DIAGNOSTIC METHODS IN VIROLOGY. Definition Viruses = non cellular organisms – – genomes - nucleic acid, - protected by a protein shell - must replicate.

DIAGNOSTIC METHODS IN DIAGNOSTIC METHODS IN VIROLOGYVIROLOGY

DefinitionDefinition

Viruses = non cellular organisms

– genomes - nucleic acid, - protected by a protein shell - must replicate inside host cells

cannot be grown on sterile media

require the presence of specific host cells

Embrionated egg Cell culture Laboratory animals

DIAGNOSTIC METHODS IN VIROLOGY

3 categories: (1) direct detection, (2) virus isolation, (3) serology.

1. Direct Examination of Specimen

– clinical specimen - examined directly for the presence of virus particles virus antigen viral nucleic acids.

– Electron Microscopy morphology / immune electron microscopy

– Light microscopy histological appearance - e.g. inclusion bodies

– Antigen detection immunofluorescence, ELISA etc.

– Molecular techniques for the direct detection of viral genomes

2. Indirect Examination = Virus isolation

– Cell Culture cytopathic effect, haemadsorption, confirmation by neutralization, interference, immunofluorescence etc.

– Eggs pocks - haemagglutination, inclusion bodies

– Animals disease or death confirmation by neutralization

3. Serology

rising titres of antibody between acute and convalescent stages of infection,

detection of IgM in primary infection.

Classical Techniques1. COMPLEMENT FIXATION TESTS (CFT) 2. HAEMAGGLUTINATION INHIBITION TESTS 2. HAEMAGGLUTINATION INHIBITION TESTS 3. IMMUNOFLUORESCENCE TECHNIQUES (IF) 3. IMMUNOFLUORESCENCE TECHNIQUES (IF) 4. NEUTRALIZATION TESTS 4. NEUTRALIZATION TESTS 5. SINGLE RADIAL HAEMOLYSIS 5. SINGLE RADIAL HAEMOLYSIS

Newer Techniques 1. RADIOIMMUNOASSAY (RIA) 1. RADIOIMMUNOASSAY (RIA) 2. ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) 2. ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) 3. PARTICLE AGGLUTINATION 3. PARTICLE AGGLUTINATION 4. WESTERN BLOT (WB) 4. WESTERN BLOT (WB)

1. Direct Examination1. Direct Examination

rapid diagnosticrapid diagnostic Result => the same/next day.Result => the same/next day.

Obs:Obs: virus isolation virus isolation serological methods serological methods

May sometimes give a rapid result. !May sometimes give a rapid result. !

1.1. Antigen Detection

Examples – IF testing of : :

nasopharyngeal aspirates for respiratory viruses nasopharyngeal aspirates for respiratory viruses e.g.. RSV, flu A, flu B, and adenoviruses, e.g.. RSV, flu A, flu B, and adenoviruses,

detection of rotavirus antigen in faeces,

detection of HSV and VZV in skin scrappings,

detection of HBsAg in serum. Usually considered as a serological test!

1.1. Antigen Detection1.1. Antigen Detection

main advantage: rapid (hours)

BUT technique - often tedious - result difficult to read and interpret, - sensitivity and specificity poor.

quality of the specimen - most important in order quality of the specimen - most important in order for the test to work properly. for the test to work properly.

1.2. Electron Microscopy (EM) basis = morphology. basis = morphology. magnification - 50,000 X magnification - 50,000 X mainly used for viral gastroenteritis mainly used for viral gastroenteritis

detecting viruses in feces: detecting viruses in feces:

– rotavirus, rotavirus, - calicivirus - calicivirus – adenovirus, adenovirus, - Norwalk-like viruses- Norwalk-like viruses– astrovirus, astrovirus,

Occasionally used for detection of viruses in skin Occasionally used for detection of viruses in skin lesions lesions (e.g. vesicles)(e.g. vesicles)– herpesviruses herpesviruses – papillomaviruses. papillomaviruses.

1.2. Electron Microscopy (EM)1.2. Electron Microscopy (EM) sensitivity and specificity enhanced by immune sensitivity and specificity enhanced by immune

electron microscopy: electron microscopy: – virus specific antibody used to agglutinate virus specific antibody used to agglutinate

virus particles =>easier to recognize. virus particles =>easier to recognize.

Problems with EM: Problems with EM: – expense - purchasing and maintaining the expense - purchasing and maintaining the

facility. facility. – sensitivity - often poor (106 v.particles/ml in sensitivity - often poor (106 v.particles/ml in

sample required for visualization) sample required for visualization) – observer - highly skilled. observer - highly skilled.

Electronmicrographs from patients suffering from Electronmicrographs from patients suffering from gastroenteritis. gastroenteritis.

From left to right: rotavirus, adenovirus, From left to right: rotavirus, adenovirus, (Courtesy of Linda M. Stannard, University of Cape Town, (Courtesy of Linda M. Stannard, University of Cape Town,

http://www.uct.ac.za/depts/mmi/stannard/emimages.html)http://www.uct.ac.za/depts/mmi/stannard/emimages.html)

Family Pox Herpes Adeno Papova Parvo Hepadna

Genome <---------------------------------------dsDNA--------------------------------------> ssDNA Partial dsDNA

Capsidsymmetry

Complex <---------------------------------------------Icosahedral-------------------------------------------------->

Envelope <-----------------Yes------------------> <-----------------------------No------------------------------> Yes

e.g. Vaccinia virus Herpes simplexvirus 2

Humanadenovirus

Papilloma Hepatitis BAdeno-Associated

MolluscumContagiosum

Animal virus classification: DNA Viruses

Plus Sense RNA Viruses

Minus Sense RNA Viruses

1.3. Light Microscopy1.3. Light Microscopy

Replicating virus => histological changes Replicating virus => histological changes (characteristic or non-specific) in infected (characteristic or non-specific) in infected cells. cells.

Viral inclusion bodies = collections of Viral inclusion bodies = collections of replicating virus particles (in nucleus or replicating virus particles (in nucleus or cytoplasm). cytoplasm). – Examples Examples

Babeş-Negri bodies in rabiesBabeş-Negri bodies in rabies cytomegalic inclusion bodies in CMV infections cytomegalic inclusion bodies in CMV infections

histology serves as an adjunct test.histology serves as an adjunct test.

RABIES VIRUSRABIES VIRUS

Babeş-Negri bodies in rabiesBabeş-Negri bodies in rabies

RabiesRabies

Rabies virus Babes –Negri Rabies virus Babes –Negri bodiesbodies

INTRANUCLEAR INCLUSIONS IN INTRANUCLEAR INCLUSIONS IN HERPESVIRUS INFECTIONSHERPESVIRUS INFECTIONS

REOVIRIDAEREOVIRIDAE

INTRACITOPLASMATIC INTRACITOPLASMATIC INCLUSIONSINCLUSIONS

INTRACITOPLASMATIC INTRACITOPLASMATIC INCLUSIONS INCLUSIONS

V.EBOLA HEPATOCITESV.EBOLA HEPATOCITES

1.4. Viral Genome Detection1.4. Viral Genome Detection molecular methods molecular methods

future direction of viral diagnosisfuture direction of viral diagnosis

a)a) Classical molecular techniques Classical molecular techniques

a)a) (dot-blot and Southern-blot) (dot-blot and Southern-blot)

– use of specific DNA/RNA probes for hybridizationuse of specific DNA/RNA probes for hybridization

– specificity depends on the conditions used for hybridization. specificity depends on the conditions used for hybridization.

– quantification of DNA/RNA present in the specimen quantification of DNA/RNA present in the specimen

– sensitivity of these techniques ~ conventional viral diagnostic sensitivity of these techniques ~ conventional viral diagnostic methods. methods.

1.4. Viral Genome Detection1.4. Viral Genome Detection b) Newer molecular techniques:b) Newer molecular techniques:- POLYMERASE CHAIN REACTION (PCR)POLYMERASE CHAIN REACTION (PCR)- LIGASE CHAIN REACTION (LCR)LIGASE CHAIN REACTION (LCR)- NUCLEIC ACID BASED AMPLIFICATION (NASBA)NUCLEIC ACID BASED AMPLIFICATION (NASBA)- BRANCHED DNA (BDNA)BRANCHED DNA (BDNA) PCR PCR extremely sensitive : extremely sensitive : 1 DNA1 DNA molecule in a molecule in a

clinical specimen. clinical specimen.

PCR – problems: PCR – problems: contamination, contamination, positive PCR result - difficult to interpret positive PCR result - difficult to interpret

latent viruses such as CMV !latent viruses such as CMV !

2. Virus Isolation2. Virus Isolation Specimen - inoculated into cell culture, Specimen - inoculated into cell culture,

eggs or animals eggs or animals Eggs and animals - difficult to handleEggs and animals - difficult to handle most viral diagnostic laboratories - cell culture only. most viral diagnostic laboratories - cell culture only.

3 types of cell cultures: 3 types of cell cultures: a.a. Primary cellsPrimary cellsb.b. Semi-continuous cellsSemi-continuous cellsc.c. Continuous cellsContinuous cells

2.1. Types of cell cultures a.a. Primary cells Primary cells cultureculture - - e.g. Monkey Kidney. e.g. Monkey Kidney.

= normal cells obtained from freshly killed adult = normal cells obtained from freshly killed adult animals. animals.

(+)(+)- best cell culture systems available best cell culture systems available - support the widest range of virusessupport the widest range of viruses

(-)(-)- can only be passaged once or twice. can only be passaged once or twice. - very expensive very expensive - difficult to obtain a reliable supply.difficult to obtain a reliable supply.

2.1. Types of cell cultures

b. Semi-continuous cells b. Semi-continuous cells – Human embryonic kidneyHuman embryonic kidney– skin fibroblasts. skin fibroblasts. – cells taken from embryonic tissuecells taken from embryonic tissue– passaged up to 50 times.passaged up to 50 times.

c. Continuous cells c. Continuous cells – HeLa, Vero, Hep2, LLC-MK2, BGM. HeLa, Vero, Hep2, LLC-MK2, BGM. – immortalized cells i.e. tumour cell lines immortalized cells i.e. tumour cell lines – passaged indefinitely. passaged indefinitely. – most easy to handle most easy to handle – range of viruses supported is often limited. range of viruses supported is often limited.

2.2. Identification of growing virus2.2. Identification of growing virus a. Cytopathic Effect (CPE) - specific or non-a. Cytopathic Effect (CPE) - specific or non-

specific specific – HSV and CMV produces a specific CPE,HSV and CMV produces a specific CPE,– enteroviruses do not produces a specific CPE.enteroviruses do not produces a specific CPE.

b. Haemadsorption b. Haemadsorption – cells acquire the ability to stick to mammalian cells acquire the ability to stick to mammalian

red blood cells. red blood cells. – mainly used for the detection of mainly used for the detection of

influenza influenza parainfluenzaviruses.parainfluenzaviruses.

Left to Right: Cytopathic effect of HSV, enterovirus 71 (ballooning), Left to Right: Cytopathic effect of HSV, enterovirus 71 (ballooning), and RSV in cell culture (syncytia formation) and RSV in cell culture (syncytia formation)

((Linda Stannard. University of Cape Town, Virology Laboratory, Yale-New Haven Hospital)Linda Stannard. University of Cape Town, Virology Laboratory, Yale-New Haven Hospital)

2.3 Problems with cell culture2.3 Problems with cell culture long period for a result (up to 4 weeks)long period for a result (up to 4 weeks)

sensitivity - often poor and depends on many sensitivity - often poor and depends on many factors, factors, – condition of the specimen, condition of the specimen, – condition of the cell sheet. condition of the cell sheet.

very susceptible to very susceptible to – bacterial contamination bacterial contamination – toxic substances in the specimen. toxic substances in the specimen.

many viruses - not grow in cell culture at all :many viruses - not grow in cell culture at all :– Hepatitis B and C, Hepatitis B and C, – Diarrhoeal viruses, Diarrhoeal viruses, – parvovirus etc. parvovirus etc.

CELL CULTURE CELL CULTURE CONTAMINATIONCONTAMINATION

CELL CULTURE INCUBATORCELL CULTURE INCUBATOR

2.4 Rapid Culture Techniques2.4 Rapid Culture Techniques

viral antigens - detected 2 to 4 days after inoculation. viral antigens - detected 2 to 4 days after inoculation.

Examples: shell vial cultures and the CMV DEAFF test. Examples: shell vial cultures and the CMV DEAFF test.

CMV DEAFF testCMV DEAFF test cell sheet grown on individual cover slips in a plastic cell sheet grown on individual cover slips in a plastic

bottle. bottle. then bottle is spun at a low speed for one hour (to then bottle is spun at a low speed for one hour (to

speed up the adsorption of the virus) speed up the adsorption of the virus) incubated for 2 to 4 days. incubated for 2 to 4 days. cover slip cover slip taken out taken out examined for the presence of CMV early antigens by examined for the presence of CMV early antigens by

IF (immunofluorescence) IF (immunofluorescence)

LeftLeft: Haemadsorption of red blood cells onto the surface of a cell : Haemadsorption of red blood cells onto the surface of a cell sheet infected by mumps virus. Also note the presence of syncytia sheet infected by mumps virus. Also note the presence of syncytia (indistinguishable from that of RSV) (Courtesy of Linda Stannard, (indistinguishable from that of RSV) (Courtesy of Linda Stannard,

University of Cape Town). University of Cape Town).

RightRight: Positive CMV DEAFF test. (Virology Laboratory, Yale-New Haven : Positive CMV DEAFF test. (Virology Laboratory, Yale-New Haven Hospital)Hospital)

CELL CULTURE PH CHECKINGCELL CULTURE PH CHECKING

CELL CULTURE RECIPIENTSCELL CULTURE RECIPIENTS

3. Serology = the mainstay of viral diagnosis3. Serology = the mainstay of viral diagnosis IgM - first antibody to appear,IgM - first antibody to appear, IgG – follow, much higher titer IgG – follow, much higher titer

Techniques:Techniques:– EIA and RIA EIA and RIA

specifically for IgM or IgG, specifically for IgM or IgG, most sensitive tests availablemost sensitive tests available

– CFT, HAI => detect total antibody (comprises CFT, HAI => detect total antibody (comprises mainly IgG) mainly IgG)

EIAs EIAs – better sensitivity, specificity and reproducibility than better sensitivity, specificity and reproducibility than

classical techniques ( CFT and HAI. )classical techniques ( CFT and HAI. )

3. Serology 3. Serology

3.1. Criteria for Primary Infection3.1. Criteria for Primary Infection

a. Significant rise in titre of IgG/total antibody a. Significant rise in titre of IgG/total antibody acute - convalescent sera acute - convalescent sera

– CFT and HAI: 4 X or greater CFT and HAI: 4 X or greater – Problem: dg. = retrospectiveProblem: dg. = retrospective

b. Presence of IgM b. Presence of IgM - EIA, RIA, and IF - EIA, RIA, and IF – rapid rapid

Problems: Problems: – interference by rheumatoid factor,interference by rheumatoid factor,– re-infection by the virus, re-infection by the virus, – unexplained persistenceunexplained persistence

Seroconversion = changing from a previously antibody Seroconversion = changing from a previously antibody negative state to a positive state.negative state to a positive state.

HIV following a needle-stick injury,HIV following a needle-stick injury, antirubella following contact with a known case.antirubella following contact with a known case.

3.2. Criteria for diagnosing re-infection/re-activation3.2. Criteria for diagnosing re-infection/re-activation

difficult to differentiate difficult to differentiate re-infection/re-activationre-infection/re-activation

important under certain situationsimportant under certain situations– rubella or toxoplasma infection rubella or toxoplasma infection

first trimester of pregnancy: first trimester of pregnancy: primary infection - high risk of fetal damageprimary infection - high risk of fetal damage re-infection not re-infection not associated with a high associated with a high

risk of fetal damagerisk of fetal damage sharp large rise in antibody titres found in sharp large rise in antibody titres found in

re-infection re-infection IgM usually low or absent in cases of re-IgM usually low or absent in cases of re-

infection/re-activation. infection/re-activation.

Serological events in primary infection and reinfection. Serological events in primary infection and reinfection. Reinfection: IgM absent or only present transiently at a low Reinfection: IgM absent or only present transiently at a low

level. level.

3.3. Limitations3.3. Limitations

Rubella and hepatitis A: Rubella and hepatitis A: – onset of clinical symptoms - development of antibodies onset of clinical symptoms - development of antibodies

=> detection of IgM or rising titres of IgG = active disease. => detection of IgM or rising titres of IgG = active disease.

Many viruses (respiratory and diarrhoeal):Many viruses (respiratory and diarrhoeal):– clinical disease clinical disease beforebefore the appearance of the appearance of

antibodies => serological diagnosis = antibodies => serological diagnosis = retrospectiveretrospective

Some viruses (HIV and rabies):Some viruses (HIV and rabies):

– clinical disease months or years clinical disease months or years afterafter seroconversion => the mere presence of seroconversion => the mere presence of antibody is sufficient to make a definitive antibody is sufficient to make a definitive diagnosis.diagnosis.

CFT in Microtiter Plate. CFT in Microtiter Plate. – Rows 1 and 2: acute and convalescent phase Rows 1 and 2: acute and convalescent phase

serum specimens, respectively. serum specimens, respectively. – The observed 4-fold increase is significant and The observed 4-fold increase is significant and

indicates infection. indicates infection.

Microplate ELISA: Microplate ELISA: – coloured wells indicate reactivitycoloured wells indicate reactivity– darker the colour, higher the reactivity darker the colour, higher the reactivity

3.4. Antibody in the CSF3.4. Antibody in the CSF

healthy person - little or no antibodies in healthy person - little or no antibodies in CSF. CSF.

viral meningitis or encephalitis,viral meningitis or encephalitis, – antibodies - produced by lymphocytesantibodies - produced by lymphocytes

finding of antibodies in the CSFfinding of antibodies in the CSF – significant when ratio between the titre of significant when ratio between the titre of

antibody in the serum and that in the CSF is antibody in the serum and that in the CSF is less than 100 (depend on an intact blood-brain less than 100 (depend on an intact blood-brain barrier !) barrier !)