 19
HUMAN RESERVOIR - CASE
A case is defined as a person in the population or study group identified as having the particular disease health disorder or condition under investigation A variety of criteria ( clinical biochemical laboratory ) may be used to identify cases Broadly the presence of infection in the host may be clinical subclinical or latent
D-r Mitova 20
A case is a risk factor hellip Infection in one person can be transmitted
to others
What is infectious disease epidemiology
D-r Mitova 21
1The clinical illness
The clinical illness maybe mild or moderate typical or atypical severe or fatal
Epidemiologically mild cases may be more important sources of infection than severe cases because they are ambulant and spread the infection wherever they go whereas severe cases usually confined to bed
D-r Mitova 22
2The subclinical cases are variously referred to as inapparent covert missed or abortive cases They are equally important sources of infection The disease agent may multiply in the host but does not manifest itself by sign and symptoms The disease agent is eliminated and contaminates the environment in the same way as clinical casesperson who are thus sick contribute more than symptomatic patient to the transmission of infection to others and what is more they do not appear in any of statistics
Subclinical cases play a dominant role in maintaining the chain of infection in the community The latent infection must be distinguished from subclinical infection In latent infection the host does not shed the infectious agent
D-r Mitova 23
The subclinical cases
Subclinical infection can be detected only by laboratory tests
Barring a few(mesles varicella) subclinical infection occurs in most infectious disease In some disease(rubella mumps polio hepatitis A and B influenza diphteria) a great deal of subclinical infection occurs
D-r Mitova 24
Latent infectionThe term latent infection must be
distinguished from subclinical infection In latent infection the host does not shed the infectious agent which lies dormant within the host without symptoms (and often without demonstrable presence in blood tissues or bodily secretions of the host) For examples latent infection occurs in herpes symplex Brill-Zincer disease
D-r Mitova 25
CARRIERS
In some disease either due to inadequate treatment or immune response the disease agent is not completely eliminated leading to a carrier state A carrier is defined as an infected person or animal that harbors a specific infectious agent in the absence of
discernible clinical disease and serves as a potential source of infection for others
D-r Mitova 26
CARRIERS
As a rule carriers are less infectious than cases but epidemiologically they are more dangerous than cases because they escape recognition and continuing as they do live a normal life among population or community they readily infect the susceptible individuals over a wider area and longer period of time under favorable conditionsthe ldquoTyphoid Maryrdquo is a classic example of a carrier-cooker Mary who gave rise to more than 1300 cases in her life time
D-r Mitova 27
The elements in a carrier state are
The presence in the body of disease agent
The absence of recognizable symptoms and signs of disease
The shedding of disease agent in the discharges or excretion
D-r Mitova 28
incubatory carriers are those who shed the infectious agent during the incubation period This usually occurs during the the last few days of incubation period As for example
Measles-the period of communicability is 4 days before the rush
Mumps-usually 4-6 days before onset of symptoms
Polio-7-10 days before onset of symptomsHepatitis B-for a month before
jaundiceabout 6 weeks after exposure
Carrier may be classified BY TYPE
D-r Mitova 29
Carrier may be classified BY TYPE
convalescent carriers that is those who continue to shed the disease agent during the period of convalescence(typhoid fever cholera diphteria bacillary disentery) In the disease clinical recovery does not coincide with bacteriological recovery
D-r Mitova 30
Carrier may be classified BY TYPE
healthy carriers they are victims of subclinical infection who have developed carrier state without suffering from overt disease but are nevertheless shedding the disease agent eg poliomyelitis cholera meningococcal meningitis salmonellosis diphtheria
D-r Mitova 31
BY DURATION
Temporary carriers are those who shed the infectious agent for short period of timeChronic carriers are those who excretes the infectious agent for indefinite periods eg typhoid fever hepatitis B dysentery etcThey are known to reintroduce disease into areas which are otherwise free of infection
D-r Mitova 32
Chronic carriers
Chronic carriers are far more important sources of infection than cases The longer the carrier state the greater the risk of community The duration of the carrier state varies with the disease In typhoid fever and hepatitis B the chronic carrier state may last for several yearsIn chronic dysentery it may last for year or longer In diphtheria the carrier state is associated with infected tonsils in typhoid fever with gall bladder disease
D-r Mitova 33
BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 9
Courses types of the Courses types of the epidemic processepidemic process
A disease is said to be sporadicsporadic when only a few cases occur here and there in a given region
OutbreakOutbreak defines as a smallusually localised epidemic
D-r Mitova 10
Courses types of the Courses types of the epidemic processepidemic process
Epidemic (from Greek epi- upon + demos Epidemic (from Greek epi- upon + demos people)-people)- outbreak of disease that affects a much greater number of people than is usual for the locality or that spreads to regions where it is ordinarily not present A disease that tends to be restricted to a particular region (endemic disease) can become epidemic if nonimmune persons are present in large numbers (as in time of war or during pilgrimages) if the infectious agent is more virulent than usual or if distribution of the disease is more easily effected Cholera and plague endemic in parts of Asia can become epidemic under the above conditions as can dysentery and many other infections Epidemics may also be caused by new disease agents in the human population such as the Ebola virus
D-r Mitova 11
Courses types of the Courses types of the epidemic processepidemic process
A pandemicpandemic (from Greek pan all + demos people) is an epidemic (an outbreak of an infectious disease) that spreads worldwide or at least across a large regionA worldwide epidemic is known as a pandemicpandemic eg the influenza pandemic of 1918 or the AIDS pandemic beginning in the 1980s
D-r Mitova 12
Examples Cholera Examples Cholera
first pandemic 1816ndash1826 Previously restricted to the Indian subcontinent the pandemic began in Bengal then spread across India by 1820 It extended as far as China and the Caspian Sea before recedingThe second pandemic (1829ndash1851) reached Europe London in 1832 Ontario Canada and New York in the same year and the Pacific coast of North America by 1834The third pandemic (1852ndash1860) mainly affected Russia with over a million deaths
D-r Mitova 13
Examples Cholera Examples Cholera
The fourth pandemic (1863ndash1875) spread mostly in Europe and AfricaIn 1866 there was an outbreak in North AmericaThe sixth pandemic (1899ndash1923) had little effect in Europe because of advances in public health but Russia was badly affected againThe seventh pandemic began in Indonesia in 1961 called El Tor after the strain and reached Bangladesh in 1963 India in 1964 and the USSR in 1966
D-r Mitova 14
Examples InfluenzaExamples Influenza
The Spanish fluSpanish flu 1918ndash1919
An estimated 17 million died in India 500000 in the United States and 200000 in the UK The virus was recently reconstructed by scientists at the CDC studying remains preserved by the Alaskan permafrost They identified it as a type of H1N1 virus
D-r Mitova 15
Courses types of the Courses types of the epidemic processepidemic process
In epidemiology an infection is said to be endemic endemic (from Greek en- in or within + demos people) in a population when that infection is maintained in the population without the need for external inputs For example chickenpox is endemic in the UK but malaria is not Every year there are a few cases of malaria acquired in the UK but these do not lead to sustained transmission in the population due to the lack of a suitable vector (mosquitoes of the genus Anopheles)
D-r Mitova 16
SOURCE AND RESERVOIR
The source of infection is defined as the person animal object or substance from which an infectious agent passes or disseminated to the host
A reservoir is defined as ldquoany person animal arthropod plant soil or substance in which an infectious agent lives and multiplies on which it depends primarily for survival and where it reproduced itself in such manner that it can be transmitted to a susceptible hostrdquo In short the reservoir is the natural habitual in which the organism metabolized and replicates
D-r Mitova 17
In Bulgaria the source of infection is defined only as the person or animal in which an infectious agent live and multiplies and from which an infectious agent passes to the host inanimate things and arthropods are defined as a vehicles of transmission
D-r Mitova 18
HUMAN RESERVOIR
By far the most important source or reservoir of infection for humans is man himself He my be a case or carrier Man is often described as him own enemy because most of the communicable disease of which man is heir to a contacted from human sources
D-r Mitova 19
HUMAN RESERVOIR - CASE
A case is defined as a person in the population or study group identified as having the particular disease health disorder or condition under investigation A variety of criteria ( clinical biochemical laboratory ) may be used to identify cases Broadly the presence of infection in the host may be clinical subclinical or latent
D-r Mitova 20
A case is a risk factor hellip Infection in one person can be transmitted
to others
What is infectious disease epidemiology
D-r Mitova 21
1The clinical illness
The clinical illness maybe mild or moderate typical or atypical severe or fatal
Epidemiologically mild cases may be more important sources of infection than severe cases because they are ambulant and spread the infection wherever they go whereas severe cases usually confined to bed
D-r Mitova 22
2The subclinical cases are variously referred to as inapparent covert missed or abortive cases They are equally important sources of infection The disease agent may multiply in the host but does not manifest itself by sign and symptoms The disease agent is eliminated and contaminates the environment in the same way as clinical casesperson who are thus sick contribute more than symptomatic patient to the transmission of infection to others and what is more they do not appear in any of statistics
Subclinical cases play a dominant role in maintaining the chain of infection in the community The latent infection must be distinguished from subclinical infection In latent infection the host does not shed the infectious agent
D-r Mitova 23
The subclinical cases
Subclinical infection can be detected only by laboratory tests
Barring a few(mesles varicella) subclinical infection occurs in most infectious disease In some disease(rubella mumps polio hepatitis A and B influenza diphteria) a great deal of subclinical infection occurs
D-r Mitova 24
Latent infectionThe term latent infection must be
distinguished from subclinical infection In latent infection the host does not shed the infectious agent which lies dormant within the host without symptoms (and often without demonstrable presence in blood tissues or bodily secretions of the host) For examples latent infection occurs in herpes symplex Brill-Zincer disease
D-r Mitova 25
CARRIERS
In some disease either due to inadequate treatment or immune response the disease agent is not completely eliminated leading to a carrier state A carrier is defined as an infected person or animal that harbors a specific infectious agent in the absence of
discernible clinical disease and serves as a potential source of infection for others
D-r Mitova 26
CARRIERS
As a rule carriers are less infectious than cases but epidemiologically they are more dangerous than cases because they escape recognition and continuing as they do live a normal life among population or community they readily infect the susceptible individuals over a wider area and longer period of time under favorable conditionsthe ldquoTyphoid Maryrdquo is a classic example of a carrier-cooker Mary who gave rise to more than 1300 cases in her life time
D-r Mitova 27
The elements in a carrier state are
The presence in the body of disease agent
The absence of recognizable symptoms and signs of disease
The shedding of disease agent in the discharges or excretion
D-r Mitova 28
incubatory carriers are those who shed the infectious agent during the incubation period This usually occurs during the the last few days of incubation period As for example
Measles-the period of communicability is 4 days before the rush
Mumps-usually 4-6 days before onset of symptoms
Polio-7-10 days before onset of symptomsHepatitis B-for a month before
jaundiceabout 6 weeks after exposure
Carrier may be classified BY TYPE
D-r Mitova 29
Carrier may be classified BY TYPE
convalescent carriers that is those who continue to shed the disease agent during the period of convalescence(typhoid fever cholera diphteria bacillary disentery) In the disease clinical recovery does not coincide with bacteriological recovery
D-r Mitova 30
Carrier may be classified BY TYPE
healthy carriers they are victims of subclinical infection who have developed carrier state without suffering from overt disease but are nevertheless shedding the disease agent eg poliomyelitis cholera meningococcal meningitis salmonellosis diphtheria
D-r Mitova 31
BY DURATION
Temporary carriers are those who shed the infectious agent for short period of timeChronic carriers are those who excretes the infectious agent for indefinite periods eg typhoid fever hepatitis B dysentery etcThey are known to reintroduce disease into areas which are otherwise free of infection
D-r Mitova 32
Chronic carriers
Chronic carriers are far more important sources of infection than cases The longer the carrier state the greater the risk of community The duration of the carrier state varies with the disease In typhoid fever and hepatitis B the chronic carrier state may last for several yearsIn chronic dysentery it may last for year or longer In diphtheria the carrier state is associated with infected tonsils in typhoid fever with gall bladder disease
D-r Mitova 33
BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 10
Courses types of the Courses types of the epidemic processepidemic process
Epidemic (from Greek epi- upon + demos Epidemic (from Greek epi- upon + demos people)-people)- outbreak of disease that affects a much greater number of people than is usual for the locality or that spreads to regions where it is ordinarily not present A disease that tends to be restricted to a particular region (endemic disease) can become epidemic if nonimmune persons are present in large numbers (as in time of war or during pilgrimages) if the infectious agent is more virulent than usual or if distribution of the disease is more easily effected Cholera and plague endemic in parts of Asia can become epidemic under the above conditions as can dysentery and many other infections Epidemics may also be caused by new disease agents in the human population such as the Ebola virus
D-r Mitova 11
Courses types of the Courses types of the epidemic processepidemic process
A pandemicpandemic (from Greek pan all + demos people) is an epidemic (an outbreak of an infectious disease) that spreads worldwide or at least across a large regionA worldwide epidemic is known as a pandemicpandemic eg the influenza pandemic of 1918 or the AIDS pandemic beginning in the 1980s
D-r Mitova 12
Examples Cholera Examples Cholera
first pandemic 1816ndash1826 Previously restricted to the Indian subcontinent the pandemic began in Bengal then spread across India by 1820 It extended as far as China and the Caspian Sea before recedingThe second pandemic (1829ndash1851) reached Europe London in 1832 Ontario Canada and New York in the same year and the Pacific coast of North America by 1834The third pandemic (1852ndash1860) mainly affected Russia with over a million deaths
D-r Mitova 13
Examples Cholera Examples Cholera
The fourth pandemic (1863ndash1875) spread mostly in Europe and AfricaIn 1866 there was an outbreak in North AmericaThe sixth pandemic (1899ndash1923) had little effect in Europe because of advances in public health but Russia was badly affected againThe seventh pandemic began in Indonesia in 1961 called El Tor after the strain and reached Bangladesh in 1963 India in 1964 and the USSR in 1966
D-r Mitova 14
Examples InfluenzaExamples Influenza
The Spanish fluSpanish flu 1918ndash1919
An estimated 17 million died in India 500000 in the United States and 200000 in the UK The virus was recently reconstructed by scientists at the CDC studying remains preserved by the Alaskan permafrost They identified it as a type of H1N1 virus
D-r Mitova 15
Courses types of the Courses types of the epidemic processepidemic process
In epidemiology an infection is said to be endemic endemic (from Greek en- in or within + demos people) in a population when that infection is maintained in the population without the need for external inputs For example chickenpox is endemic in the UK but malaria is not Every year there are a few cases of malaria acquired in the UK but these do not lead to sustained transmission in the population due to the lack of a suitable vector (mosquitoes of the genus Anopheles)
D-r Mitova 16
SOURCE AND RESERVOIR
The source of infection is defined as the person animal object or substance from which an infectious agent passes or disseminated to the host
A reservoir is defined as ldquoany person animal arthropod plant soil or substance in which an infectious agent lives and multiplies on which it depends primarily for survival and where it reproduced itself in such manner that it can be transmitted to a susceptible hostrdquo In short the reservoir is the natural habitual in which the organism metabolized and replicates
D-r Mitova 17
In Bulgaria the source of infection is defined only as the person or animal in which an infectious agent live and multiplies and from which an infectious agent passes to the host inanimate things and arthropods are defined as a vehicles of transmission
D-r Mitova 18
HUMAN RESERVOIR
By far the most important source or reservoir of infection for humans is man himself He my be a case or carrier Man is often described as him own enemy because most of the communicable disease of which man is heir to a contacted from human sources
D-r Mitova 19
HUMAN RESERVOIR - CASE
A case is defined as a person in the population or study group identified as having the particular disease health disorder or condition under investigation A variety of criteria ( clinical biochemical laboratory ) may be used to identify cases Broadly the presence of infection in the host may be clinical subclinical or latent
D-r Mitova 20
A case is a risk factor hellip Infection in one person can be transmitted
to others
What is infectious disease epidemiology
D-r Mitova 21
1The clinical illness
The clinical illness maybe mild or moderate typical or atypical severe or fatal
Epidemiologically mild cases may be more important sources of infection than severe cases because they are ambulant and spread the infection wherever they go whereas severe cases usually confined to bed
D-r Mitova 22
2The subclinical cases are variously referred to as inapparent covert missed or abortive cases They are equally important sources of infection The disease agent may multiply in the host but does not manifest itself by sign and symptoms The disease agent is eliminated and contaminates the environment in the same way as clinical casesperson who are thus sick contribute more than symptomatic patient to the transmission of infection to others and what is more they do not appear in any of statistics
Subclinical cases play a dominant role in maintaining the chain of infection in the community The latent infection must be distinguished from subclinical infection In latent infection the host does not shed the infectious agent
D-r Mitova 23
The subclinical cases
Subclinical infection can be detected only by laboratory tests
Barring a few(mesles varicella) subclinical infection occurs in most infectious disease In some disease(rubella mumps polio hepatitis A and B influenza diphteria) a great deal of subclinical infection occurs
D-r Mitova 24
Latent infectionThe term latent infection must be
distinguished from subclinical infection In latent infection the host does not shed the infectious agent which lies dormant within the host without symptoms (and often without demonstrable presence in blood tissues or bodily secretions of the host) For examples latent infection occurs in herpes symplex Brill-Zincer disease
D-r Mitova 25
CARRIERS
In some disease either due to inadequate treatment or immune response the disease agent is not completely eliminated leading to a carrier state A carrier is defined as an infected person or animal that harbors a specific infectious agent in the absence of
discernible clinical disease and serves as a potential source of infection for others
D-r Mitova 26
CARRIERS
As a rule carriers are less infectious than cases but epidemiologically they are more dangerous than cases because they escape recognition and continuing as they do live a normal life among population or community they readily infect the susceptible individuals over a wider area and longer period of time under favorable conditionsthe ldquoTyphoid Maryrdquo is a classic example of a carrier-cooker Mary who gave rise to more than 1300 cases in her life time
D-r Mitova 27
The elements in a carrier state are
The presence in the body of disease agent
The absence of recognizable symptoms and signs of disease
The shedding of disease agent in the discharges or excretion
D-r Mitova 28
incubatory carriers are those who shed the infectious agent during the incubation period This usually occurs during the the last few days of incubation period As for example
Measles-the period of communicability is 4 days before the rush
Mumps-usually 4-6 days before onset of symptoms
Polio-7-10 days before onset of symptomsHepatitis B-for a month before
jaundiceabout 6 weeks after exposure
Carrier may be classified BY TYPE
D-r Mitova 29
Carrier may be classified BY TYPE
convalescent carriers that is those who continue to shed the disease agent during the period of convalescence(typhoid fever cholera diphteria bacillary disentery) In the disease clinical recovery does not coincide with bacteriological recovery
D-r Mitova 30
Carrier may be classified BY TYPE
healthy carriers they are victims of subclinical infection who have developed carrier state without suffering from overt disease but are nevertheless shedding the disease agent eg poliomyelitis cholera meningococcal meningitis salmonellosis diphtheria
D-r Mitova 31
BY DURATION
Temporary carriers are those who shed the infectious agent for short period of timeChronic carriers are those who excretes the infectious agent for indefinite periods eg typhoid fever hepatitis B dysentery etcThey are known to reintroduce disease into areas which are otherwise free of infection
D-r Mitova 32
Chronic carriers
Chronic carriers are far more important sources of infection than cases The longer the carrier state the greater the risk of community The duration of the carrier state varies with the disease In typhoid fever and hepatitis B the chronic carrier state may last for several yearsIn chronic dysentery it may last for year or longer In diphtheria the carrier state is associated with infected tonsils in typhoid fever with gall bladder disease
D-r Mitova 33
BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 11
Courses types of the Courses types of the epidemic processepidemic process
A pandemicpandemic (from Greek pan all + demos people) is an epidemic (an outbreak of an infectious disease) that spreads worldwide or at least across a large regionA worldwide epidemic is known as a pandemicpandemic eg the influenza pandemic of 1918 or the AIDS pandemic beginning in the 1980s
D-r Mitova 12
Examples Cholera Examples Cholera
first pandemic 1816ndash1826 Previously restricted to the Indian subcontinent the pandemic began in Bengal then spread across India by 1820 It extended as far as China and the Caspian Sea before recedingThe second pandemic (1829ndash1851) reached Europe London in 1832 Ontario Canada and New York in the same year and the Pacific coast of North America by 1834The third pandemic (1852ndash1860) mainly affected Russia with over a million deaths
D-r Mitova 13
Examples Cholera Examples Cholera
The fourth pandemic (1863ndash1875) spread mostly in Europe and AfricaIn 1866 there was an outbreak in North AmericaThe sixth pandemic (1899ndash1923) had little effect in Europe because of advances in public health but Russia was badly affected againThe seventh pandemic began in Indonesia in 1961 called El Tor after the strain and reached Bangladesh in 1963 India in 1964 and the USSR in 1966
D-r Mitova 14
Examples InfluenzaExamples Influenza
The Spanish fluSpanish flu 1918ndash1919
An estimated 17 million died in India 500000 in the United States and 200000 in the UK The virus was recently reconstructed by scientists at the CDC studying remains preserved by the Alaskan permafrost They identified it as a type of H1N1 virus
D-r Mitova 15
Courses types of the Courses types of the epidemic processepidemic process
In epidemiology an infection is said to be endemic endemic (from Greek en- in or within + demos people) in a population when that infection is maintained in the population without the need for external inputs For example chickenpox is endemic in the UK but malaria is not Every year there are a few cases of malaria acquired in the UK but these do not lead to sustained transmission in the population due to the lack of a suitable vector (mosquitoes of the genus Anopheles)
D-r Mitova 16
SOURCE AND RESERVOIR
The source of infection is defined as the person animal object or substance from which an infectious agent passes or disseminated to the host
A reservoir is defined as ldquoany person animal arthropod plant soil or substance in which an infectious agent lives and multiplies on which it depends primarily for survival and where it reproduced itself in such manner that it can be transmitted to a susceptible hostrdquo In short the reservoir is the natural habitual in which the organism metabolized and replicates
D-r Mitova 17
In Bulgaria the source of infection is defined only as the person or animal in which an infectious agent live and multiplies and from which an infectious agent passes to the host inanimate things and arthropods are defined as a vehicles of transmission
D-r Mitova 18
HUMAN RESERVOIR
By far the most important source or reservoir of infection for humans is man himself He my be a case or carrier Man is often described as him own enemy because most of the communicable disease of which man is heir to a contacted from human sources
D-r Mitova 19
HUMAN RESERVOIR - CASE
A case is defined as a person in the population or study group identified as having the particular disease health disorder or condition under investigation A variety of criteria ( clinical biochemical laboratory ) may be used to identify cases Broadly the presence of infection in the host may be clinical subclinical or latent
D-r Mitova 20
A case is a risk factor hellip Infection in one person can be transmitted
to others
What is infectious disease epidemiology
D-r Mitova 21
1The clinical illness
The clinical illness maybe mild or moderate typical or atypical severe or fatal
Epidemiologically mild cases may be more important sources of infection than severe cases because they are ambulant and spread the infection wherever they go whereas severe cases usually confined to bed
D-r Mitova 22
2The subclinical cases are variously referred to as inapparent covert missed or abortive cases They are equally important sources of infection The disease agent may multiply in the host but does not manifest itself by sign and symptoms The disease agent is eliminated and contaminates the environment in the same way as clinical casesperson who are thus sick contribute more than symptomatic patient to the transmission of infection to others and what is more they do not appear in any of statistics
Subclinical cases play a dominant role in maintaining the chain of infection in the community The latent infection must be distinguished from subclinical infection In latent infection the host does not shed the infectious agent
D-r Mitova 23
The subclinical cases
Subclinical infection can be detected only by laboratory tests
Barring a few(mesles varicella) subclinical infection occurs in most infectious disease In some disease(rubella mumps polio hepatitis A and B influenza diphteria) a great deal of subclinical infection occurs
D-r Mitova 24
Latent infectionThe term latent infection must be
distinguished from subclinical infection In latent infection the host does not shed the infectious agent which lies dormant within the host without symptoms (and often without demonstrable presence in blood tissues or bodily secretions of the host) For examples latent infection occurs in herpes symplex Brill-Zincer disease
D-r Mitova 25
CARRIERS
In some disease either due to inadequate treatment or immune response the disease agent is not completely eliminated leading to a carrier state A carrier is defined as an infected person or animal that harbors a specific infectious agent in the absence of
discernible clinical disease and serves as a potential source of infection for others
D-r Mitova 26
CARRIERS
As a rule carriers are less infectious than cases but epidemiologically they are more dangerous than cases because they escape recognition and continuing as they do live a normal life among population or community they readily infect the susceptible individuals over a wider area and longer period of time under favorable conditionsthe ldquoTyphoid Maryrdquo is a classic example of a carrier-cooker Mary who gave rise to more than 1300 cases in her life time
D-r Mitova 27
The elements in a carrier state are
The presence in the body of disease agent
The absence of recognizable symptoms and signs of disease
The shedding of disease agent in the discharges or excretion
D-r Mitova 28
incubatory carriers are those who shed the infectious agent during the incubation period This usually occurs during the the last few days of incubation period As for example
Measles-the period of communicability is 4 days before the rush
Mumps-usually 4-6 days before onset of symptoms
Polio-7-10 days before onset of symptomsHepatitis B-for a month before
jaundiceabout 6 weeks after exposure
Carrier may be classified BY TYPE
D-r Mitova 29
Carrier may be classified BY TYPE
convalescent carriers that is those who continue to shed the disease agent during the period of convalescence(typhoid fever cholera diphteria bacillary disentery) In the disease clinical recovery does not coincide with bacteriological recovery
D-r Mitova 30
Carrier may be classified BY TYPE
healthy carriers they are victims of subclinical infection who have developed carrier state without suffering from overt disease but are nevertheless shedding the disease agent eg poliomyelitis cholera meningococcal meningitis salmonellosis diphtheria
D-r Mitova 31
BY DURATION
Temporary carriers are those who shed the infectious agent for short period of timeChronic carriers are those who excretes the infectious agent for indefinite periods eg typhoid fever hepatitis B dysentery etcThey are known to reintroduce disease into areas which are otherwise free of infection
D-r Mitova 32
Chronic carriers
Chronic carriers are far more important sources of infection than cases The longer the carrier state the greater the risk of community The duration of the carrier state varies with the disease In typhoid fever and hepatitis B the chronic carrier state may last for several yearsIn chronic dysentery it may last for year or longer In diphtheria the carrier state is associated with infected tonsils in typhoid fever with gall bladder disease
D-r Mitova 33
BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 12
Examples Cholera Examples Cholera
first pandemic 1816ndash1826 Previously restricted to the Indian subcontinent the pandemic began in Bengal then spread across India by 1820 It extended as far as China and the Caspian Sea before recedingThe second pandemic (1829ndash1851) reached Europe London in 1832 Ontario Canada and New York in the same year and the Pacific coast of North America by 1834The third pandemic (1852ndash1860) mainly affected Russia with over a million deaths
D-r Mitova 13
Examples Cholera Examples Cholera
The fourth pandemic (1863ndash1875) spread mostly in Europe and AfricaIn 1866 there was an outbreak in North AmericaThe sixth pandemic (1899ndash1923) had little effect in Europe because of advances in public health but Russia was badly affected againThe seventh pandemic began in Indonesia in 1961 called El Tor after the strain and reached Bangladesh in 1963 India in 1964 and the USSR in 1966
D-r Mitova 14
Examples InfluenzaExamples Influenza
The Spanish fluSpanish flu 1918ndash1919
An estimated 17 million died in India 500000 in the United States and 200000 in the UK The virus was recently reconstructed by scientists at the CDC studying remains preserved by the Alaskan permafrost They identified it as a type of H1N1 virus
D-r Mitova 15
Courses types of the Courses types of the epidemic processepidemic process
In epidemiology an infection is said to be endemic endemic (from Greek en- in or within + demos people) in a population when that infection is maintained in the population without the need for external inputs For example chickenpox is endemic in the UK but malaria is not Every year there are a few cases of malaria acquired in the UK but these do not lead to sustained transmission in the population due to the lack of a suitable vector (mosquitoes of the genus Anopheles)
D-r Mitova 16
SOURCE AND RESERVOIR
The source of infection is defined as the person animal object or substance from which an infectious agent passes or disseminated to the host
A reservoir is defined as ldquoany person animal arthropod plant soil or substance in which an infectious agent lives and multiplies on which it depends primarily for survival and where it reproduced itself in such manner that it can be transmitted to a susceptible hostrdquo In short the reservoir is the natural habitual in which the organism metabolized and replicates
D-r Mitova 17
In Bulgaria the source of infection is defined only as the person or animal in which an infectious agent live and multiplies and from which an infectious agent passes to the host inanimate things and arthropods are defined as a vehicles of transmission
D-r Mitova 18
HUMAN RESERVOIR
By far the most important source or reservoir of infection for humans is man himself He my be a case or carrier Man is often described as him own enemy because most of the communicable disease of which man is heir to a contacted from human sources
D-r Mitova 19
HUMAN RESERVOIR - CASE
A case is defined as a person in the population or study group identified as having the particular disease health disorder or condition under investigation A variety of criteria ( clinical biochemical laboratory ) may be used to identify cases Broadly the presence of infection in the host may be clinical subclinical or latent
D-r Mitova 20
A case is a risk factor hellip Infection in one person can be transmitted
to others
What is infectious disease epidemiology
D-r Mitova 21
1The clinical illness
The clinical illness maybe mild or moderate typical or atypical severe or fatal
Epidemiologically mild cases may be more important sources of infection than severe cases because they are ambulant and spread the infection wherever they go whereas severe cases usually confined to bed
D-r Mitova 22
2The subclinical cases are variously referred to as inapparent covert missed or abortive cases They are equally important sources of infection The disease agent may multiply in the host but does not manifest itself by sign and symptoms The disease agent is eliminated and contaminates the environment in the same way as clinical casesperson who are thus sick contribute more than symptomatic patient to the transmission of infection to others and what is more they do not appear in any of statistics
Subclinical cases play a dominant role in maintaining the chain of infection in the community The latent infection must be distinguished from subclinical infection In latent infection the host does not shed the infectious agent
D-r Mitova 23
The subclinical cases
Subclinical infection can be detected only by laboratory tests
Barring a few(mesles varicella) subclinical infection occurs in most infectious disease In some disease(rubella mumps polio hepatitis A and B influenza diphteria) a great deal of subclinical infection occurs
D-r Mitova 24
Latent infectionThe term latent infection must be
distinguished from subclinical infection In latent infection the host does not shed the infectious agent which lies dormant within the host without symptoms (and often without demonstrable presence in blood tissues or bodily secretions of the host) For examples latent infection occurs in herpes symplex Brill-Zincer disease
D-r Mitova 25
CARRIERS
In some disease either due to inadequate treatment or immune response the disease agent is not completely eliminated leading to a carrier state A carrier is defined as an infected person or animal that harbors a specific infectious agent in the absence of
discernible clinical disease and serves as a potential source of infection for others
D-r Mitova 26
CARRIERS
As a rule carriers are less infectious than cases but epidemiologically they are more dangerous than cases because they escape recognition and continuing as they do live a normal life among population or community they readily infect the susceptible individuals over a wider area and longer period of time under favorable conditionsthe ldquoTyphoid Maryrdquo is a classic example of a carrier-cooker Mary who gave rise to more than 1300 cases in her life time
D-r Mitova 27
The elements in a carrier state are
The presence in the body of disease agent
The absence of recognizable symptoms and signs of disease
The shedding of disease agent in the discharges or excretion
D-r Mitova 28
incubatory carriers are those who shed the infectious agent during the incubation period This usually occurs during the the last few days of incubation period As for example
Measles-the period of communicability is 4 days before the rush
Mumps-usually 4-6 days before onset of symptoms
Polio-7-10 days before onset of symptomsHepatitis B-for a month before
jaundiceabout 6 weeks after exposure
Carrier may be classified BY TYPE
D-r Mitova 29
Carrier may be classified BY TYPE
convalescent carriers that is those who continue to shed the disease agent during the period of convalescence(typhoid fever cholera diphteria bacillary disentery) In the disease clinical recovery does not coincide with bacteriological recovery
D-r Mitova 30
Carrier may be classified BY TYPE
healthy carriers they are victims of subclinical infection who have developed carrier state without suffering from overt disease but are nevertheless shedding the disease agent eg poliomyelitis cholera meningococcal meningitis salmonellosis diphtheria
D-r Mitova 31
BY DURATION
Temporary carriers are those who shed the infectious agent for short period of timeChronic carriers are those who excretes the infectious agent for indefinite periods eg typhoid fever hepatitis B dysentery etcThey are known to reintroduce disease into areas which are otherwise free of infection
D-r Mitova 32
Chronic carriers
Chronic carriers are far more important sources of infection than cases The longer the carrier state the greater the risk of community The duration of the carrier state varies with the disease In typhoid fever and hepatitis B the chronic carrier state may last for several yearsIn chronic dysentery it may last for year or longer In diphtheria the carrier state is associated with infected tonsils in typhoid fever with gall bladder disease
D-r Mitova 33
BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 13
Examples Cholera Examples Cholera
The fourth pandemic (1863ndash1875) spread mostly in Europe and AfricaIn 1866 there was an outbreak in North AmericaThe sixth pandemic (1899ndash1923) had little effect in Europe because of advances in public health but Russia was badly affected againThe seventh pandemic began in Indonesia in 1961 called El Tor after the strain and reached Bangladesh in 1963 India in 1964 and the USSR in 1966
D-r Mitova 14
Examples InfluenzaExamples Influenza
The Spanish fluSpanish flu 1918ndash1919
An estimated 17 million died in India 500000 in the United States and 200000 in the UK The virus was recently reconstructed by scientists at the CDC studying remains preserved by the Alaskan permafrost They identified it as a type of H1N1 virus
D-r Mitova 15
Courses types of the Courses types of the epidemic processepidemic process
In epidemiology an infection is said to be endemic endemic (from Greek en- in or within + demos people) in a population when that infection is maintained in the population without the need for external inputs For example chickenpox is endemic in the UK but malaria is not Every year there are a few cases of malaria acquired in the UK but these do not lead to sustained transmission in the population due to the lack of a suitable vector (mosquitoes of the genus Anopheles)
D-r Mitova 16
SOURCE AND RESERVOIR
The source of infection is defined as the person animal object or substance from which an infectious agent passes or disseminated to the host
A reservoir is defined as ldquoany person animal arthropod plant soil or substance in which an infectious agent lives and multiplies on which it depends primarily for survival and where it reproduced itself in such manner that it can be transmitted to a susceptible hostrdquo In short the reservoir is the natural habitual in which the organism metabolized and replicates
D-r Mitova 17
In Bulgaria the source of infection is defined only as the person or animal in which an infectious agent live and multiplies and from which an infectious agent passes to the host inanimate things and arthropods are defined as a vehicles of transmission
D-r Mitova 18
HUMAN RESERVOIR
By far the most important source or reservoir of infection for humans is man himself He my be a case or carrier Man is often described as him own enemy because most of the communicable disease of which man is heir to a contacted from human sources
D-r Mitova 19
HUMAN RESERVOIR - CASE
A case is defined as a person in the population or study group identified as having the particular disease health disorder or condition under investigation A variety of criteria ( clinical biochemical laboratory ) may be used to identify cases Broadly the presence of infection in the host may be clinical subclinical or latent
D-r Mitova 20
A case is a risk factor hellip Infection in one person can be transmitted
to others
What is infectious disease epidemiology
D-r Mitova 21
1The clinical illness
The clinical illness maybe mild or moderate typical or atypical severe or fatal
Epidemiologically mild cases may be more important sources of infection than severe cases because they are ambulant and spread the infection wherever they go whereas severe cases usually confined to bed
D-r Mitova 22
2The subclinical cases are variously referred to as inapparent covert missed or abortive cases They are equally important sources of infection The disease agent may multiply in the host but does not manifest itself by sign and symptoms The disease agent is eliminated and contaminates the environment in the same way as clinical casesperson who are thus sick contribute more than symptomatic patient to the transmission of infection to others and what is more they do not appear in any of statistics
Subclinical cases play a dominant role in maintaining the chain of infection in the community The latent infection must be distinguished from subclinical infection In latent infection the host does not shed the infectious agent
D-r Mitova 23
The subclinical cases
Subclinical infection can be detected only by laboratory tests
Barring a few(mesles varicella) subclinical infection occurs in most infectious disease In some disease(rubella mumps polio hepatitis A and B influenza diphteria) a great deal of subclinical infection occurs
D-r Mitova 24
Latent infectionThe term latent infection must be
distinguished from subclinical infection In latent infection the host does not shed the infectious agent which lies dormant within the host without symptoms (and often without demonstrable presence in blood tissues or bodily secretions of the host) For examples latent infection occurs in herpes symplex Brill-Zincer disease
D-r Mitova 25
CARRIERS
In some disease either due to inadequate treatment or immune response the disease agent is not completely eliminated leading to a carrier state A carrier is defined as an infected person or animal that harbors a specific infectious agent in the absence of
discernible clinical disease and serves as a potential source of infection for others
D-r Mitova 26
CARRIERS
As a rule carriers are less infectious than cases but epidemiologically they are more dangerous than cases because they escape recognition and continuing as they do live a normal life among population or community they readily infect the susceptible individuals over a wider area and longer period of time under favorable conditionsthe ldquoTyphoid Maryrdquo is a classic example of a carrier-cooker Mary who gave rise to more than 1300 cases in her life time
D-r Mitova 27
The elements in a carrier state are
The presence in the body of disease agent
The absence of recognizable symptoms and signs of disease
The shedding of disease agent in the discharges or excretion
D-r Mitova 28
incubatory carriers are those who shed the infectious agent during the incubation period This usually occurs during the the last few days of incubation period As for example
Measles-the period of communicability is 4 days before the rush
Mumps-usually 4-6 days before onset of symptoms
Polio-7-10 days before onset of symptomsHepatitis B-for a month before
jaundiceabout 6 weeks after exposure
Carrier may be classified BY TYPE
D-r Mitova 29
Carrier may be classified BY TYPE
convalescent carriers that is those who continue to shed the disease agent during the period of convalescence(typhoid fever cholera diphteria bacillary disentery) In the disease clinical recovery does not coincide with bacteriological recovery
D-r Mitova 30
Carrier may be classified BY TYPE
healthy carriers they are victims of subclinical infection who have developed carrier state without suffering from overt disease but are nevertheless shedding the disease agent eg poliomyelitis cholera meningococcal meningitis salmonellosis diphtheria
D-r Mitova 31
BY DURATION
Temporary carriers are those who shed the infectious agent for short period of timeChronic carriers are those who excretes the infectious agent for indefinite periods eg typhoid fever hepatitis B dysentery etcThey are known to reintroduce disease into areas which are otherwise free of infection
D-r Mitova 32
Chronic carriers
Chronic carriers are far more important sources of infection than cases The longer the carrier state the greater the risk of community The duration of the carrier state varies with the disease In typhoid fever and hepatitis B the chronic carrier state may last for several yearsIn chronic dysentery it may last for year or longer In diphtheria the carrier state is associated with infected tonsils in typhoid fever with gall bladder disease
D-r Mitova 33
BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 14
Examples InfluenzaExamples Influenza
The Spanish fluSpanish flu 1918ndash1919
An estimated 17 million died in India 500000 in the United States and 200000 in the UK The virus was recently reconstructed by scientists at the CDC studying remains preserved by the Alaskan permafrost They identified it as a type of H1N1 virus
D-r Mitova 15
Courses types of the Courses types of the epidemic processepidemic process
In epidemiology an infection is said to be endemic endemic (from Greek en- in or within + demos people) in a population when that infection is maintained in the population without the need for external inputs For example chickenpox is endemic in the UK but malaria is not Every year there are a few cases of malaria acquired in the UK but these do not lead to sustained transmission in the population due to the lack of a suitable vector (mosquitoes of the genus Anopheles)
D-r Mitova 16
SOURCE AND RESERVOIR
The source of infection is defined as the person animal object or substance from which an infectious agent passes or disseminated to the host
A reservoir is defined as ldquoany person animal arthropod plant soil or substance in which an infectious agent lives and multiplies on which it depends primarily for survival and where it reproduced itself in such manner that it can be transmitted to a susceptible hostrdquo In short the reservoir is the natural habitual in which the organism metabolized and replicates
D-r Mitova 17
In Bulgaria the source of infection is defined only as the person or animal in which an infectious agent live and multiplies and from which an infectious agent passes to the host inanimate things and arthropods are defined as a vehicles of transmission
D-r Mitova 18
HUMAN RESERVOIR
By far the most important source or reservoir of infection for humans is man himself He my be a case or carrier Man is often described as him own enemy because most of the communicable disease of which man is heir to a contacted from human sources
D-r Mitova 19
HUMAN RESERVOIR - CASE
A case is defined as a person in the population or study group identified as having the particular disease health disorder or condition under investigation A variety of criteria ( clinical biochemical laboratory ) may be used to identify cases Broadly the presence of infection in the host may be clinical subclinical or latent
D-r Mitova 20
A case is a risk factor hellip Infection in one person can be transmitted
to others
What is infectious disease epidemiology
D-r Mitova 21
1The clinical illness
The clinical illness maybe mild or moderate typical or atypical severe or fatal
Epidemiologically mild cases may be more important sources of infection than severe cases because they are ambulant and spread the infection wherever they go whereas severe cases usually confined to bed
D-r Mitova 22
2The subclinical cases are variously referred to as inapparent covert missed or abortive cases They are equally important sources of infection The disease agent may multiply in the host but does not manifest itself by sign and symptoms The disease agent is eliminated and contaminates the environment in the same way as clinical casesperson who are thus sick contribute more than symptomatic patient to the transmission of infection to others and what is more they do not appear in any of statistics
Subclinical cases play a dominant role in maintaining the chain of infection in the community The latent infection must be distinguished from subclinical infection In latent infection the host does not shed the infectious agent
D-r Mitova 23
The subclinical cases
Subclinical infection can be detected only by laboratory tests
Barring a few(mesles varicella) subclinical infection occurs in most infectious disease In some disease(rubella mumps polio hepatitis A and B influenza diphteria) a great deal of subclinical infection occurs
D-r Mitova 24
Latent infectionThe term latent infection must be
distinguished from subclinical infection In latent infection the host does not shed the infectious agent which lies dormant within the host without symptoms (and often without demonstrable presence in blood tissues or bodily secretions of the host) For examples latent infection occurs in herpes symplex Brill-Zincer disease
D-r Mitova 25
CARRIERS
In some disease either due to inadequate treatment or immune response the disease agent is not completely eliminated leading to a carrier state A carrier is defined as an infected person or animal that harbors a specific infectious agent in the absence of
discernible clinical disease and serves as a potential source of infection for others
D-r Mitova 26
CARRIERS
As a rule carriers are less infectious than cases but epidemiologically they are more dangerous than cases because they escape recognition and continuing as they do live a normal life among population or community they readily infect the susceptible individuals over a wider area and longer period of time under favorable conditionsthe ldquoTyphoid Maryrdquo is a classic example of a carrier-cooker Mary who gave rise to more than 1300 cases in her life time
D-r Mitova 27
The elements in a carrier state are
The presence in the body of disease agent
The absence of recognizable symptoms and signs of disease
The shedding of disease agent in the discharges or excretion
D-r Mitova 28
incubatory carriers are those who shed the infectious agent during the incubation period This usually occurs during the the last few days of incubation period As for example
Measles-the period of communicability is 4 days before the rush
Mumps-usually 4-6 days before onset of symptoms
Polio-7-10 days before onset of symptomsHepatitis B-for a month before
jaundiceabout 6 weeks after exposure
Carrier may be classified BY TYPE
D-r Mitova 29
Carrier may be classified BY TYPE
convalescent carriers that is those who continue to shed the disease agent during the period of convalescence(typhoid fever cholera diphteria bacillary disentery) In the disease clinical recovery does not coincide with bacteriological recovery
D-r Mitova 30
Carrier may be classified BY TYPE
healthy carriers they are victims of subclinical infection who have developed carrier state without suffering from overt disease but are nevertheless shedding the disease agent eg poliomyelitis cholera meningococcal meningitis salmonellosis diphtheria
D-r Mitova 31
BY DURATION
Temporary carriers are those who shed the infectious agent for short period of timeChronic carriers are those who excretes the infectious agent for indefinite periods eg typhoid fever hepatitis B dysentery etcThey are known to reintroduce disease into areas which are otherwise free of infection
D-r Mitova 32
Chronic carriers
Chronic carriers are far more important sources of infection than cases The longer the carrier state the greater the risk of community The duration of the carrier state varies with the disease In typhoid fever and hepatitis B the chronic carrier state may last for several yearsIn chronic dysentery it may last for year or longer In diphtheria the carrier state is associated with infected tonsils in typhoid fever with gall bladder disease
D-r Mitova 33
BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 15
Courses types of the Courses types of the epidemic processepidemic process
In epidemiology an infection is said to be endemic endemic (from Greek en- in or within + demos people) in a population when that infection is maintained in the population without the need for external inputs For example chickenpox is endemic in the UK but malaria is not Every year there are a few cases of malaria acquired in the UK but these do not lead to sustained transmission in the population due to the lack of a suitable vector (mosquitoes of the genus Anopheles)
D-r Mitova 16
SOURCE AND RESERVOIR
The source of infection is defined as the person animal object or substance from which an infectious agent passes or disseminated to the host
A reservoir is defined as ldquoany person animal arthropod plant soil or substance in which an infectious agent lives and multiplies on which it depends primarily for survival and where it reproduced itself in such manner that it can be transmitted to a susceptible hostrdquo In short the reservoir is the natural habitual in which the organism metabolized and replicates
D-r Mitova 17
In Bulgaria the source of infection is defined only as the person or animal in which an infectious agent live and multiplies and from which an infectious agent passes to the host inanimate things and arthropods are defined as a vehicles of transmission
D-r Mitova 18
HUMAN RESERVOIR
By far the most important source or reservoir of infection for humans is man himself He my be a case or carrier Man is often described as him own enemy because most of the communicable disease of which man is heir to a contacted from human sources
D-r Mitova 19
HUMAN RESERVOIR - CASE
A case is defined as a person in the population or study group identified as having the particular disease health disorder or condition under investigation A variety of criteria ( clinical biochemical laboratory ) may be used to identify cases Broadly the presence of infection in the host may be clinical subclinical or latent
D-r Mitova 20
A case is a risk factor hellip Infection in one person can be transmitted
to others
What is infectious disease epidemiology
D-r Mitova 21
1The clinical illness
The clinical illness maybe mild or moderate typical or atypical severe or fatal
Epidemiologically mild cases may be more important sources of infection than severe cases because they are ambulant and spread the infection wherever they go whereas severe cases usually confined to bed
D-r Mitova 22
2The subclinical cases are variously referred to as inapparent covert missed or abortive cases They are equally important sources of infection The disease agent may multiply in the host but does not manifest itself by sign and symptoms The disease agent is eliminated and contaminates the environment in the same way as clinical casesperson who are thus sick contribute more than symptomatic patient to the transmission of infection to others and what is more they do not appear in any of statistics
Subclinical cases play a dominant role in maintaining the chain of infection in the community The latent infection must be distinguished from subclinical infection In latent infection the host does not shed the infectious agent
D-r Mitova 23
The subclinical cases
Subclinical infection can be detected only by laboratory tests
Barring a few(mesles varicella) subclinical infection occurs in most infectious disease In some disease(rubella mumps polio hepatitis A and B influenza diphteria) a great deal of subclinical infection occurs
D-r Mitova 24
Latent infectionThe term latent infection must be
distinguished from subclinical infection In latent infection the host does not shed the infectious agent which lies dormant within the host without symptoms (and often without demonstrable presence in blood tissues or bodily secretions of the host) For examples latent infection occurs in herpes symplex Brill-Zincer disease
D-r Mitova 25
CARRIERS
In some disease either due to inadequate treatment or immune response the disease agent is not completely eliminated leading to a carrier state A carrier is defined as an infected person or animal that harbors a specific infectious agent in the absence of
discernible clinical disease and serves as a potential source of infection for others
D-r Mitova 26
CARRIERS
As a rule carriers are less infectious than cases but epidemiologically they are more dangerous than cases because they escape recognition and continuing as they do live a normal life among population or community they readily infect the susceptible individuals over a wider area and longer period of time under favorable conditionsthe ldquoTyphoid Maryrdquo is a classic example of a carrier-cooker Mary who gave rise to more than 1300 cases in her life time
D-r Mitova 27
The elements in a carrier state are
The presence in the body of disease agent
The absence of recognizable symptoms and signs of disease
The shedding of disease agent in the discharges or excretion
D-r Mitova 28
incubatory carriers are those who shed the infectious agent during the incubation period This usually occurs during the the last few days of incubation period As for example
Measles-the period of communicability is 4 days before the rush
Mumps-usually 4-6 days before onset of symptoms
Polio-7-10 days before onset of symptomsHepatitis B-for a month before
jaundiceabout 6 weeks after exposure
Carrier may be classified BY TYPE
D-r Mitova 29
Carrier may be classified BY TYPE
convalescent carriers that is those who continue to shed the disease agent during the period of convalescence(typhoid fever cholera diphteria bacillary disentery) In the disease clinical recovery does not coincide with bacteriological recovery
D-r Mitova 30
Carrier may be classified BY TYPE
healthy carriers they are victims of subclinical infection who have developed carrier state without suffering from overt disease but are nevertheless shedding the disease agent eg poliomyelitis cholera meningococcal meningitis salmonellosis diphtheria
D-r Mitova 31
BY DURATION
Temporary carriers are those who shed the infectious agent for short period of timeChronic carriers are those who excretes the infectious agent for indefinite periods eg typhoid fever hepatitis B dysentery etcThey are known to reintroduce disease into areas which are otherwise free of infection
D-r Mitova 32
Chronic carriers
Chronic carriers are far more important sources of infection than cases The longer the carrier state the greater the risk of community The duration of the carrier state varies with the disease In typhoid fever and hepatitis B the chronic carrier state may last for several yearsIn chronic dysentery it may last for year or longer In diphtheria the carrier state is associated with infected tonsils in typhoid fever with gall bladder disease
D-r Mitova 33
BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 16
SOURCE AND RESERVOIR
The source of infection is defined as the person animal object or substance from which an infectious agent passes or disseminated to the host
A reservoir is defined as ldquoany person animal arthropod plant soil or substance in which an infectious agent lives and multiplies on which it depends primarily for survival and where it reproduced itself in such manner that it can be transmitted to a susceptible hostrdquo In short the reservoir is the natural habitual in which the organism metabolized and replicates
D-r Mitova 17
In Bulgaria the source of infection is defined only as the person or animal in which an infectious agent live and multiplies and from which an infectious agent passes to the host inanimate things and arthropods are defined as a vehicles of transmission
D-r Mitova 18
HUMAN RESERVOIR
By far the most important source or reservoir of infection for humans is man himself He my be a case or carrier Man is often described as him own enemy because most of the communicable disease of which man is heir to a contacted from human sources
D-r Mitova 19
HUMAN RESERVOIR - CASE
A case is defined as a person in the population or study group identified as having the particular disease health disorder or condition under investigation A variety of criteria ( clinical biochemical laboratory ) may be used to identify cases Broadly the presence of infection in the host may be clinical subclinical or latent
D-r Mitova 20
A case is a risk factor hellip Infection in one person can be transmitted
to others
What is infectious disease epidemiology
D-r Mitova 21
1The clinical illness
The clinical illness maybe mild or moderate typical or atypical severe or fatal
Epidemiologically mild cases may be more important sources of infection than severe cases because they are ambulant and spread the infection wherever they go whereas severe cases usually confined to bed
D-r Mitova 22
2The subclinical cases are variously referred to as inapparent covert missed or abortive cases They are equally important sources of infection The disease agent may multiply in the host but does not manifest itself by sign and symptoms The disease agent is eliminated and contaminates the environment in the same way as clinical casesperson who are thus sick contribute more than symptomatic patient to the transmission of infection to others and what is more they do not appear in any of statistics
Subclinical cases play a dominant role in maintaining the chain of infection in the community The latent infection must be distinguished from subclinical infection In latent infection the host does not shed the infectious agent
D-r Mitova 23
The subclinical cases
Subclinical infection can be detected only by laboratory tests
Barring a few(mesles varicella) subclinical infection occurs in most infectious disease In some disease(rubella mumps polio hepatitis A and B influenza diphteria) a great deal of subclinical infection occurs
D-r Mitova 24
Latent infectionThe term latent infection must be
distinguished from subclinical infection In latent infection the host does not shed the infectious agent which lies dormant within the host without symptoms (and often without demonstrable presence in blood tissues or bodily secretions of the host) For examples latent infection occurs in herpes symplex Brill-Zincer disease
D-r Mitova 25
CARRIERS
In some disease either due to inadequate treatment or immune response the disease agent is not completely eliminated leading to a carrier state A carrier is defined as an infected person or animal that harbors a specific infectious agent in the absence of
discernible clinical disease and serves as a potential source of infection for others
D-r Mitova 26
CARRIERS
As a rule carriers are less infectious than cases but epidemiologically they are more dangerous than cases because they escape recognition and continuing as they do live a normal life among population or community they readily infect the susceptible individuals over a wider area and longer period of time under favorable conditionsthe ldquoTyphoid Maryrdquo is a classic example of a carrier-cooker Mary who gave rise to more than 1300 cases in her life time
D-r Mitova 27
The elements in a carrier state are
The presence in the body of disease agent
The absence of recognizable symptoms and signs of disease
The shedding of disease agent in the discharges or excretion
D-r Mitova 28
incubatory carriers are those who shed the infectious agent during the incubation period This usually occurs during the the last few days of incubation period As for example
Measles-the period of communicability is 4 days before the rush
Mumps-usually 4-6 days before onset of symptoms
Polio-7-10 days before onset of symptomsHepatitis B-for a month before
jaundiceabout 6 weeks after exposure
Carrier may be classified BY TYPE
D-r Mitova 29
Carrier may be classified BY TYPE
convalescent carriers that is those who continue to shed the disease agent during the period of convalescence(typhoid fever cholera diphteria bacillary disentery) In the disease clinical recovery does not coincide with bacteriological recovery
D-r Mitova 30
Carrier may be classified BY TYPE
healthy carriers they are victims of subclinical infection who have developed carrier state without suffering from overt disease but are nevertheless shedding the disease agent eg poliomyelitis cholera meningococcal meningitis salmonellosis diphtheria
D-r Mitova 31
BY DURATION
Temporary carriers are those who shed the infectious agent for short period of timeChronic carriers are those who excretes the infectious agent for indefinite periods eg typhoid fever hepatitis B dysentery etcThey are known to reintroduce disease into areas which are otherwise free of infection
D-r Mitova 32
Chronic carriers
Chronic carriers are far more important sources of infection than cases The longer the carrier state the greater the risk of community The duration of the carrier state varies with the disease In typhoid fever and hepatitis B the chronic carrier state may last for several yearsIn chronic dysentery it may last for year or longer In diphtheria the carrier state is associated with infected tonsils in typhoid fever with gall bladder disease
D-r Mitova 33
BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 17
In Bulgaria the source of infection is defined only as the person or animal in which an infectious agent live and multiplies and from which an infectious agent passes to the host inanimate things and arthropods are defined as a vehicles of transmission
D-r Mitova 18
HUMAN RESERVOIR
By far the most important source or reservoir of infection for humans is man himself He my be a case or carrier Man is often described as him own enemy because most of the communicable disease of which man is heir to a contacted from human sources
D-r Mitova 19
HUMAN RESERVOIR - CASE
A case is defined as a person in the population or study group identified as having the particular disease health disorder or condition under investigation A variety of criteria ( clinical biochemical laboratory ) may be used to identify cases Broadly the presence of infection in the host may be clinical subclinical or latent
D-r Mitova 20
A case is a risk factor hellip Infection in one person can be transmitted
to others
What is infectious disease epidemiology
D-r Mitova 21
1The clinical illness
The clinical illness maybe mild or moderate typical or atypical severe or fatal
Epidemiologically mild cases may be more important sources of infection than severe cases because they are ambulant and spread the infection wherever they go whereas severe cases usually confined to bed
D-r Mitova 22
2The subclinical cases are variously referred to as inapparent covert missed or abortive cases They are equally important sources of infection The disease agent may multiply in the host but does not manifest itself by sign and symptoms The disease agent is eliminated and contaminates the environment in the same way as clinical casesperson who are thus sick contribute more than symptomatic patient to the transmission of infection to others and what is more they do not appear in any of statistics
Subclinical cases play a dominant role in maintaining the chain of infection in the community The latent infection must be distinguished from subclinical infection In latent infection the host does not shed the infectious agent
D-r Mitova 23
The subclinical cases
Subclinical infection can be detected only by laboratory tests
Barring a few(mesles varicella) subclinical infection occurs in most infectious disease In some disease(rubella mumps polio hepatitis A and B influenza diphteria) a great deal of subclinical infection occurs
D-r Mitova 24
Latent infectionThe term latent infection must be
distinguished from subclinical infection In latent infection the host does not shed the infectious agent which lies dormant within the host without symptoms (and often without demonstrable presence in blood tissues or bodily secretions of the host) For examples latent infection occurs in herpes symplex Brill-Zincer disease
D-r Mitova 25
CARRIERS
In some disease either due to inadequate treatment or immune response the disease agent is not completely eliminated leading to a carrier state A carrier is defined as an infected person or animal that harbors a specific infectious agent in the absence of
discernible clinical disease and serves as a potential source of infection for others
D-r Mitova 26
CARRIERS
As a rule carriers are less infectious than cases but epidemiologically they are more dangerous than cases because they escape recognition and continuing as they do live a normal life among population or community they readily infect the susceptible individuals over a wider area and longer period of time under favorable conditionsthe ldquoTyphoid Maryrdquo is a classic example of a carrier-cooker Mary who gave rise to more than 1300 cases in her life time
D-r Mitova 27
The elements in a carrier state are
The presence in the body of disease agent
The absence of recognizable symptoms and signs of disease
The shedding of disease agent in the discharges or excretion
D-r Mitova 28
incubatory carriers are those who shed the infectious agent during the incubation period This usually occurs during the the last few days of incubation period As for example
Measles-the period of communicability is 4 days before the rush
Mumps-usually 4-6 days before onset of symptoms
Polio-7-10 days before onset of symptomsHepatitis B-for a month before
jaundiceabout 6 weeks after exposure
Carrier may be classified BY TYPE
D-r Mitova 29
Carrier may be classified BY TYPE
convalescent carriers that is those who continue to shed the disease agent during the period of convalescence(typhoid fever cholera diphteria bacillary disentery) In the disease clinical recovery does not coincide with bacteriological recovery
D-r Mitova 30
Carrier may be classified BY TYPE
healthy carriers they are victims of subclinical infection who have developed carrier state without suffering from overt disease but are nevertheless shedding the disease agent eg poliomyelitis cholera meningococcal meningitis salmonellosis diphtheria
D-r Mitova 31
BY DURATION
Temporary carriers are those who shed the infectious agent for short period of timeChronic carriers are those who excretes the infectious agent for indefinite periods eg typhoid fever hepatitis B dysentery etcThey are known to reintroduce disease into areas which are otherwise free of infection
D-r Mitova 32
Chronic carriers
Chronic carriers are far more important sources of infection than cases The longer the carrier state the greater the risk of community The duration of the carrier state varies with the disease In typhoid fever and hepatitis B the chronic carrier state may last for several yearsIn chronic dysentery it may last for year or longer In diphtheria the carrier state is associated with infected tonsils in typhoid fever with gall bladder disease
D-r Mitova 33
BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 18
HUMAN RESERVOIR
By far the most important source or reservoir of infection for humans is man himself He my be a case or carrier Man is often described as him own enemy because most of the communicable disease of which man is heir to a contacted from human sources
D-r Mitova 19
HUMAN RESERVOIR - CASE
A case is defined as a person in the population or study group identified as having the particular disease health disorder or condition under investigation A variety of criteria ( clinical biochemical laboratory ) may be used to identify cases Broadly the presence of infection in the host may be clinical subclinical or latent
D-r Mitova 20
A case is a risk factor hellip Infection in one person can be transmitted
to others
What is infectious disease epidemiology
D-r Mitova 21
1The clinical illness
The clinical illness maybe mild or moderate typical or atypical severe or fatal
Epidemiologically mild cases may be more important sources of infection than severe cases because they are ambulant and spread the infection wherever they go whereas severe cases usually confined to bed
D-r Mitova 22
2The subclinical cases are variously referred to as inapparent covert missed or abortive cases They are equally important sources of infection The disease agent may multiply in the host but does not manifest itself by sign and symptoms The disease agent is eliminated and contaminates the environment in the same way as clinical casesperson who are thus sick contribute more than symptomatic patient to the transmission of infection to others and what is more they do not appear in any of statistics
Subclinical cases play a dominant role in maintaining the chain of infection in the community The latent infection must be distinguished from subclinical infection In latent infection the host does not shed the infectious agent
D-r Mitova 23
The subclinical cases
Subclinical infection can be detected only by laboratory tests
Barring a few(mesles varicella) subclinical infection occurs in most infectious disease In some disease(rubella mumps polio hepatitis A and B influenza diphteria) a great deal of subclinical infection occurs
D-r Mitova 24
Latent infectionThe term latent infection must be
distinguished from subclinical infection In latent infection the host does not shed the infectious agent which lies dormant within the host without symptoms (and often without demonstrable presence in blood tissues or bodily secretions of the host) For examples latent infection occurs in herpes symplex Brill-Zincer disease
D-r Mitova 25
CARRIERS
In some disease either due to inadequate treatment or immune response the disease agent is not completely eliminated leading to a carrier state A carrier is defined as an infected person or animal that harbors a specific infectious agent in the absence of
discernible clinical disease and serves as a potential source of infection for others
D-r Mitova 26
CARRIERS
As a rule carriers are less infectious than cases but epidemiologically they are more dangerous than cases because they escape recognition and continuing as they do live a normal life among population or community they readily infect the susceptible individuals over a wider area and longer period of time under favorable conditionsthe ldquoTyphoid Maryrdquo is a classic example of a carrier-cooker Mary who gave rise to more than 1300 cases in her life time
D-r Mitova 27
The elements in a carrier state are
The presence in the body of disease agent
The absence of recognizable symptoms and signs of disease
The shedding of disease agent in the discharges or excretion
D-r Mitova 28
incubatory carriers are those who shed the infectious agent during the incubation period This usually occurs during the the last few days of incubation period As for example
Measles-the period of communicability is 4 days before the rush
Mumps-usually 4-6 days before onset of symptoms
Polio-7-10 days before onset of symptomsHepatitis B-for a month before
jaundiceabout 6 weeks after exposure
Carrier may be classified BY TYPE
D-r Mitova 29
Carrier may be classified BY TYPE
convalescent carriers that is those who continue to shed the disease agent during the period of convalescence(typhoid fever cholera diphteria bacillary disentery) In the disease clinical recovery does not coincide with bacteriological recovery
D-r Mitova 30
Carrier may be classified BY TYPE
healthy carriers they are victims of subclinical infection who have developed carrier state without suffering from overt disease but are nevertheless shedding the disease agent eg poliomyelitis cholera meningococcal meningitis salmonellosis diphtheria
D-r Mitova 31
BY DURATION
Temporary carriers are those who shed the infectious agent for short period of timeChronic carriers are those who excretes the infectious agent for indefinite periods eg typhoid fever hepatitis B dysentery etcThey are known to reintroduce disease into areas which are otherwise free of infection
D-r Mitova 32
Chronic carriers
Chronic carriers are far more important sources of infection than cases The longer the carrier state the greater the risk of community The duration of the carrier state varies with the disease In typhoid fever and hepatitis B the chronic carrier state may last for several yearsIn chronic dysentery it may last for year or longer In diphtheria the carrier state is associated with infected tonsils in typhoid fever with gall bladder disease
D-r Mitova 33
BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 19
HUMAN RESERVOIR - CASE
A case is defined as a person in the population or study group identified as having the particular disease health disorder or condition under investigation A variety of criteria ( clinical biochemical laboratory ) may be used to identify cases Broadly the presence of infection in the host may be clinical subclinical or latent
D-r Mitova 20
A case is a risk factor hellip Infection in one person can be transmitted
to others
What is infectious disease epidemiology
D-r Mitova 21
1The clinical illness
The clinical illness maybe mild or moderate typical or atypical severe or fatal
Epidemiologically mild cases may be more important sources of infection than severe cases because they are ambulant and spread the infection wherever they go whereas severe cases usually confined to bed
D-r Mitova 22
2The subclinical cases are variously referred to as inapparent covert missed or abortive cases They are equally important sources of infection The disease agent may multiply in the host but does not manifest itself by sign and symptoms The disease agent is eliminated and contaminates the environment in the same way as clinical casesperson who are thus sick contribute more than symptomatic patient to the transmission of infection to others and what is more they do not appear in any of statistics
Subclinical cases play a dominant role in maintaining the chain of infection in the community The latent infection must be distinguished from subclinical infection In latent infection the host does not shed the infectious agent
D-r Mitova 23
The subclinical cases
Subclinical infection can be detected only by laboratory tests
Barring a few(mesles varicella) subclinical infection occurs in most infectious disease In some disease(rubella mumps polio hepatitis A and B influenza diphteria) a great deal of subclinical infection occurs
D-r Mitova 24
Latent infectionThe term latent infection must be
distinguished from subclinical infection In latent infection the host does not shed the infectious agent which lies dormant within the host without symptoms (and often without demonstrable presence in blood tissues or bodily secretions of the host) For examples latent infection occurs in herpes symplex Brill-Zincer disease
D-r Mitova 25
CARRIERS
In some disease either due to inadequate treatment or immune response the disease agent is not completely eliminated leading to a carrier state A carrier is defined as an infected person or animal that harbors a specific infectious agent in the absence of
discernible clinical disease and serves as a potential source of infection for others
D-r Mitova 26
CARRIERS
As a rule carriers are less infectious than cases but epidemiologically they are more dangerous than cases because they escape recognition and continuing as they do live a normal life among population or community they readily infect the susceptible individuals over a wider area and longer period of time under favorable conditionsthe ldquoTyphoid Maryrdquo is a classic example of a carrier-cooker Mary who gave rise to more than 1300 cases in her life time
D-r Mitova 27
The elements in a carrier state are
The presence in the body of disease agent
The absence of recognizable symptoms and signs of disease
The shedding of disease agent in the discharges or excretion
D-r Mitova 28
incubatory carriers are those who shed the infectious agent during the incubation period This usually occurs during the the last few days of incubation period As for example
Measles-the period of communicability is 4 days before the rush
Mumps-usually 4-6 days before onset of symptoms
Polio-7-10 days before onset of symptomsHepatitis B-for a month before
jaundiceabout 6 weeks after exposure
Carrier may be classified BY TYPE
D-r Mitova 29
Carrier may be classified BY TYPE
convalescent carriers that is those who continue to shed the disease agent during the period of convalescence(typhoid fever cholera diphteria bacillary disentery) In the disease clinical recovery does not coincide with bacteriological recovery
D-r Mitova 30
Carrier may be classified BY TYPE
healthy carriers they are victims of subclinical infection who have developed carrier state without suffering from overt disease but are nevertheless shedding the disease agent eg poliomyelitis cholera meningococcal meningitis salmonellosis diphtheria
D-r Mitova 31
BY DURATION
Temporary carriers are those who shed the infectious agent for short period of timeChronic carriers are those who excretes the infectious agent for indefinite periods eg typhoid fever hepatitis B dysentery etcThey are known to reintroduce disease into areas which are otherwise free of infection
D-r Mitova 32
Chronic carriers
Chronic carriers are far more important sources of infection than cases The longer the carrier state the greater the risk of community The duration of the carrier state varies with the disease In typhoid fever and hepatitis B the chronic carrier state may last for several yearsIn chronic dysentery it may last for year or longer In diphtheria the carrier state is associated with infected tonsils in typhoid fever with gall bladder disease
D-r Mitova 33
BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 20
A case is a risk factor hellip Infection in one person can be transmitted
to others
What is infectious disease epidemiology
D-r Mitova 21
1The clinical illness
The clinical illness maybe mild or moderate typical or atypical severe or fatal
Epidemiologically mild cases may be more important sources of infection than severe cases because they are ambulant and spread the infection wherever they go whereas severe cases usually confined to bed
D-r Mitova 22
2The subclinical cases are variously referred to as inapparent covert missed or abortive cases They are equally important sources of infection The disease agent may multiply in the host but does not manifest itself by sign and symptoms The disease agent is eliminated and contaminates the environment in the same way as clinical casesperson who are thus sick contribute more than symptomatic patient to the transmission of infection to others and what is more they do not appear in any of statistics
Subclinical cases play a dominant role in maintaining the chain of infection in the community The latent infection must be distinguished from subclinical infection In latent infection the host does not shed the infectious agent
D-r Mitova 23
The subclinical cases
Subclinical infection can be detected only by laboratory tests
Barring a few(mesles varicella) subclinical infection occurs in most infectious disease In some disease(rubella mumps polio hepatitis A and B influenza diphteria) a great deal of subclinical infection occurs
D-r Mitova 24
Latent infectionThe term latent infection must be
distinguished from subclinical infection In latent infection the host does not shed the infectious agent which lies dormant within the host without symptoms (and often without demonstrable presence in blood tissues or bodily secretions of the host) For examples latent infection occurs in herpes symplex Brill-Zincer disease
D-r Mitova 25
CARRIERS
In some disease either due to inadequate treatment or immune response the disease agent is not completely eliminated leading to a carrier state A carrier is defined as an infected person or animal that harbors a specific infectious agent in the absence of
discernible clinical disease and serves as a potential source of infection for others
D-r Mitova 26
CARRIERS
As a rule carriers are less infectious than cases but epidemiologically they are more dangerous than cases because they escape recognition and continuing as they do live a normal life among population or community they readily infect the susceptible individuals over a wider area and longer period of time under favorable conditionsthe ldquoTyphoid Maryrdquo is a classic example of a carrier-cooker Mary who gave rise to more than 1300 cases in her life time
D-r Mitova 27
The elements in a carrier state are
The presence in the body of disease agent
The absence of recognizable symptoms and signs of disease
The shedding of disease agent in the discharges or excretion
D-r Mitova 28
incubatory carriers are those who shed the infectious agent during the incubation period This usually occurs during the the last few days of incubation period As for example
Measles-the period of communicability is 4 days before the rush
Mumps-usually 4-6 days before onset of symptoms
Polio-7-10 days before onset of symptomsHepatitis B-for a month before
jaundiceabout 6 weeks after exposure
Carrier may be classified BY TYPE
D-r Mitova 29
Carrier may be classified BY TYPE
convalescent carriers that is those who continue to shed the disease agent during the period of convalescence(typhoid fever cholera diphteria bacillary disentery) In the disease clinical recovery does not coincide with bacteriological recovery
D-r Mitova 30
Carrier may be classified BY TYPE
healthy carriers they are victims of subclinical infection who have developed carrier state without suffering from overt disease but are nevertheless shedding the disease agent eg poliomyelitis cholera meningococcal meningitis salmonellosis diphtheria
D-r Mitova 31
BY DURATION
Temporary carriers are those who shed the infectious agent for short period of timeChronic carriers are those who excretes the infectious agent for indefinite periods eg typhoid fever hepatitis B dysentery etcThey are known to reintroduce disease into areas which are otherwise free of infection
D-r Mitova 32
Chronic carriers
Chronic carriers are far more important sources of infection than cases The longer the carrier state the greater the risk of community The duration of the carrier state varies with the disease In typhoid fever and hepatitis B the chronic carrier state may last for several yearsIn chronic dysentery it may last for year or longer In diphtheria the carrier state is associated with infected tonsils in typhoid fever with gall bladder disease
D-r Mitova 33
BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 21
1The clinical illness
The clinical illness maybe mild or moderate typical or atypical severe or fatal
Epidemiologically mild cases may be more important sources of infection than severe cases because they are ambulant and spread the infection wherever they go whereas severe cases usually confined to bed
D-r Mitova 22
2The subclinical cases are variously referred to as inapparent covert missed or abortive cases They are equally important sources of infection The disease agent may multiply in the host but does not manifest itself by sign and symptoms The disease agent is eliminated and contaminates the environment in the same way as clinical casesperson who are thus sick contribute more than symptomatic patient to the transmission of infection to others and what is more they do not appear in any of statistics
Subclinical cases play a dominant role in maintaining the chain of infection in the community The latent infection must be distinguished from subclinical infection In latent infection the host does not shed the infectious agent
D-r Mitova 23
The subclinical cases
Subclinical infection can be detected only by laboratory tests
Barring a few(mesles varicella) subclinical infection occurs in most infectious disease In some disease(rubella mumps polio hepatitis A and B influenza diphteria) a great deal of subclinical infection occurs
D-r Mitova 24
Latent infectionThe term latent infection must be
distinguished from subclinical infection In latent infection the host does not shed the infectious agent which lies dormant within the host without symptoms (and often without demonstrable presence in blood tissues or bodily secretions of the host) For examples latent infection occurs in herpes symplex Brill-Zincer disease
D-r Mitova 25
CARRIERS
In some disease either due to inadequate treatment or immune response the disease agent is not completely eliminated leading to a carrier state A carrier is defined as an infected person or animal that harbors a specific infectious agent in the absence of
discernible clinical disease and serves as a potential source of infection for others
D-r Mitova 26
CARRIERS
As a rule carriers are less infectious than cases but epidemiologically they are more dangerous than cases because they escape recognition and continuing as they do live a normal life among population or community they readily infect the susceptible individuals over a wider area and longer period of time under favorable conditionsthe ldquoTyphoid Maryrdquo is a classic example of a carrier-cooker Mary who gave rise to more than 1300 cases in her life time
D-r Mitova 27
The elements in a carrier state are
The presence in the body of disease agent
The absence of recognizable symptoms and signs of disease
The shedding of disease agent in the discharges or excretion
D-r Mitova 28
incubatory carriers are those who shed the infectious agent during the incubation period This usually occurs during the the last few days of incubation period As for example
Measles-the period of communicability is 4 days before the rush
Mumps-usually 4-6 days before onset of symptoms
Polio-7-10 days before onset of symptomsHepatitis B-for a month before
jaundiceabout 6 weeks after exposure
Carrier may be classified BY TYPE
D-r Mitova 29
Carrier may be classified BY TYPE
convalescent carriers that is those who continue to shed the disease agent during the period of convalescence(typhoid fever cholera diphteria bacillary disentery) In the disease clinical recovery does not coincide with bacteriological recovery
D-r Mitova 30
Carrier may be classified BY TYPE
healthy carriers they are victims of subclinical infection who have developed carrier state without suffering from overt disease but are nevertheless shedding the disease agent eg poliomyelitis cholera meningococcal meningitis salmonellosis diphtheria
D-r Mitova 31
BY DURATION
Temporary carriers are those who shed the infectious agent for short period of timeChronic carriers are those who excretes the infectious agent for indefinite periods eg typhoid fever hepatitis B dysentery etcThey are known to reintroduce disease into areas which are otherwise free of infection
D-r Mitova 32
Chronic carriers
Chronic carriers are far more important sources of infection than cases The longer the carrier state the greater the risk of community The duration of the carrier state varies with the disease In typhoid fever and hepatitis B the chronic carrier state may last for several yearsIn chronic dysentery it may last for year or longer In diphtheria the carrier state is associated with infected tonsils in typhoid fever with gall bladder disease
D-r Mitova 33
BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 22
2The subclinical cases are variously referred to as inapparent covert missed or abortive cases They are equally important sources of infection The disease agent may multiply in the host but does not manifest itself by sign and symptoms The disease agent is eliminated and contaminates the environment in the same way as clinical casesperson who are thus sick contribute more than symptomatic patient to the transmission of infection to others and what is more they do not appear in any of statistics
Subclinical cases play a dominant role in maintaining the chain of infection in the community The latent infection must be distinguished from subclinical infection In latent infection the host does not shed the infectious agent
D-r Mitova 23
The subclinical cases
Subclinical infection can be detected only by laboratory tests
Barring a few(mesles varicella) subclinical infection occurs in most infectious disease In some disease(rubella mumps polio hepatitis A and B influenza diphteria) a great deal of subclinical infection occurs
D-r Mitova 24
Latent infectionThe term latent infection must be
distinguished from subclinical infection In latent infection the host does not shed the infectious agent which lies dormant within the host without symptoms (and often without demonstrable presence in blood tissues or bodily secretions of the host) For examples latent infection occurs in herpes symplex Brill-Zincer disease
D-r Mitova 25
CARRIERS
In some disease either due to inadequate treatment or immune response the disease agent is not completely eliminated leading to a carrier state A carrier is defined as an infected person or animal that harbors a specific infectious agent in the absence of
discernible clinical disease and serves as a potential source of infection for others
D-r Mitova 26
CARRIERS
As a rule carriers are less infectious than cases but epidemiologically they are more dangerous than cases because they escape recognition and continuing as they do live a normal life among population or community they readily infect the susceptible individuals over a wider area and longer period of time under favorable conditionsthe ldquoTyphoid Maryrdquo is a classic example of a carrier-cooker Mary who gave rise to more than 1300 cases in her life time
D-r Mitova 27
The elements in a carrier state are
The presence in the body of disease agent
The absence of recognizable symptoms and signs of disease
The shedding of disease agent in the discharges or excretion
D-r Mitova 28
incubatory carriers are those who shed the infectious agent during the incubation period This usually occurs during the the last few days of incubation period As for example
Measles-the period of communicability is 4 days before the rush
Mumps-usually 4-6 days before onset of symptoms
Polio-7-10 days before onset of symptomsHepatitis B-for a month before
jaundiceabout 6 weeks after exposure
Carrier may be classified BY TYPE
D-r Mitova 29
Carrier may be classified BY TYPE
convalescent carriers that is those who continue to shed the disease agent during the period of convalescence(typhoid fever cholera diphteria bacillary disentery) In the disease clinical recovery does not coincide with bacteriological recovery
D-r Mitova 30
Carrier may be classified BY TYPE
healthy carriers they are victims of subclinical infection who have developed carrier state without suffering from overt disease but are nevertheless shedding the disease agent eg poliomyelitis cholera meningococcal meningitis salmonellosis diphtheria
D-r Mitova 31
BY DURATION
Temporary carriers are those who shed the infectious agent for short period of timeChronic carriers are those who excretes the infectious agent for indefinite periods eg typhoid fever hepatitis B dysentery etcThey are known to reintroduce disease into areas which are otherwise free of infection
D-r Mitova 32
Chronic carriers
Chronic carriers are far more important sources of infection than cases The longer the carrier state the greater the risk of community The duration of the carrier state varies with the disease In typhoid fever and hepatitis B the chronic carrier state may last for several yearsIn chronic dysentery it may last for year or longer In diphtheria the carrier state is associated with infected tonsils in typhoid fever with gall bladder disease
D-r Mitova 33
BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 23
The subclinical cases
Subclinical infection can be detected only by laboratory tests
Barring a few(mesles varicella) subclinical infection occurs in most infectious disease In some disease(rubella mumps polio hepatitis A and B influenza diphteria) a great deal of subclinical infection occurs
D-r Mitova 24
Latent infectionThe term latent infection must be
distinguished from subclinical infection In latent infection the host does not shed the infectious agent which lies dormant within the host without symptoms (and often without demonstrable presence in blood tissues or bodily secretions of the host) For examples latent infection occurs in herpes symplex Brill-Zincer disease
D-r Mitova 25
CARRIERS
In some disease either due to inadequate treatment or immune response the disease agent is not completely eliminated leading to a carrier state A carrier is defined as an infected person or animal that harbors a specific infectious agent in the absence of
discernible clinical disease and serves as a potential source of infection for others
D-r Mitova 26
CARRIERS
As a rule carriers are less infectious than cases but epidemiologically they are more dangerous than cases because they escape recognition and continuing as they do live a normal life among population or community they readily infect the susceptible individuals over a wider area and longer period of time under favorable conditionsthe ldquoTyphoid Maryrdquo is a classic example of a carrier-cooker Mary who gave rise to more than 1300 cases in her life time
D-r Mitova 27
The elements in a carrier state are
The presence in the body of disease agent
The absence of recognizable symptoms and signs of disease
The shedding of disease agent in the discharges or excretion
D-r Mitova 28
incubatory carriers are those who shed the infectious agent during the incubation period This usually occurs during the the last few days of incubation period As for example
Measles-the period of communicability is 4 days before the rush
Mumps-usually 4-6 days before onset of symptoms
Polio-7-10 days before onset of symptomsHepatitis B-for a month before
jaundiceabout 6 weeks after exposure
Carrier may be classified BY TYPE
D-r Mitova 29
Carrier may be classified BY TYPE
convalescent carriers that is those who continue to shed the disease agent during the period of convalescence(typhoid fever cholera diphteria bacillary disentery) In the disease clinical recovery does not coincide with bacteriological recovery
D-r Mitova 30
Carrier may be classified BY TYPE
healthy carriers they are victims of subclinical infection who have developed carrier state without suffering from overt disease but are nevertheless shedding the disease agent eg poliomyelitis cholera meningococcal meningitis salmonellosis diphtheria
D-r Mitova 31
BY DURATION
Temporary carriers are those who shed the infectious agent for short period of timeChronic carriers are those who excretes the infectious agent for indefinite periods eg typhoid fever hepatitis B dysentery etcThey are known to reintroduce disease into areas which are otherwise free of infection
D-r Mitova 32
Chronic carriers
Chronic carriers are far more important sources of infection than cases The longer the carrier state the greater the risk of community The duration of the carrier state varies with the disease In typhoid fever and hepatitis B the chronic carrier state may last for several yearsIn chronic dysentery it may last for year or longer In diphtheria the carrier state is associated with infected tonsils in typhoid fever with gall bladder disease
D-r Mitova 33
BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 24
Latent infectionThe term latent infection must be
distinguished from subclinical infection In latent infection the host does not shed the infectious agent which lies dormant within the host without symptoms (and often without demonstrable presence in blood tissues or bodily secretions of the host) For examples latent infection occurs in herpes symplex Brill-Zincer disease
D-r Mitova 25
CARRIERS
In some disease either due to inadequate treatment or immune response the disease agent is not completely eliminated leading to a carrier state A carrier is defined as an infected person or animal that harbors a specific infectious agent in the absence of
discernible clinical disease and serves as a potential source of infection for others
D-r Mitova 26
CARRIERS
As a rule carriers are less infectious than cases but epidemiologically they are more dangerous than cases because they escape recognition and continuing as they do live a normal life among population or community they readily infect the susceptible individuals over a wider area and longer period of time under favorable conditionsthe ldquoTyphoid Maryrdquo is a classic example of a carrier-cooker Mary who gave rise to more than 1300 cases in her life time
D-r Mitova 27
The elements in a carrier state are
The presence in the body of disease agent
The absence of recognizable symptoms and signs of disease
The shedding of disease agent in the discharges or excretion
D-r Mitova 28
incubatory carriers are those who shed the infectious agent during the incubation period This usually occurs during the the last few days of incubation period As for example
Measles-the period of communicability is 4 days before the rush
Mumps-usually 4-6 days before onset of symptoms
Polio-7-10 days before onset of symptomsHepatitis B-for a month before
jaundiceabout 6 weeks after exposure
Carrier may be classified BY TYPE
D-r Mitova 29
Carrier may be classified BY TYPE
convalescent carriers that is those who continue to shed the disease agent during the period of convalescence(typhoid fever cholera diphteria bacillary disentery) In the disease clinical recovery does not coincide with bacteriological recovery
D-r Mitova 30
Carrier may be classified BY TYPE
healthy carriers they are victims of subclinical infection who have developed carrier state without suffering from overt disease but are nevertheless shedding the disease agent eg poliomyelitis cholera meningococcal meningitis salmonellosis diphtheria
D-r Mitova 31
BY DURATION
Temporary carriers are those who shed the infectious agent for short period of timeChronic carriers are those who excretes the infectious agent for indefinite periods eg typhoid fever hepatitis B dysentery etcThey are known to reintroduce disease into areas which are otherwise free of infection
D-r Mitova 32
Chronic carriers
Chronic carriers are far more important sources of infection than cases The longer the carrier state the greater the risk of community The duration of the carrier state varies with the disease In typhoid fever and hepatitis B the chronic carrier state may last for several yearsIn chronic dysentery it may last for year or longer In diphtheria the carrier state is associated with infected tonsils in typhoid fever with gall bladder disease
D-r Mitova 33
BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 25
CARRIERS
In some disease either due to inadequate treatment or immune response the disease agent is not completely eliminated leading to a carrier state A carrier is defined as an infected person or animal that harbors a specific infectious agent in the absence of
discernible clinical disease and serves as a potential source of infection for others
D-r Mitova 26
CARRIERS
As a rule carriers are less infectious than cases but epidemiologically they are more dangerous than cases because they escape recognition and continuing as they do live a normal life among population or community they readily infect the susceptible individuals over a wider area and longer period of time under favorable conditionsthe ldquoTyphoid Maryrdquo is a classic example of a carrier-cooker Mary who gave rise to more than 1300 cases in her life time
D-r Mitova 27
The elements in a carrier state are
The presence in the body of disease agent
The absence of recognizable symptoms and signs of disease
The shedding of disease agent in the discharges or excretion
D-r Mitova 28
incubatory carriers are those who shed the infectious agent during the incubation period This usually occurs during the the last few days of incubation period As for example
Measles-the period of communicability is 4 days before the rush
Mumps-usually 4-6 days before onset of symptoms
Polio-7-10 days before onset of symptomsHepatitis B-for a month before
jaundiceabout 6 weeks after exposure
Carrier may be classified BY TYPE
D-r Mitova 29
Carrier may be classified BY TYPE
convalescent carriers that is those who continue to shed the disease agent during the period of convalescence(typhoid fever cholera diphteria bacillary disentery) In the disease clinical recovery does not coincide with bacteriological recovery
D-r Mitova 30
Carrier may be classified BY TYPE
healthy carriers they are victims of subclinical infection who have developed carrier state without suffering from overt disease but are nevertheless shedding the disease agent eg poliomyelitis cholera meningococcal meningitis salmonellosis diphtheria
D-r Mitova 31
BY DURATION
Temporary carriers are those who shed the infectious agent for short period of timeChronic carriers are those who excretes the infectious agent for indefinite periods eg typhoid fever hepatitis B dysentery etcThey are known to reintroduce disease into areas which are otherwise free of infection
D-r Mitova 32
Chronic carriers
Chronic carriers are far more important sources of infection than cases The longer the carrier state the greater the risk of community The duration of the carrier state varies with the disease In typhoid fever and hepatitis B the chronic carrier state may last for several yearsIn chronic dysentery it may last for year or longer In diphtheria the carrier state is associated with infected tonsils in typhoid fever with gall bladder disease
D-r Mitova 33
BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 26
CARRIERS
As a rule carriers are less infectious than cases but epidemiologically they are more dangerous than cases because they escape recognition and continuing as they do live a normal life among population or community they readily infect the susceptible individuals over a wider area and longer period of time under favorable conditionsthe ldquoTyphoid Maryrdquo is a classic example of a carrier-cooker Mary who gave rise to more than 1300 cases in her life time
D-r Mitova 27
The elements in a carrier state are
The presence in the body of disease agent
The absence of recognizable symptoms and signs of disease
The shedding of disease agent in the discharges or excretion
D-r Mitova 28
incubatory carriers are those who shed the infectious agent during the incubation period This usually occurs during the the last few days of incubation period As for example
Measles-the period of communicability is 4 days before the rush
Mumps-usually 4-6 days before onset of symptoms
Polio-7-10 days before onset of symptomsHepatitis B-for a month before
jaundiceabout 6 weeks after exposure
Carrier may be classified BY TYPE
D-r Mitova 29
Carrier may be classified BY TYPE
convalescent carriers that is those who continue to shed the disease agent during the period of convalescence(typhoid fever cholera diphteria bacillary disentery) In the disease clinical recovery does not coincide with bacteriological recovery
D-r Mitova 30
Carrier may be classified BY TYPE
healthy carriers they are victims of subclinical infection who have developed carrier state without suffering from overt disease but are nevertheless shedding the disease agent eg poliomyelitis cholera meningococcal meningitis salmonellosis diphtheria
D-r Mitova 31
BY DURATION
Temporary carriers are those who shed the infectious agent for short period of timeChronic carriers are those who excretes the infectious agent for indefinite periods eg typhoid fever hepatitis B dysentery etcThey are known to reintroduce disease into areas which are otherwise free of infection
D-r Mitova 32
Chronic carriers
Chronic carriers are far more important sources of infection than cases The longer the carrier state the greater the risk of community The duration of the carrier state varies with the disease In typhoid fever and hepatitis B the chronic carrier state may last for several yearsIn chronic dysentery it may last for year or longer In diphtheria the carrier state is associated with infected tonsils in typhoid fever with gall bladder disease
D-r Mitova 33
BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 27
The elements in a carrier state are
The presence in the body of disease agent
The absence of recognizable symptoms and signs of disease
The shedding of disease agent in the discharges or excretion
D-r Mitova 28
incubatory carriers are those who shed the infectious agent during the incubation period This usually occurs during the the last few days of incubation period As for example
Measles-the period of communicability is 4 days before the rush
Mumps-usually 4-6 days before onset of symptoms
Polio-7-10 days before onset of symptomsHepatitis B-for a month before
jaundiceabout 6 weeks after exposure
Carrier may be classified BY TYPE
D-r Mitova 29
Carrier may be classified BY TYPE
convalescent carriers that is those who continue to shed the disease agent during the period of convalescence(typhoid fever cholera diphteria bacillary disentery) In the disease clinical recovery does not coincide with bacteriological recovery
D-r Mitova 30
Carrier may be classified BY TYPE
healthy carriers they are victims of subclinical infection who have developed carrier state without suffering from overt disease but are nevertheless shedding the disease agent eg poliomyelitis cholera meningococcal meningitis salmonellosis diphtheria
D-r Mitova 31
BY DURATION
Temporary carriers are those who shed the infectious agent for short period of timeChronic carriers are those who excretes the infectious agent for indefinite periods eg typhoid fever hepatitis B dysentery etcThey are known to reintroduce disease into areas which are otherwise free of infection
D-r Mitova 32
Chronic carriers
Chronic carriers are far more important sources of infection than cases The longer the carrier state the greater the risk of community The duration of the carrier state varies with the disease In typhoid fever and hepatitis B the chronic carrier state may last for several yearsIn chronic dysentery it may last for year or longer In diphtheria the carrier state is associated with infected tonsils in typhoid fever with gall bladder disease
D-r Mitova 33
BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 28
incubatory carriers are those who shed the infectious agent during the incubation period This usually occurs during the the last few days of incubation period As for example
Measles-the period of communicability is 4 days before the rush
Mumps-usually 4-6 days before onset of symptoms
Polio-7-10 days before onset of symptomsHepatitis B-for a month before
jaundiceabout 6 weeks after exposure
Carrier may be classified BY TYPE
D-r Mitova 29
Carrier may be classified BY TYPE
convalescent carriers that is those who continue to shed the disease agent during the period of convalescence(typhoid fever cholera diphteria bacillary disentery) In the disease clinical recovery does not coincide with bacteriological recovery
D-r Mitova 30
Carrier may be classified BY TYPE
healthy carriers they are victims of subclinical infection who have developed carrier state without suffering from overt disease but are nevertheless shedding the disease agent eg poliomyelitis cholera meningococcal meningitis salmonellosis diphtheria
D-r Mitova 31
BY DURATION
Temporary carriers are those who shed the infectious agent for short period of timeChronic carriers are those who excretes the infectious agent for indefinite periods eg typhoid fever hepatitis B dysentery etcThey are known to reintroduce disease into areas which are otherwise free of infection
D-r Mitova 32
Chronic carriers
Chronic carriers are far more important sources of infection than cases The longer the carrier state the greater the risk of community The duration of the carrier state varies with the disease In typhoid fever and hepatitis B the chronic carrier state may last for several yearsIn chronic dysentery it may last for year or longer In diphtheria the carrier state is associated with infected tonsils in typhoid fever with gall bladder disease
D-r Mitova 33
BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 29
Carrier may be classified BY TYPE
convalescent carriers that is those who continue to shed the disease agent during the period of convalescence(typhoid fever cholera diphteria bacillary disentery) In the disease clinical recovery does not coincide with bacteriological recovery
D-r Mitova 30
Carrier may be classified BY TYPE
healthy carriers they are victims of subclinical infection who have developed carrier state without suffering from overt disease but are nevertheless shedding the disease agent eg poliomyelitis cholera meningococcal meningitis salmonellosis diphtheria
D-r Mitova 31
BY DURATION
Temporary carriers are those who shed the infectious agent for short period of timeChronic carriers are those who excretes the infectious agent for indefinite periods eg typhoid fever hepatitis B dysentery etcThey are known to reintroduce disease into areas which are otherwise free of infection
D-r Mitova 32
Chronic carriers
Chronic carriers are far more important sources of infection than cases The longer the carrier state the greater the risk of community The duration of the carrier state varies with the disease In typhoid fever and hepatitis B the chronic carrier state may last for several yearsIn chronic dysentery it may last for year or longer In diphtheria the carrier state is associated with infected tonsils in typhoid fever with gall bladder disease
D-r Mitova 33
BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 30
Carrier may be classified BY TYPE
healthy carriers they are victims of subclinical infection who have developed carrier state without suffering from overt disease but are nevertheless shedding the disease agent eg poliomyelitis cholera meningococcal meningitis salmonellosis diphtheria
D-r Mitova 31
BY DURATION
Temporary carriers are those who shed the infectious agent for short period of timeChronic carriers are those who excretes the infectious agent for indefinite periods eg typhoid fever hepatitis B dysentery etcThey are known to reintroduce disease into areas which are otherwise free of infection
D-r Mitova 32
Chronic carriers
Chronic carriers are far more important sources of infection than cases The longer the carrier state the greater the risk of community The duration of the carrier state varies with the disease In typhoid fever and hepatitis B the chronic carrier state may last for several yearsIn chronic dysentery it may last for year or longer In diphtheria the carrier state is associated with infected tonsils in typhoid fever with gall bladder disease
D-r Mitova 33
BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 31
BY DURATION
Temporary carriers are those who shed the infectious agent for short period of timeChronic carriers are those who excretes the infectious agent for indefinite periods eg typhoid fever hepatitis B dysentery etcThey are known to reintroduce disease into areas which are otherwise free of infection
D-r Mitova 32
Chronic carriers
Chronic carriers are far more important sources of infection than cases The longer the carrier state the greater the risk of community The duration of the carrier state varies with the disease In typhoid fever and hepatitis B the chronic carrier state may last for several yearsIn chronic dysentery it may last for year or longer In diphtheria the carrier state is associated with infected tonsils in typhoid fever with gall bladder disease
D-r Mitova 33
BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 32
Chronic carriers
Chronic carriers are far more important sources of infection than cases The longer the carrier state the greater the risk of community The duration of the carrier state varies with the disease In typhoid fever and hepatitis B the chronic carrier state may last for several yearsIn chronic dysentery it may last for year or longer In diphtheria the carrier state is associated with infected tonsils in typhoid fever with gall bladder disease
D-r Mitova 33
BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 33
BY PORTAL OF EXIT OF THE IFECTIOUS AGENT
UrinaryIntestinalRespiratoryOthers
In typhoid fever the urinary carrier is more dangerous than an intestinal carrier
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 34
EARLY DIAGNOSIS IS NEEDED FOR
The treatment of patientFor epidemiological investigationTo study the time place and person distributionFor the institution of prevention and control measures
The first step in the control of a communicable disease is its rapid identification
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 35
Gastro Intestinal Fecal Oral Route
Transmission by the fecal-oral route is the second most important mode of transmission after the respiratory tract
1048698 excreted by the feces1048698 transmitted to the oralportal of entry through1048698 contaminated food1048698 contaminatedwater milk drinks1048698 hands1048698 flies
Viruses with envelopes do not survive exposure to hydrochloric acid in the stomach bile acids in the duodenum salts and enzymes of the gut Smallenterovirus without envelope(Norwalk agent rotavirus polio and coxsackie are able to resist HepatitisA and E are also transmitted by thefecal oral route
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 36
Transmission by gastrointestinal routeFecal oral route
1048698 Typhoid fever1048698 Shigella1048698 Cholera1048698 Polio1048698 Coxsackie Echo Reo1048698 Norwalk agent1048698 Rotavirus1048698 Hepatitis A Hepatitis E
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 37
Intestinal diseasesHuman Pathogens Enterobacteriaceae as a group were originally
divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans The pathogenic genera were Salmonella and Shigella However it is now known that E coli causes at least five types of gastrointestinal disease in humans Pathogenicity in E coli strains is due to the presence of one or more virulence factors including invasiveness factors (invasins) heat-labile and heat-stable enterotoxins verotoxins and colonization factors or adhesins
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 38
Intestinal diseases
Yersinia enterocolitica causes diarrhea probably by a combination of invasiveness and the presence of a heat-stable enterotoxin Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 39
Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae Transmission to humans is by water or food The natural reservoir of the organism is not known It was long assumed to be humans but some evidence suggests that it is the aquatic environment
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 40
S aureus causes food poisoning by releasing enterotoxins into food and toxic shock syndrome by release of superantigens into the blood stream
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 41
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 42
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 43
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 44
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 45
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 46
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 47
Staphylococcal food poisoning (staphyloenterotoxicosis staphyloenterotoxemia) is the name of the condition caused by the enterotoxins which some strains of S aureus produceThe onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute depending on individual susceptibility to the toxin the amount of contaminated food eaten the amount of toxin in the food ingested and the general health of the victim The most common symptoms are nausea vomiting retching abdominal cramping and prostration Some individuals may not always demonstrate all the symptoms associated with the illness Recovery generally takes two days However it us not unusual for complete recovery to take three days and sometimes longer in severe cases
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 48
Normal flora FECES ANALYSIS
Staphylococcus aureus only the predominance of S aureus and CandidaCandida albicansEcoliProteusKlebsiellaEnterobacterSerratiaCitrobacterPseudomonas aeruginosa exceptionallyenteropathogenic and in this case in a pure culture
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 49
Intestinal diseases FECES ANALYSIS
Collection of stools-A fresh specimen of stool should be collected for laboratory examination Sample should be collected before the person is treated withantibiotics After the stools been collected there occurs within a few hours an acidification which is noxious for some bacteria speciesThe feces analysis should begin within 2 to 3 hours after the samples have been taken
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 50
MICROSCOPIC EXAMINATION Before diluting a fragment of the
stools in distilled water observeThere consistency liquid soft moldenThe possible presence of
blood mucus or glairs
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 51
DIRECT EXAMINATION
Observe the possible presence of leucocytes suspicion of invading bacteriaInvestigate the presence of bacilli with monotrichous motility suspicion of Campylobacter or Vibrio
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 52
After Gram strainNote the percentage of Gram+ and Gram- bacteria the flora is normally polymorphous and composed ofabout 20 Gram+ and 80 Gram-Investigate the possible presence of curved Gram- rods
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 53
The following flora are abnormal
Predominance of Gram + cocci suspicion of staphylococcal enterocolitisAbundant yeastMonomorphous aspect presence of Gram- bacilli only
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 54
Laboratory diagnosis
of shigellosis
and salmonellas
salmonella
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 55
Diagnosis of staphylococcal foodborne illness
Incriminated foods should be collected and examined for staphylococci The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains toxin
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 56
LABORATORY DIAGNOSIS OF CHOLERA If a microscope with dark field illumination is available it may be possible to diagnose about 80 of the cases within a few minutes and more cases after 5-6 hours of incubation in alkaline peptone water In the dark field the vibrios evoke the image of many shooting stars in a dark sky In motility cases on mixing with polyvalent anti-cholera serum the organism are presumed to be cholera vibriosCulture method Bile Salt Agar mediumIn liquid choleric stools V cholerae is a pure culture In stools from healthy carriers V cholerae is found small quantities mixed with the commensal flora
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 57
LABORATORY DIAGNOSIS OF CHOLERA
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 58
LABORATORY DIAGNOSIS
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 59
Diagnostic studies
The diagnosis of pertussis is based on a characteristic history and physical examination If a child presents with a history of paroxysmal cough for greater than 1 week associated with vomiting whoops or cyanosis the child is suspected of having pertussisThe best method for making the diagnosis of pertussis is culture the organism from the nasopharynxFor culture andor PCR tests it is still recommended to take nasopharyngeal secretion with a bent charcoal swab
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 60
Isolation requires special media (chocolate agar or Bordet-Gengou medium) The organism requires both the X and V factors for growth especially for initial isolation
Bpertussis Bordet-Gengou medium
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 61
MENINGOCOCCAL DISEASE
Bacterial meningitis most commonly result from the dissemination of microorganisms from a distant site of infection the most common pathogenic agents in approximately 95 of bacterial meningitis cases are H influenzae Streptococcus pneumoniae and N meningitidis
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 62
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 63
At least 16 serogroups characterized by differences in the polyside capsule have been identified Groups A B and C account about 90 of meningococcal disease
Nasopharyngeal carriage of meningococci is relatively common among healthy young children 5~15 whereas the correspond ding figure in adult population is about 1
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 64
N meningitidis
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 65
Diphtheria (Greek for ldquoleatherrdquo (διφθερα dipthera)
Clinical Features Respiratory diphtheria presents as a sore throat with low-grade fever and an adherent membrane of the tonsils pharynx or nose Neck swelling is usually present in severe disease Cutaneous diphtheria presents as infected skin lesions which lack a characteristic appearance
Etiologic Agent Toxin-producing strains of Corynebacterium diphtheriae
Transmission Direct person- to-person transmission by intimate respiratory and physical contact Cutaneous lesions are important in transmission
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 66
Diphtheria
Pharyngeal diphtheria with membranes covering the tonsils and uvula
C diphtheriae
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 67
A throat swab should be cultured on Lofflerrsquos medium a tellurite plate and a blood agar plate Specimens should be transported to the laboratory immediately The laboratory should be notified ahead of time of a suspected diagnosis of diphtheria If the specimen cannot be transported immediately to the laboratory a transport medium such as Amies may be used If more than 24 hours will elapse before receipt in the laboratory specimens should be shipped at 4o C by courier to the receiving laboratory The typical gray-color of tellurium in the colony is a telltale diagnostic criterion
Diphtheria-laboratory diagnosis
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 68
Viral hepatitisThe term hepatitis refer to any condition in which the liverbecomes inflamed and its cells degenerate and die1 Viral causes of hepatitisbull Hepatitis A virusbull Hepatitis B virusbull Hepatitis C virusbull Hepatitis D virusbull Hepatitis E virusbull Others Ebstein-barrbull virus rubella cytomegalovirus herpes virus varicella zoster
and coxackie B2 Bacterial causes of hepatitis include many organisms that
cause septicaemia among them E coli3 Chemical cause of hepatitis include
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 69
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 70
Source ofvirus
feces bloodblood-derived
body fluids
bloodblood-derived
body fluids
bloodblood-derived
body fluids
feces
Route oftransmission
fecal-oral percutaneouspermucosal
percutaneouspermucosal
percutaneouspermucosal
fecal-oral
Chronicinfection
no yes yes yes no
Prevention prepost-exposure
immunization
prepost-exposure
immunization
blood donorscreening
risk behaviormodification
prepost-exposure
immunizationrisk behaviormodification
ensure safedrinking
water
Type of HepatitisA B C D E
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 71
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 72
Laboratory Diagnosis
Acute infection is diagnosed by the detection of HAV-IgM in serum by EIA
Past Infection ie immunity is determined by the detection of HAV-IgG by EIA
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 73
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 74
High ModerateLowNot
Detectable
blood semen urineserum vaginal fluid feces
wound exudates saliva sweat
tearsbreastmilk
Concentration of Hepatitis B Virus in Various Body Fluids
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 75
Diagnosis A battery of serological tests are used for the diagnosis of acute
and chronic hepatitis B infection
HBsAg - used as a general marker of infection
HBsAb - used to document recovery andor immunity to HBV infection
anti-HBc IgM - marker of acute infection
anti-HBcIgG - past or chronic infection
HBeAg - indicates active replication of virus and therefore infectiveness
Anti-Hbe - virus no longer replicating However the patient can still be positive for HBsAg which is made by integrated HBV
HBV-DNA - indicates active replication of virus more accurate than HBeAg especially in cases of escape mutants Used mainly for monitoring response to therapy
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 76
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 77
Transfusion or transplant from infected donor
Injecting drug use
Hemodialysis (yrs on treatment)
Accidental injuries with needlessharps
Sexualhousehold exposure to anti-HCV-positive contact
Multiple sex partners
Birth to HCV-infected mother
Risk Factors Associated with
Transmission of HCV
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 78
Laboratory Diagnosis
HCV antibody - generally used to diagnose hepatitis C infection Not useful in the acute phase as it takes at least 4 weeks after infection before antibody appears
HCV-RNA - various techniques are available eg PCR and branched DNA May be used to diagnose HCV infection in the acute phase However its main use is in monitoring the response to antiviral therapy
HCV-antigen - an EIA for HCV antigen is available It is used in the same capacity as HCV-RNA tests but is much easier to carry out
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 79
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 80
Coinfectionndash severe acute diseasendash low risk of chronic infection
Superinfectionndash usually develop chronic HDV infectionndash high risk of severe chronic liver diseasendash may present as an acute hepatitis
Hepatitis D - Clinical Features
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 81
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 82
Incubation period Average 40 days
Range 15-60 days
Case-fatality rate Overall 1-3Pregnant
women 15-25
Illness severity Increased with age Chronic sequelae None identified
Hepatitis E - Clinical Features
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 83
Diagnostic Methods in Virology
1 Direct Examination
2 Indirect Examination (Virus
Isolation)
3 Serology
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 84
Direct Examination
1 Antigen Detection immunofluorescence ELISA etc
2 Electron Microscopy morphology of virus particles
immune electron microscopy
3 Light Microscopy histological appearance
inclusion bodies
4 Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR)
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 85
Indirect Examination
1 Cell Culture cytopathic effect (CPE) haemabsorption
immunofluorescence
2 Eggs pocks on CAM
haemagglutination
inclusion bodies
3 Animals disease or death
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 86
SerologyDetection of rising titres of antibody between acute and convalescent stages of infection or the detection of IgM in primary infection
Classical Techniques Newer Techniques
1 Complement fixation tests (CFT) 1 Radioimmunoassay (RIA)2 Haemagglutination inhibition tests 2 Enzyme linked immunosorbent assay (EIA)3 Immunofluorescence techniques (IF) 3 Particle agglutination4 Neutralization tests 4 Western Blot (WB)5 Counter-immunoelectrophoresis 5 RIBA Line immunoassay
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 87
ELISA for HIV antibody
Microplate ELISA for HIV antibody coloured wells indicate reactivity
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 88
Western Blot
HIV-1 Western BlotLane1 Positive Control
Lane 2 Negative Control
Sample A Negative
Sample B Indeterminate
Sample C Positive
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 89
Polymerase Chain Reaction (1)
PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique
It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest
These oligonucleotides act as primers for the thermostable Taq polymerase Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC) annealing of primers to their complementary sequences (50oC) and primer extension (72oC) result in the exponential production of the specific target fragment
Further sensitivity and specificity may be obtained by the nested PCR
Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis hybridization with a specific oligonucleotide probe restriction enzyme analysis or DNA sequencing
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 90
Polymerase Chain Reaction (2)
Advantages of PCR Extremely high sensitivity may detect down to one viral genome per sample volume Easy to set up Fast turnaround time
Disadvantages of PCR Extremely liable to contamination High degree of operator skill required Not easy to set up a quantitative assay A positive result may be difficult to interpret especially with latent viruses such as
CMV where any seropositive person will have virus present in their blood irrespective whether they have disease or not
These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate
quantification of results However these assays are very expensive
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 91
Schematic of PCR
Each cycle doubles the copy number of the target
D-r Mitova 92
Some Pathogens that Cross the Placenta
D-r Mitova 92
Some Pathogens that Cross the Placenta
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