Infectious Disease

Infectious diseaseAn infectious disease is a clinically evident illness resulting from the presence of pathogenic

microbial agents, including pathogenic viruses, pathogenic bacteria, fungi, protozoa, multicellular parasites,

and aberrant proteins known as prions. These pathogens are able to cause disease in animals and/or

plants. Infectious pathologies are also called communicable diseases or transmissible diseases due to

their potential of transmission from one person or species to another by a replicating agent (as opposed to

a toxin]

Transmission of an infectious disease may occur through one or more of diverse pathways including

physical contact with infected individuals. These infecting agents may also be transmitted through liquids,

food, body fluids, contaminated objects, airborne inhalation, or through vector-borne spread.

[2]Transmissible diseases which occur through contact with an ill person or their secretions, or objects

touched by them, are especially infective, and are sometimes referred to as contagious diseases. Infectious

(communicable) diseases which usually require a more specialized route of infection, such as vector

transmission, blood or needle transmission, or sexual transmission, are usually not regarded as

contagious, and thus not are not as amenable to medical quarantine of victims.

The term infectivity describes the ability of an organism to enter, survive and multiply in the host, while

the infectiousness of a disease indicates the comparative ease with which the disease is transmitted to

other hosts.[3] An infection however, is not synonymous with an infectious disease, as an infection may not

cause important clinical symptoms or impair host function.[2]

Classification

Among the almost infinite varieties of microorganisms, relatively few cause disease in otherwise healthy

individuals.[4] Infectious disease results from the interplay between those few pathogens and the defenses

of the hosts they infect. The appearance and severity of disease resulting from any pathogen depends

upon the ability of that pathogen to damage the host as well as the ability of the host to resist the pathogen.

Infectious microorganisms, or microbes, are therefore classified as either primary pathogens or

as opportunistic pathogens according to the status of host defenses.

Primary pathogens cause disease as a result of their presence or activity within the normal, healthy host,

and their intrinsic virulence (the severity of the disease they cause) is, in part, a necessary consequence of

their need to reproduce and spread. Many of the most common primary pathogens of humans only infect

humans, however many serious diseases are caused by organisms acquired from the environment or

which infect non-human hosts.

Organisms which cause an infectious disease in a host with depressed resistance are classified

as opportunistic pathogens. Opportunistic disease may be caused by microbes that are ordinarily in contact

with the host, such as pathogenic bacteria or fungi in the gastrointestinal or the upper respiratory tract, and

they may also result from (otherwise innocuous) microbes acquired from other hosts (as in Clostridium

difficile colitis) or from the environment as a result of traumatic introduction (as in surgical wound infections

or compound fractures). An opportunistic disease requires impairment of host defenses, which may occur

as a result of genetic defects (such as Chronic granulomatous disease), exposure to antimicrobial drugs

or immunosuppressive chemicals (as might occur followingpoisoning or cancer chemotherapy), exposure

to ionizing radiation, or as a result of an infectious disease with immunosuppressive activity (such as

with measles, malaria or HIV disease). Primary pathogens may also cause more severe disease in a host

with depressed resistance than would normally occur in an immunosufficient host.[2]

One way of proving that a given disease is "infectious", is to satisfy Koch's postulates (first proposed

by Robert Koch), which demands that the infectious agent be identified only in patients and not in healthy

controls, and that patients who contract the agent also develop the disease. These postulates were first

used in the discovery that Mycobacteria species cause tuberculosis. Koch's postulates cannot be met

ethically for many human diseases because they require experimental infection of a healthy individual with

a pathogen produced as a pure culture. Often, even diseases that are quite clearly infectious do not meet

the infectious criteria. For example, Treponema pallidum , the causative spirochete of syphilis, cannot

be cultured in vitro - however the organism can be cultured in rabbittestes. It is less clear that a pure culture

comes from an animal source serving as host than it is when derived from microbes derived from plate

culture. Epidemiology is another important tool used to study disease in a population. For infectious

diseases it helps to determine if a disease outbreak is sporadic (occasional occurrence), endemic (regular

cases often occurring in a region), epidemic (an unusually high number of cases in a region),

or pandemic (a global epidemic).

Transmission

Washing one's hands, a form of hygiene, is the number one way to prevent the spread of infectious disease.

An infectious disease is transmitted from some source. Defining the means of transmission plays an

important part in understanding the biology of an infectious agent, and in addressing the disease it causes.

Transmission may occur through several different mechanisms. Respiratory diseases andmeningitis are

commonly acquired by contact with aerosolized droplets, spread by sneezing, coughing, talking, kissing or

even singing. Gastrointestinaldiseases are often acquired by ingesting contaminated food and

water. Sexually transmitted diseases are acquired through contact with bodily fluids, generally as a result of

sexual activity. Some infectious agents may be spread as a result of contact with a contaminated,

inanimate object (known as afomite), such as a coin passed from one person to another, while other

diseases penetrate the skin directly.[2]

Transmission of infectious diseases may also involve a vector. Vectors may be mechanical or biological. A

mechanical vector picks up an infectious agent on the outside of its body and transmits it in a passive

manner. An example of a mechanical vector is a housefly, which lands on cow dung, contaminating its

appendages with bacteria from the feces, and then lands on food prior to consumption. The pathogen

never enters the body of the fly.

Culex mosquitos (Culex quinquefasciatus  shown) are biological vectors that transmit West Nile Virus.

In contrast, biological vectors harbor pathogens within their bodies and deliver pathogens to new hosts in

an active manner, usually a bite. Biological vectors are often responsible for serious blood-borne diseases,

such as malaria,viral encephalitis, Chagas disease , Lyme disease and African sleeping sickness. Biological

vectors are usually, though not exclusively, arthropods, such as mosquitoes, ticks, fleas and lice. Vectors

are often required in the life cycle of a pathogen. A common strategy used to control vector borne infectious

diseases is to interrupt the life cycle of a pathogen by killing the vector.

The relationship between virulence and transmission is complex, and has important consequences for the

long term evolution of a pathogen. Since it takes many generations for a microbe and a new host species

to co-evolve, an emerging pathogen may hit its earliest victims especially hard. It is usually in the first wave

of a new disease that death rates are highest. If a disease is rapidly fatal, the host may die before the

microbe can get passed along to another host. However, this cost may be overwhelmed by the short term

benefit of higher infectiousness if transmission is linked to virulence, as it is for instance in the case of

cholera (the explosive diarrhea aids the bacterium in finding new hosts) or many respiratory infections

(sneezing and coughing create infectious aerosols).

Prevention

One of the ways to prevent or slow down the transmission of infectious diseases is to recognize the

different characteristics of various diseases.[5] Some critical disease characteristics that should be

evaluated include virulence, distance traveled by victims, and level of contagiousness. The human strains

of Ebola virus, for example, incapacitate its victims extremely quickly and kills them soon after. As a result,

the victims of this disease do not have the opportunity to travel very far from the initial infection zone.

[6] Also, this virus must spread through skin lesions or permeable membranes such as the eye. Thus, the

initial stage of Ebola is not very contagious since its victims experience only internal hemorrhaging. As a

result of the above features, the spread of Ebola is very rapid and usually stays within a relatively confined

geographical area. In contrast, Human Immunodeficiency Virus (HIV) kills its victims very slowly by

attacking their immune system.[2] As a result, many of its victims transmit the virus to other individuals

before even realizing that they are carrying the disease. Also, the relatively low virulence allows its victims

to travel long distances, increasing the likelihood of anepidemic.

Another effective way to decrease the transmission rate of infectious diseases is to recognize the effects

of small-world networks.[5] In epidemics, there are often extensive interactions within hubs or groups of

infected individuals and other interactions within discrete hubs of susceptible individuals. Despite the low

interaction between discrete hubs, the disease can jump to and spread in a susceptible hub via a single or

few interactions with an infected hub. Thus, infection rates in small-world networks can be reduced

somewhat if interactions between individuals within infected hubs are eliminated (Figure 1). However,

infection rates can be drastically reduced if the main focus is on the prevention of transmission jumps

between hubs. The use of needle exchange programs in areas with a high density of drug users with HIV is

an example of the successful implementation of this treatment method. [6] Another example is the use of

ring culling or vaccination of potentially susceptible livestock in adjacent farms to prevent the spread of

the foot-and-mouth virus in 2001.[7]

General methods to prevent transmission of pathogens may include disinfection  pest control edit

Immunity

Mary Mallon (a.k.a Typhoid Mary) was an asymptomatic carrier of typhoid fever. Over the course of her career as a

cook, she infected 53 people, three of whom died.

Infection with most pathogens does not result in death of the host and the offending organism is ultimately

cleared after the symptoms of the disease have waned. [4] This process requires immune mechanisms to kill

or inactivate the inoculum of the pathogen. Specific acquired immunity against infectious diseases may be

mediated by antibodies and/or T lymphocytes. Immunity mediated by these two factors may be manifested

by:

a direct effect upon a pathogen, such as antibody-initiated complement-

dependent bacteriolysis, opsonoization, phagocytosis and killing, as

occurs for some bacteria,

neutralization of viruses so that these organisms cannot enter cells,

or by T lymphocytes which will kill a cell parasitized by a microorganism.

The immune system response to a microorganism often causes symptoms such as a

high fever and inflammation, and has the potential to be more devastating than direct damage caused by a

microbe.[2]

Resistance to infection (immunity) may be acquired following a disease, by asymptomatic carriage of the

pathogen, by harboring an organism with a similar structure (crossreacting), or by vaccination. Knowledge

of the protective antigens and specific acquired host immune factors is more complete for primary

pathogens than for opportunistic pathogens.

Immune resistance to an infectious disease requires a critical level of either antigen-specific antibodies

and/or T cells when the host encounters the pathogen. Some individuals develop natural serum antibodies

to the surface polysaccharides of some agents although they have had little or no contact with the agent,

these natural antibodies confer specific protection to adults and are passively transmitted to newborns.

Host genetic factors

The clearance of the pathogens, either treatment-induced or spontaneous, can be influenced by the

genetic variants carried by the individual patients. For instance, for genotype 1 hepatitis C treated

with Pegylated interferon-alpha-2a  or Pegylated interferon-alpha-2b  (brand names Pegasys or PEG-Intron)

combined with ribavirin, it has been shown that genetic polymorphisms near the human IL28B gene,

encoding interferon lambda 3, are associated with significant differences in the treatment-induced

clearance of the virus. This finding, originally reported in Nature,[8] showed that genotype 1 hepatitis C

patients carrying certain genetic variant alleles near the IL28B gene are more possibly to achieve sustained

virological response after the treatment than others. Later report from Nature[9] demonstrated that the same

genetic variants are also associated with the natural clearance of the genotype 1 hepatitis C virus.

Diagnosis

Diagnosis of infectious disease sometimes involves identifying an infectious agent either directly or

indirectly. In practice most minor infectious diseases such as warts, cutaneous abscesses,respiratory

system infections and diarrheal diseases are diagnosed by their clinical presentation. Conclusions about

the cause of the disease are based upon the likelihood that a patient came in contact with a particular

agent, the presence of a microbe in a community, and other epidemiological considerations. Given

sufficient effort, all known infectious agents can be specifically identified. The benefits of identification,

however, are often greatly outweighed by the cost, as often there is no specific treatment, the cause is

obvious, or the outcome of an infection is benign.

Specific identification of an infectious agent is usually only determined when such identification can aid in

the treatment or prevention of the disease, or to advance knowledge of the course of an illness prior to the

development of effective therapeutic or preventative measures. For example, in the early 1980s, prior to

the appearance of AZT for the treatment of AIDS, the course of the disease was closely followed by

monitoring the composition of patient blood samples, even though the outcome would not offer the patient

any further treatment options. In part, these studies on the appearance ofHIV in specific communities

permitted the advancement of hypotheses as to the route of transmission of the virus. By understanding

how the disease was transmitted, resources could be targeted to the communities at greatest risk in

campaigns aimed at reducing the number of new infections. The specific serological diagnostic

identification, and later genotypic or molecular identification, of HIV also enabled the development of

hypotheses as to the temporal and geographical origins of the virus, as well as a myriad of other

hypothesis.[2] The development of molecular diagnostic tools have enabled physicians and researchers to

monitor the efficacy of treatment with anti-retroviral drugs. Molecular diagnostics are now commonly used

to identify HIV in healthy people long before the onset of illness and have been used to demonstrate the

existence of people who are genetically resistant to HIV infection. Thus, while there still is no cure for AIDS,

there is great therapeutic and predictive benefit to identifying the virus and monitoring the virus levels within

the blood of infected individuals, both for the patient and for the community at large.

Diagnosis of infectious disease is nearly always initiated by medical history and physical examination. More

detailed identification techniques involve the culture of infectious agents isolated from a patient. Culture

allows identification of infectious organisms by examining their microscopic features, by detecting the

presence of substances produced by pathogens, and by directly identifying an organism by its genotype.

Other techniques (such as X-rays, CAT scans, PET scans or NMR) are used to produce images of internal

abnormalities resulting from the growth of an infectious agent. The images are useful in detection of, for

example, a bone abscess or a spongiform encephalopathy produced by a prion.

Microbial culture

Four nutrient agar plates growing colonies of common Gram negativebacteria.

Microbiological culture is a principal tool used to diagnose infectious disease. In a microbial culture,

a growth medium is provided for a specific agent. A sample taken from potentially diseased tissue or fluid is

then tested for the presence of an infectious agent able to grow within that medium. Most pathogenic

bacteria are easily grown on nutrient agar, a form of solid medium that supplies carbohydrates and proteins

necessary for growth of a bacterium, along with copious amounts of water. A single bacterium will grow into

a visible mound on the surface of the plate called a colony, which may be separated from other colonies or

melded together into a "lawn". The size, color, shape and form of a colony is characteristic of the bacterial

species, its specific genetic makeup (its strain), and the environment which supports its growth. Other

ingredients are often added to the plate to aid in identification. Plates may contain substances that permit

the growth of some bacteria and not others, or that change color in response to certain bacteria and not

others. Bacteriological plates such as these are commonly used in the clinical identification of infectious

bacterium. Microbial culture may also be used in the identification of viruses: the medium in this case being

cells grown in culture that the virus can infect, and then alter or kill. In the case of viral identification, a

region of dead cells results from viral growth, and is called a "plaque". Eukaryotic parasites may also be

grown in culture as a means of identifying a particular agent.

In the absence of suitable plate culture techniques, some microbes require culture within live animals.

Bacteria such as Mycobacterium leprae and T. pallidumcan be grown in animals, although serological and

microscopic techniques make the use of live animals unnecessary. Viruses are also usually identified using

alternatives to growth in culture or animals. Some viruses may be grown in embryonated eggs. Another

useful identification method is Xenodiagnosis, or the use of a vector to support the growth of an infectious

agent. Chagas disease  is the most significant example, because it is difficult to directly demonstrate the

presence of the causative agent, Trypanosoma cruzi  in a patient, which therefore makes it difficult to

definitively make a diagnosis. In this case, xenodiagnosis involves the use of the vector of the Chagas

agent T. cruzi, an uninfected triatomine bug, which takes a blood meal from a person suspected of having

been infected. The bug is later inspected for growth of T. cruzi within its gut.

Microscopy

Another principal tool in the diagnosis of infectious disease is microscopy. Virtually all of the culture

techniques discussed above rely, at some point, on microscopic examination for definitive identification of

the infectious agent. Microscopy may be carried out with simple instruments, such as the compound light

microscope, or with instruments as complex as an electron microscope. Samples obtained from patients

may be viewed directly under the light microscope, and can often rapidly lead to identification. Microscopy

is often also used in conjunction with biochemical stainingtechniques, and can be made exquisitely specific

when used in combination with antibody based techniques. For example, the use of antibodies made

artificially fluorescent (fluorescently labeled antibodies) can be directed to bind to and identify a

specific antigens present on a pathogen. A fluorescence microscope is then used to detect fluorescently

labeled antibodies bound to internalized antigens within clinical samples or cultured cells. This technique is

especially useful in the diagnosis of viral diseases, where the light microscope is incapable of identifying a

virus directly.

Other microscopic procedures may also aid in identifying infectious agents. Almost all cells readily stain

with a number of basic dyes due to the electrostatic attraction between negatively charged cellular

molecules and the positive charge on the dye. A cell is normally transparent under a microscope, and using

a stain increases the contrast of a cell with its background. Staining a cell with a dye such as Giemsa stain

or crystal violet allows a microscopist to describe its size, shape, internal and external components and its

associations with other cells. The response of bacteria to different staining procedures is used in

the taxonomic classification of microbes as well. Two methods, the Gram stain and the acid-fast stain, are

the standard approaches used to classify bacteria and to diagnosis of disease. The Gram stain identifies

the bacterial groups Firmicutes and Actinobacteria, both of which contain many significant human

pathogens. The acid-fast staining procedure identifies the Actinobacterial

genera Mycobacterium and Nocardia.

Biochemical tests

Biochemical tests used in the identification of infectious agents include the detection

of metabolic or enzymatic products characteristic of a particular infectious agent. Since bacteria

fermentcarbohydrates in patterns characteristic of their genus and species, the detection

of fermentation products is commonly used in bacterial identification. Acids, alcohols and gases are usually

detected in these tests when bacteria are grown in selective liquid or solid media.

The isolation of enzymes from infected tissue can also provide the basis of a biochemical diagnosis of an

infectious disease. For example, humans can make neither RNA replicases nor reverse transcriptase, and

the presence of these enzymes are characteristic of specific types of viral infections. The ability of the viral

protein hemagglutinin to bind red blood cells together into a detectable matrix may also be characterized as

a biochemical test for viral infection, although strictly speaking hemagglutinin is not an enzyme and has no

metabolic function.

Serological methods are highly sensitive, specific and often extremely rapid tests used to identify

microorganisms. These tests are based upon the ability of an antibody to bind specifically to an antigen.

The antigen, usually a protein or carbohydrate made by an infectious agent, is bound by the antibody. This

binding then sets off a chain of events that can be visibly obvious in various ways, dependent upon the test.

For example, "Strep throat" is often diagnosed within minutes, and is based on the appearance of antigens

made by the causative agent, S. pyogenes, that is retrieved from a patients throat with a cotton swab.

Serological tests, if available, are usually the preferred route of identification, however the tests are costly

to develop and the reagents used in the test often requirerefrigeration. Some serological methods are

extremely costly, although when commonly used, such as with the "strep test", they can be inexpensive.[2]

Complex serological techniques have been developed into what are known as Immunoassays.

Immunoassays can use the basic antibody – antigen binding as the basis to produce an electro - magnetic

or particle radiation signal, which can be detected by some form of instrumentation. Signal of unknowns

can be compared to that of standards allowing quantitation of the target antigen. To aid in the diagnosis of

infectious diseases, immunoassays can detect or measure antigens from either infectious agents or

proteins generated by an infected organism in response to a foreign agent. For example, immunoassay A

may detect the presence of a surface protein from a virus particle. Immunoassay B on the other hand may

detect or measure antibodies produced by an organism’s immune system which are made to neutralize and

allow the destruction of the virus.

Instrumentation can be used to read extremely small signals created by secondary reactions linked to the

antibody – antigen binding. Instrumentation can control sampling, reagent use, reaction times, signal

detection, calculation of results, and data management to yield a cost effective automated process for

diagnosis of infectious disease.

Molecular diagnostics

Technologies based upon the polymerase chain reaction (PCR) method will become nearly ubiquitous gold

standards of diagnostics of the near future, for several reasons. First, the catalog of infectious agents has

grown to the point that virtually all of the significant infectious agents of the human population have been

identified. Second, an infectious agent must grow within the human body to cause disease; essentially it

must amplify its own nucleic acids in order to cause a disease. This amplification of nucleic acid in infected

tissue offers an opportunity to detect the infectious agent by using PCR. Third, the essential tools for

directing PCR, primers, are derived from the genomes of infectious agents, and with time those genomes

will be known, if they are not already.

Thus, the technological ability to detect any infectious agent rapidly and specifically are currently available.

The only remaining blockades to the use of PCR as a standard tool of diagnosis are in its cost and

application, neither of which is insurmountable. The diagnosis of a few diseases will not benefit from the

development of PCR methods, such as some of the clostridial diseases (tetanus andbotulism). These

diseases are fundamentally biological poisonings by relatively small numbers of infectious bacteria that

produce extremely potent neurotoxins. A significant proliferation of the infectious agent does not occur, this

limits the ability of PCR to detect the presence of any bacteria.

Epidemiology

Disability-adjusted life year for infectious and parasitic diseases per 100,000 inhabitants in 2004.[10]

      no data      ≤250      250-500      500-1000      1000-2000      2000-3000      3000-4000      4000-5000      5000-6250      6250-12500      12500-

25000      25000-50000      ≥50000

The World Health Organization collects information on global deaths by International Classification of

Disease (ICD) code categories. The following table lists the top infectious disease killers which caused

more than 100,000 deaths in 2002 (estimated). 1993 data is included for comparison.

Worldwide mortality due to infectious diseases[11][12]

Rank Cause of death Deaths 2002Percentage ofall deaths

Deaths 1993 1993 Rank

N/A All infectious diseases 14.7 million 25.9% 16.4 million 32.2%

1 Lower respiratory infections [13] 3.9 million 6.9% 4.1 million 1

2 HIV/AIDS 2.8 million 4.9% 0.7 million 7

3 Diarrheal diseases [14] 1.8 million 3.2% 3.0 million 2

4 Tuberculosis (TB) 1.6 million 2.7% 2.7 million 3

5 Malaria 1.3 million 2.2% 2.0 million 4

6 Measles 0.6 million 1.1% 1.1 million 5

7 Pertussis 0.29 million 0.5% 0.36 million 7

8 Tetanus 0.21 million 0.4% 0.15 million 12

9 Meningitis 0.17 million 0.3% 0.25 million 8

10 Syphilis 0.16 million 0.3% 0.19 million 11

11 Hepatitis B 0.10 million 0.2% 0.93 million 6

12-17 Tropical diseases (6)[15] 0.13 million 0.2% 0.53 million 9, 10, 16-18

Note: Other causes of death include maternal and perinatal conditions (5.2%), nutritional deficiencies (0.9%),noncommunicable conditions (58.8%), and injuries (9.1%).

The top three single agent/disease killers are HIV/AIDS, TB and malaria. While the number of deaths due

to nearly every disease have decreased, deaths due to HIV/AIDS have increased fourfold. Childhood

diseases include pertussis, poliomyelitis, diphtheria, measles and tetanus. Children also make up a large

percentage of lower respiratory and diarrheal deaths.

Historic pandemics

A young Bangladeshi girl infected with smallpox (1973). Due to the development of the smallpox vaccine, the disease

was officially eradicated in 1979.

A pandemic (or global epidemic) is a disease that affects people over an extensive geographical area.

Plague of Justinian , from 541 to 750, killed between 50% and 60% of

Europe's population.[16]

The Black Death of 1347 to 1352 killed 25 million in Europe over 5 years

(estimated to be between 25 and 50% of the populations of Europe, Asia,

and Africa - the world population at the time was 500 million).

The introduction of smallpox, measles, and typhus to the areas of Central

and South America by European explorers during the 15th and 16th

centuries caused pandemics among the native inhabitants. Between 1518

and 1568 disease pandemics are said to have caused the population

of Mexico to fall from 20 million to 3 million.[17]

The first European influenza epidemic occurred between 1556 and 1560,

with an estimated mortality rate of 20%.[17]

Smallpox  killed an estimated 60 million Europeans during the 18th

century[18] (approximately 400,000 per year).[19] Up to 30% of those

infected, including 80% of the children under 5 years of age, died from the

disease, and one third of the survivors went blind.[20]

In the 19th century, tuberculosis killed an estimated one-quarter of the

adult population of Europe;[21] by 1918 one in six deaths in France were

still caused by TB.

The Influenza Pandemic of 1918 (or the Spanish Flu) killed 25-50 million

people (about 2% of world population of 1.7 billion).

[22] Today Influenza kills about 250,000 to 500,000 worldwide each year.

Emerging diseases

In most cases, microorganisms live in harmony with their hosts via mutual or commensal interactions.

Diseases can emerge when existing parasites become pathogenic or when new pathogenic parasites enter

a new host.

1. Coevolution  between parasite and host can lead to hosts

becoming resistant to the parasites or the parasites may evolve

greater virulence, leading toimmunopathological disease.

2. Human activity is involved with many emerging infectious diseases,

such as environmental change enabling a parasite to occupy

new niches. When that happens, a pathogen that had been confined

to a remote habitat has a wider distribution and possibly a new host

organism. Parasites jumping from nonhuman to human hosts are

known as zoonoses. Under disease invasion, when a parasite

invades a new host species, it may become pathogenic in the new

host.[23]

Several human activities have led to the emergence and spread of new diseases, [23] see also Globalization

and Disease and Wildlife disease:

Encroachment on wildlife habitats. The construction of new villages and

housing developments in rural areas force animals to live in dense

populations, creating opportunities for microbes to mutate and emerge.[24]

Changes in agriculture. The introduction of new crops attracts new crop

pests and the microbes they carry to farming communities, exposing

people to unfamiliar diseases.

The destruction of rain forests. As countries make use of their rain forests,

by building roads through forests and clearing areas for settlement or

commercial ventures, people encounter insects and other animals

harboring previously unknown microorganisms.

Uncontrolled urbanization. The rapid growth of cities in many developing

countries tends to concentrate large numbers of people into crowded

areas with poor sanitation. These conditions foster transmission of

contagious diseases.

Modern transport. Ships and other cargo carriers often harbor unintended

"passengers", that can spread diseases to faraway destinations. While

with international jet-airplane travel, people infected with a disease can

carry it to distant lands, or home to their families, before their first

symptoms appear.

Pollution  of the environment. Changes in the climate (such as global

warming) can cause microorganisms to adapt and create new strains,

which can give them an evolution advantage.

History

East German postage stamps depicting four antique microscopes. Advancements in microscopy were essential to the

early study of infectious diseases.

When the Black Death bubonic plague reached al-Andalus in the 14th century, Ibn Khatima and Ibn al-

Khatib hypothesized that infectious diseases are caused by "contagious entities" which enter the human

body.[25] Such ideas became more popular in Europe during the Renaissance, particularly through the

writing of the Italian monk Girolamo Fracastoro .[26]

Anton van Leeuwenhoek (1632–1723) advanced the science of microscopy by being the first to observe

microorganisms, allowing for easy visualization of bacteria.

In the mid-19th century John Snow and William Budd did important work demonstrating the contagiousness

of typhoid and cholera through contaminated water. Both are credited with decreasing epidemics of cholera

in their towns by implementing measures to prevent contamination of water.[27]

Louis Pasteur proved beyond doubt that certain diseases are caused by infectious agents, and developed

a vaccine for rabies.

Robert Koch, provided the study of infectious diseases with a scientific basis known as Koch's postulates.

Edward Jenner, Jonas Salk and Albert Sabin developed effective vaccines for smallpox and polio, which

would later result in the eradication and near-eradication of these diseases, respectively.

Alexander Fleming discovered the world's first antibiotic Penicillin which Florey and Chain then developed.

Gerhard Domagk developed sulphonamides, the first broad spectrum synthetic antibacterial drugs.

Medical specialists

The medical treatment of infectious diseases falls into the medical field of Infectiology and in some cases

the study of propagation pertains to the field ofEpidemiology. Generally, infections are initially diagnosed

by primary care physicians or internal medicine specialists. For example, an

"uncomplicated"pneumonia will generally be treated by the internist or the pulmonologist (lung

physician).The work of the infectiologist therefore entails working with both patients and general

practitioners, as well as laboratory scientists, immunologists, bacteriologists and other specialists.

An infectious disease team may be alerted when:

The disease has not been definitively diagnosed after an initial workup

The patient is immunocompromised (for example, in AIDS or

after chemotherapy);

The infectious agent is of an uncommon nature (e.g. tropical diseases);

The disease has not responded to first line antibiotics;

The disease might be dangerous to other patients, and the patient might

have to be isolated

PRENATAL DIAGNOSIS OF MONOGENIC DISORDERS BY DNA ANALYSIS:

WHAT, WHY, WHO, AND HOW?M.A. Morris

Division of Medical Genetics, Department of Genetics and Microbiology,

University Medical Centre, 1211 Geneva 4, Switzerland

Introduction 

Fifteen years after the first prenatal DNA diagnosis was carried out, by Kan and Dozy in 1978 (for sickle cell anaemia (7)), the DNA-based diagnosis of monogenic disorders is finally becoming considered a routine service, in general because of the rapid advance of knowledge in the field of human molecular genetics. More particularly, the invention and development of the polymerase chain reaction (PCR) has made prenatal detection faster, cheaper, and more sensitive. Prenatal DNA testing is commonly provided by specialized laboratories attached to clinical cytogenetic services, whose experience in genetic counselling is of great importance.

What?

At present, prenatal DNA testing is restricted to monogenic disorders—those diseases in which the clinical phenotype is a direct consequence of the mutation of a single gene. In the long term, it is possible that diagnosis will also become available for multigenic diseases, when their genetic aetiology is better understood.

Tests are available for the majority of the common monogenic disorders, as well as for a great number of rarer ones. Table 1 shows a selection of seven diseases for which prenatal testing is available and commonly requested. All of these diseases, with the exception of congenital adrenal hyperplasia, are relatively common and severe. Cystic fibrosis is the most common lethal genetic disease of childhood in populations of white European origin, fragile X syndrome is one of the most common causes of mental retardation in males, and both sickle cell anaemia and type I (Portuguese-type) familial amyloidotic polyneuropathy (FAP1) are lethal diseases which reach endemic levels in some regions.

It is no longer meaningful (nor indeed possible) to produce an exhaustive catalogue of diseases for which DNA testing is available. In the most recent comprehensive review of this subject, Connor (2) lists forty-seven diseases for which DNA-based prenatal diagnosis had been reported, and a further sixty-nine for which testing would be technically possible if desired. In the last year, this list has expanded by perhaps one quarter.

For DNA testing to be made available for a particular disease, only two basic conditions must be fulfilled:

1. A precise clinical diagnosis must be possible.2. The defective gene must either have been accurately mapped to a region of a chromosome or, ideally,

cloned and characterized.

The former point initially seems self-evident, but incorporates the important concept of genetic heterogeneity: a disease phenotype can be caused by defects in different genes. In the worst cases, different genetic defects simply cannot be distinguished on clinical grounds, potentially leading to the wrong gene being analysed. Furthermore, the clinician must always be ready to reconsider diagnoses that were made years earlier, perhaps because new genes have been found; this can be difficult or impossible if no patients are available for examination and if the clinical documentation is incomplete.

The latter point illustrates one of the major reasons for the recent rapid increase in the clinical significance of molecular genetics: more and more human genes are being characterized and their roles in disease investigated, providing the basic tools for clinical diagnosis. In particular, one of the principal aims of the Human Genome Project is the production of a map of all human genes, which will be a tool of immeasurable value in clinical medecine (1).

Why?

In general, two conditions are required to justify prenatal diagnosis of any sort:

1. The risk of disease should be greater than the risk of the test.2. A " useful action " should be available when the result is obtained.

Ethical considerations are of great importance in prenatal diagnosis, because for the majority of disorders termination is the intended course of action; the interested reader is referred to the excellent study of Fletcher and Wertz (5).

Prenatal DNA diagnosis is not a screening test

At present, prenatal DNA analysis is a test performed exclusively on indication—in contrast to physical, cytogenetic, and enzymatic examinations, it is not regarded as a screening test.

Prenatal diagnosis is generally contraindicated when the risk of performing the test exceeds the risk of the disease being present. The nature of the molecular genetics techniques used in prenatal diagnosis imposes a high degree of specificity—one test examines one (defined) disorder. Given the incidence of most monogenic disorders, it is evidently only in exceptional circumstances that the risk to a fetus of inheriting a defined disease will exceed the risk of performing the test (which necessarily involves amniocentesis, chorionic villus sampling (CVS), or fetal blood sampling), and therefore that DNA diagnosis is indicated.

In contrast, the more general tests currently employed in prenatal screening can detect the effects of a wide range of different disorders; although each one of these may be relatively infrequent, the cumulative risk is often significant, notably with increased maternal age.

Two situations can be foreseen where prenatal DNA screening might be justified. Firstly, for the rare situation where lethal genetic diseases are endemic in local populations, such as FAP1 in northern Sweden or in certain regions of Portugal (about 1 in 30 affected), and sickle cell anaemia in some regions of the Mediterranean (1 in 3 carriers).

Secondly, amongst women who have requested amniocentesis or CVS for the diagnosis of chromosomal anomalies and have concomitantly accepted the risk of an invasive technique. The incidences cited in Table 1 show that, amongst 10’000 cytogenetic prenatal diagnoses (e.g. for maternal age) in Switzerland, there will be 5 fetuses with cystic fibrosis, 4 males with fragile X syndrome, and 1-2 males with Duchenne muscular dystrophy. In certain populations, there might in addition be 30 with sickle cell anaemia or FAP1. In all these cases, the result of " cytogenetically normal " would be given.

In conclusion, in some situations there may be a clear argument for offering a DNA-based screening of a few monogenic disorders, selected according to their frequency in the population in question.

The purpose of a prenatal diagnosis is to act on the result

The procedure of a prenatal diagnosis, for a monogenic disorder or for any other, should be undertaken only in the aim of taking positive action in the case of an unfavourable result. For the great majority of disorders, at present the only possible action is termination. The parents should be counselled about the different courses of action before starting the diagnostic procedure, to allow them to take an informed decision.

In the future, many diseases may be susceptible to treatment in utero, but at present this is unfortunately a rare option. One common disorder amenable to such treatment is congenital adrenal hyperplasia, where prenatal diagnosis has a proven positive value in avoiding the virilizing effects associated with the defect of the enzyme steroid 21-hydroxylase.

If neither termination nor in utero treatment is being considered, there is rarely an indication for prenatal diagnosis.

Who?

It depends on the risk

As was described above, prenatal diagnosis is generally indicated for parents for whom the risk of having an affected fetus are greater than the risk of undergoing the test, or in the rare cases where an in utero treatment is available. Consequently, the major consideration when answering the above question is the precise risk of having an affected child.

Three factors are involved in defining the genetic risk for a couple:

1. The mode of transmission of the disorder.2. For recessive disorders only, the population frequency of (asymptomatic) carriers.3. The results of DNA testing of the parents (rather than the fetus).

Example: Cystic fibrosis. To illustrate the practical application of risk calculations in combination with DNA testing, we will consider a family with one member affected by cystic fibrosis (CF), an autosomal recessive disorder which is very common in populations of white European origin (Table 2).

The medical genetics of CF are well known:

autosomal recessive transmission; carrier frequency: about 1 in 23 people; incidence (at birth) 1 in 2000; gene " CFTR " on chromosome 7; well-characterized; over 200 mutations known; one very common mutation:  F508 (70% of all mutations); about 80% of mutations can be routinely detected.

One boy of the family is affected by the disease (individual III-1). The known autosomal recessive inheritance of the disease indicates that his parents (II-1 and II-2) are obligate carriers of mutations of the CFTR gene. The risk ® for each subsequent child of this couple is a function of four probabilities:

p(father carrier) x p(mother carrier) x p(father transmits) x p(mother transmits)

R = 1 x 1 x ½ x ½ = ¼

With such a high risk, a prenatal diagnosis would of course be indicated. But is a prenatal similarly indicated for the aunt of the affected child and her husband (II-3 and II-4)?

The prior risk that the aunt is a carrier—before any DNA analysis has been performed—is approximately ½, and the risk of her husband is that of the normal population. Consequently,

Rprior = ½ x 1/23 x ½ x ½ = 1/184

The prior risk for the fetus is perhaps low in absolute terms, but is nonetheless sufficiently high (over ten times that of the normal population) to provoke concern.

DNA testing of the parents is useful to modify this risk, and to give a better indication of the options in terms of prenatal diagnosis. Let us suppose that the most common mutations of CFTR are tested in the couple: the aunt II-3 is shown to be a carrier, but her husband III-4 is not. These mutations account for 80% of all CFTR mutations (in terms of frequency), and so the residual risk that the husband is a carrier is only 20% of the prior risk.

Rresidual = 1 x (1/23 x 20/100) x ½ x ½ = 1/460

Even though the one of the couple is a proven carrier, testing of her husband has led to a reduced calculated risk for the fetus, and to a greatly reduced indication for prenatal testing.

These calculations can be utilised for other autosomal recessive disorders, simply by modifying the carrier frequency.

It should also be noted that, in the case of the couple II-3 and II-4, it is not possible to guarantee an informative prenatal diagnosis, because although no mutation has been found in the father, the presence of an undetected mutation cannot be excluded. There are two possible outcomes to a prenatal diagnosis:

1. If the mother is shown not to have transmitted her mutation, the fetus cannot be affected.2. If the mother does transmit her mutation, the statistical risk that the fetus be affected increases, but no

further diagnosis can be performed.

The former result is obviously of positive value, and the latter equally obviously has a considerable negative psychological effect. Despite this, in our experience approximately half of couples request prenatal diagnosis in these circumstances after being informed of the possible outcomes.

How?

Two different types of analysis are used for in DNA diagnosis.

  DIRECT ANALYSIS INDIRECT ANALYSIS

Prerequisites: gene cloned;mutation characterized.

accurate diagnosis;gene localized;linked markers available;DNA from index case available.

Advantages: 100% accuracy;speed;DNA from index case not essential.

very many disorders.

Disadvantages: gene must be well characterized. accuracy <100%.

Techniques: Southern blot (deletions, duplications, expanding repeats);

Southern blot (RFLP);

PCR (point mutations, deletions, expanding repeats).

PCR (microsatellites RFLP).

Direct analysis is the method of choice

Direct analysis of the mutation responsible for a disease permits a rapid diagnosis, with 100% accuracy, and generally at reasonable cost. In diseases with a unique mutation, such as sickle cell anaemia, the test can be used without any preliminary family studies. In those diseases with multiple mutations, such as cystic fibrosis, it is necessary to study an affected index patient (or carrier), to identify the particular mutation(s) which will be sought during the prenatal diagnosis.

Example: sickle cell anaemia. Fig. 1 shows a family with one child affected by sickle cell anaemia, who is by definition homozygous for the mutant gene haemoglobin (HB) S. The parents are heterozygous, with one mutant and one normal gene. Future pregnancies for this couple can be tested immediately, by directly testing for the presence or absence of the mutation in a CVS or amniocentesis, with the polymerase chain reaction (PCR).

Briefly, the ·ß-globin gene is amplified to near-purity with the PCR, and then tested with the enzyme Dde1. This enzyme recognizes the DNA sequence of the normal gene and cuts it into two fragments (two bands on the gel), but is unable to recognize the mutant gene, leaving it uncut (one band). The pattern of the fragments after gel electrophoresis provides the diagnosis. The result can be obtained within 24 hours of the reception of the sample.

Indirect analysis can be used for many disorders, but is <100% accurate

Direct analysis is frequently impossible, either because the disease gene is not completely characterized or because the exact mutation in a family cannot be identified. In such cases, indirect analysis is the only option, on the condition that the position of the gene on the chromosomes is known and that a linked marker is available. A linked marker is a sequence of DNA—perhaps a gene, perhaps a sequence which codes for nothing—which is physically near to the gene of interest, and which is polymorphic in the population. It may have only two different forms (such as the Rhesus blood group), or many (for example the HLA system of major histocompatibility antigens).

Fig. 2 shows an indirect analysis of a family with a child with the autosomal recessive disease congenital adrenal hyperplasia (CAH). The disease gene, steroid 21-hydroxylase, has many different mutations, and so indirect testing is generally necessary. 21-hydroxylase is adjacent to the HLA genes on chromosome 6, providing a perfect polymorphic marker for diagnosis.

In this family, three different variants of HLA (alleles) are present. Because the HLA genes are so closely linked to the disease gene, the HLA alleles in the affected child can be used as labels to identify the mutant chromosomes in the parents. Thus the father’s mutation is associated with B7, and the mother’s with B2. DNA analysis of the fetal HLA genes indicates that the fetus has inherited a B7 allele from each parent, and by inference a mutant 21-hydroxylase gene from the father but a normal one from the mother. The diagnosis is therefore that the fetus is an unaffected carrier of CAH.

Unfortunately, this analysis carries a small risk of error. During meiosis, every chromosome undergoes at least one crossover event with its homologue. Because the marker used in the diagnosis is not precisely at the site of the mutation, there is a possibility that during the maternal meiosis there was a crossover between the HLA and

21-hydroxylase genes. The outcome of such a recombination would be a pair of chromosomes with the B2 allele on the healthy chromosome and the B7 on the mutant, and subsequently a false diagnosis.

This risk is proportional to the distance between the polymorphic marker and the disease gene, and so it is important to use the most closely-linked markers available. The frequency of recombination should be determined before using a marker, to permit an accurate assessment of the risk of error (in the above example, the risk of error is approximately 1%). In addition, this risk can be almost eliminated by using a marker to each side of the gene: in this case, an error could only arise if two crossovers, one just to each side of the gene, were to occur, which is very unlikely.

Finally, it must be noted that it is essential to have a DNA sample from an affected individual to perform an indirect analysis, to determine which " label " is associated with the mutation in each family member. It is consequently often necessary to maintain a " DNA bank " of samples from affected patients, in case a diagnosis will ever be required in a family.

Concluding remarks

Prenatal diagnosis of monogenic disease is now accepted as a routine service in clinical genetics, although it should be noted that the analyses are technically demanding and not generally amenable to a " kit " approach but require a specialized laboratory. Many monogenic diseases can be tested now, and more are being added to the list every month. The message to the practising clinician is: if in doubt, ask!

DNA technology is also being applied to cytogenetic analyses, to increase their sensitivity and informativity. Fluorescent in situ hybridization can be used clinically to identify marker chromosomes, to characterize complex rearrangements, to detect microdeletions, or simply to provide rapid detection of chromosomal aneuploidies without cell culture (8). In addition, analysis of DNA polymorphisms has been used in the author’s laboratory to exclude uniparental disomy in some families with balanced translocations (4).

In the future, it is possible that DNA analysis will be offered as a screening test for several of the most frequent disorders, particularly in pregnancies where chromosomal analysis has already been requested.

In the very long term, prenatal diagnosis in very high risk pregnancies may be superseded by preimplantation diagnosis, in which single cells from a number of embryos are tested, and only non-affected embryos implanted. This approach has already been successfully used for cystic fibrosis (6).

I would like to thank my colleagues in the Division of Medical Genetics for their help and for many interesting discussions. The views expressed in this article are the views of the author, and not necessarily policy of this Division.

References

1. Antonarakis, S.E. (1993): Trends Genet., 9:142-147.2. Connor, J.M. (1992): In: Prenatal Diagnosis and Screening, edited by D.J.H. Brock, C.H. Rodeck, and

M.A. Ferguson-Smith, pp. 515-547. Churchill Livingstone, Edinburgh.3. Davies, K. (1990): Nature, 348:110-111.4. Engel, E., and DeLozier-Blanchet, C.D. (1991): Am. J. Med. Genet., 40:432-439.5. Fletcher, J.C., and Wertz, D.C. (1992): In: Prenatal Diagnosis and Screening, edited by D.J.H. Brock,

C.H. Rodeck and M.A. Ferguson-Smith, pp. 741-754. Churchill Livingstone, Edinburgh.6. Handyside, A.H., Lesko, J.G., Tarìn, J.J., Winston, R.M.L., and Hughes, M.R. (1992): N. Eng. J. Med.,

327:905-909.7. Kan, Y.W., and Dozy, A.M. (1978): Lancet, ii:910-912.8. Ledbetter, D.H. (1992): Hum. Molec. Genet., 1:297-299.9. Mandel, J-L. (1993): Nature Genet., 4:8-9.10. Miller, W.L., and Morel, Y. (1989): Ann. Rev. Genet., 23:371-393.11. Morris, M.A., Nichols, W., and Benson, M. (1991): Am. J. Med. Genet., 39:123-124.12. Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A., and Arnheim, N.

(1986): Science, 230:1350-1354.13. Ward, P.A., Hejtmancik, J.F., Witkowski, J.A., Baumbach, L.L., Gunnell, S., Speer, J., Hawley, P.,

Tantravahi, U., and Caskey, C.T. (1989): Am. J. Hum. Genet., 44:270-281.

Diabetes mellitus: Excerpt from Professional Guide to Diseases

Diabetes mellitus (DM) is a chronic disease of absolute or relative insulin deficiency or resistance

characterized by disturbances in carbohydrate, protein, and fat metabolism. A leading cause of

death by disease in the United States, this syndrome is a contributing factor in about 50% of

myocardial infarctions and about 75% of strokes as well as in renal failure and peripheral

vascular disease. It’s also the leading cause of new blindness.

DM occurs in four forms classified by etiology: type 1, type 2, other specific types, and

gestational diabetes mellitus (GDM). Type 1 is further subdivided into immune-mediated

diabetes and idiopathic diabetes. Those who were previously in the type 1 diabetes group fall

into this group.Children and adolescents with type 1 immune-mediated diabetes rapidly develop

ketoacidosis, but most adults with this type experience only modest fasting hyperglycemia unless

they develop an infection or experience another stressor. Patients with type 1 idiopathic diabetes

are prone to ketoacidosis.

Most patients with type 2 diabetes are obese. The “other specific types” category includes

people who have diabetes as a result of a genetic defect, endocrinopathies, or exposure to

certain drugs or chemicals. GDM occurs during pregnancy. In this type of diabetes, glucose

tolerance levels usually return to normal after delivery.

Causes and incidence

DM affects an estimated 6% of the population of the United States, about half of whom are

undiagnosed. Incidence is greater in females and rises with age. Type 2 accounts for 90% of

cases.

In type 1 diabetes, pancreatic beta-cell destruction or a primary defect in beta-cell function

results in failure to release insulin and ineffective glucose transport. Type 1 immune-mediated

diabetes is caused by cell-mediated destruction of pancreatic beta cells. The rate of beta-cell

destruction is usually higher in children than in adults. The idiopathic form of type 1 diabetes has

no known cause. Patients with this form have no evidence of autoimmunity and don’t produce

insulin.

In type 2 diabetes, beta cells release insulin, but receptors are insulin-resistant and glucose

transport is variable and ineffective. Risk factors for type 2 diabetes include:

❑ obesity (even an increased percentage of body fat primarily in the abdominal region); risk

decreases with weight and drug therapy

❑ lack of physical activity

❑ history of GDM

❑ hypertension

❑ Black, Hispanic, Pacific Islander, Asian American, Native American origin

❑ strong family history of diabetes

❑ older than age 45

❑ high-density lipoprotein cholesterol of less than 35 or triglyceride of greater than 250

❑ Seriously impaired glucose tolerance (IGT) test.

ELDER TIP As the body ages, the cells become more resistant to insulin, thus reducing the older

adult’s ability to metabolize glucose. In addition, the release of insulin from the pancreatic beta

cells is reduced and delayed. These combined processes result in hyperglycemia. In the older

patient, sudden concentrations of glucose cause increased and more prolonged hyperglycemia.

The “other specific types” of DM result from various conditions (such as a genetic defect of the

beta cells or endocrinopathies) or from use of or exposure to certain drugs or chemicals. GDM is

considered present whenever a patient has any degree of abnormal glucose during pregnancy.

This form may result from weight gain and increased levels of estrogen and placental hormones,

which antagonize insulin.

Insulin transports glucose into the cell for use as energy and storage as glycogen. It also

stimulates protein synthesis and free fatty acid storage in the fat deposits. Insulin deficiency

compromises the body tissues’access to essential nutrients for fuel and storage.

Signs and symptoms

Diabetes may begin dramatically with ketoacidosis or insidiously. Its most common symptom is

fatigue from energy deficiency and a catabolic state. Insulin deficiency causes hyperglycemia,

which pulls fluid from body tissues, causing osmotic diuresis, polyuria, dehydration, polydipsia,

dry mucous membranes, poor skin turgor and, in most patients, unexplained weight loss.

ELDER TIP Because their thirst mechanism functions less effectively, older adults may not

report polydipsia, a hallmark of diabetes in younger adults.

In ketoacidosis and hyperosmolar hyperglycemic nonketotic syndrome, dehydration may cause

hypovolemia and shock. Wasting of glucose in the urine usually produces weight loss and

hunger in type 1 diabetes, even if the patient eats voraciously.

Long-term effects of diabetes may include retinopathy, nephropathy, atherosclerosis, and

peripheral and autonomic neuropathy. Peripheral neuropathy usually affects the hands and feet

and may cause numbness or pain. Autonomic neuropathy may manifest itself in several ways,

including gastroparesis (leading to delayed gastric emptying and a feeling of nausea and fullness

after meals), nocturnal diarrhea, impotence, and orthostatic hypotension.

Because hyperglycemia impairs the patient’s resistance to infection, diabetes may result in skin

and urinary tract infections (UTIs) and vaginitis. Glucose content of the epidermis and urine

encourages bacterial growth.

Diagnosis

According to the American Diabetes Association (ADA), DM can be diagnosed if any of the

following exist:

❑ symptoms of diabetes (polyuria, polydipsia, and unexplained weight loss) plus a random (non-

fasting) blood glucose level greater than or equal to 200 mg/dl accompanied by symptoms of

diabetes.

❑ a fasting blood glucose level (no caloric intake for at least 8 hours) greater than or equal to

126 mg/dl.

❑ a plasma glucose value in the 2-hour sample of the oral glucose tolerance test greater than or

equal to 200 mg/dl. This test should be performed after a glucose load dose of 75 g of anhydrous

glucose.

If results are questionable, the diagnosis should be confirmed by a repeat test on a different day.

The ADA also recommends the following testing guidelines:

❑ Test every 3 years: people age 45 or older without symptoms

❑ Test immediately: people with the classic symptoms

❑ High-risk groups should be tested frequently: Individuals with impaired glucose tolerance

usually have normal blood levels unless challenged by a glucose load, such as a piece of pie or

glass of orange juice. Two hours after a glucose load, the glucose level ranges from 140 to 199

mg/dl. These individuals have an abnormal fasting glucose level between 110 and 125 mg/dl.

Because the fasting plasma glucose test is sufficient to make the diagnosis of diabetes, it

replaces the oral glucose tolerance test. (See Classifying blood glucose levels.)

An ophthalmologic examination may show diabetic retinopathy. Other diagnostic and monitoring

tests include urinalysis for acetone and blood testing for glycosylated hemoglobin (Hb A1C), which

reflects recent glucose cortisol.

Treatment

Effective treatment normalizes blood glucose and decreases complications using insulin

replacement, diet, and exercise. Current forms of insulin replacement include single-dose,

mixed-dose, split-mixed dose, and multiple-dose regimens. The multiple-dose regimens may use

an insulin pump. Insulin may be rapid acting, intermediate acting, long acting, or a combination

of rapid acting and intermediate acting; it may be standard or purified, and it may be derived from

beef, pork, or human sources. Purified human insulin is used commonly today. Pancreas

transplantation is experimental and requires chronic immunosuppression.

Successful treatment requires an extensive dietary education. The patient’s diet is specifically

tailored to include the right amount and combination of foods. Almost all foods may be eaten

occasionally. The diet should address dietary prescriptions as well as personal and cultural

preferences to improve adherence and control. For the obese patient with type 2 diabetes,

weight reduction is a goal. In type 1 diabetes, the calorie allotment may be high, depending on

growth stage and activity level.

Type 2 diabetes may require oral antidiabetic drugs to stimulate endogenous insulin production,

increase insulin sensitivity at the cellular level, and suppress hepatic gluconeogenesis.

Five types of drugs have been used to treat diabetes. Sulfonylureas stimulate pancreatic insulin

release, increase tissue sensitivity to insulin, and require insulin’s presence to work. Meglitinides

cause immediate, brief release of insulin and are taken immediately before meals. Biguanides

decrease hepatic glucose production and increase tissue sensitivity to insulin. Alpha-glucosidase

inhibitors slow the breakdown of glucose and decrease postprandial glucose peaks. The

thiazolidinediones enhance the action of insulin; however, insulin must be present for them to

work. These drugs also reduce insulin resistance by decreasing hepatic glucose production and

increasing glucose uptake. They have also been shown to lower blood pressure in diabetic

hypertensive patients. Cholesterol and triglyceride levels may also be reduced.

Treatment of long-term diabetic complications may include transplantation or dialysis for renal

failure, photocoagulation for retinopathy, and vascular surgery for large-vessel disease.

Meticulous blood glucose control is essential.

Alert  Any patient with a wound that has lasted more than 8 weeks and who has tried standard

wound care and revascularization without improvement should consider hyperbaric oxygen

therapy. This treatment may speed healing by allowing more oxygen to get to the wound and

may therefore result in fewer amputations.

Keeping glucose at near-normal levels for 5 years or more reduces both the onset and

progression of retinopathy, nephropathy, and neuropathy. In type 2 diabetes, blood pressure

control as well as smoking cessation reduces the onset and progression of complications,

including cardiovascular disease.

Special considerations

Stress the importance of complying with the prescribed treatment program. Tailor your teaching

to the patient’s needs, abilities, and developmental stage. Include diet; purpose, administration,

and possible adverse effects of medication; exercise; monitoring; hygiene; and the prevention

and recognition of hypoglycemia and hyperglycemia. Stress the effect of blood glucose control

on long-term health.

Alert  Watch for acute complications of diabetic therapy, especially hypoglycemia (vagueness,

slow cerebration, dizziness, weakness, pallor, tachycardia, diaphoresis, seizures, and coma);

immediately give carbohydrates, ideally in the form of fruit juice, glucose tablets, honey or, if the

patient is unconscious, glucagon or dextrose I.V.

Alert  Be alert for signs of ketoacidosis (acetone breath, dehydration, weak and rapid pulse, and

Kussmaul’s respirations) and hyperosmolar coma (polyuria, thirst, neurologic abnormalities, and

stupor). These hyperglycemic crises require I.V. fluids, insulin and, usually, potassium

replacement.

❑ Monitor diabetes control by obtaining blood glucose, glycohemoglobulin, lipid levels, and

blood pressure measurements regularly.

❑ Watch for diabetic effects on the cardiovascular system, such as cerebrovascular, coronary

artery, and peripheral vascular impairment, and on the peripheral and autonomic nervous

systems. Treat all injuries, cuts, and blisters (particularly on the legs or feet) meticulously. Be

alert for signs of UTI and renal disease.

❑ Urge regular ophthalmologic examinations to detect diabetic retinopathy.

❑ Assess for signs of diabetic neuropathy (numbness or pain in hands and feet, footdrop,

neurogenic bladder). Stress the need for personal safety precautions because decreased

sensation can mask injuries. Minimize complications by maintaining strict blood glucose control.

❑ Teach the patient to care for his feet by washing them daily, drying carefully between toes,

and inspecting for corns, calluses, redness, swelling, bruises, and breaks in the skin. Urge him to

report changes to the physician. Advise him to wear nonconstricting shoes and to avoid walking

barefoot. Instruct him to use over-the-counter athlete’s foot remedies and seek professional care

should athlete’s foot not improve.

❑ Teach the patient how to manage his diabetes when he has a minor illness, such as a cold,

flu, or upset stomach.

❑ To delay the clinical onset of diabetes, teach people at high risk to avoid risk factors. Advise

genetic counseling for young adult diabetics who are planning families.

❑ Further information may be obtained from the Juvenile Diabetes Foundation, the ADA, and the

American Association of Diabetes Educators.

Pictures

http://www.wrongdiagnosis.com/a/all/book-diseases-7a.htm

Polymerase chain reactionFrom Wikipedia, the free encyclopedia

"PCR" redirects here. For other uses, see PCR (disambiguation).

A strip of eight PCR tubes, each containing a 100 μL reaction mixture

The polymerase chain reaction (PCR) is a scientific technique in molecular biology to amplify a single or

a few copies of a piece of DNAacross several orders of magnitude, generating thousands to millions of

copies of a particular DNA sequence. The method relies on thermal cycling, consisting of cycles of

repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the

DNA. Primers(short DNA fragments) containing sequences complementary to the target region along with

a DNA polymerase (after which the method is named) are key components to enable selective and

repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication,

setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be

extensively modified to perform a wide array of genetic manipulations.

Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase , an enzyme

originally isolated from the bacteriumThermus aquaticus. This DNA polymerase enzymatically assembles a

new DNA strand from DNA building blocks, the nucleotides, by using single-stranded DNA as a template

and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis.

The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR

sample to a defined series of temperature steps. These thermal cycling steps are necessary first to

physically separate the two strands in a DNA double helix at a high temperature in a process called DNA

melting. At a lower temperature, each strand is then used as the template in DNA synthesis by the DNA

polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use

of primers that are complementary to the DNA region targeted for amplification under specific thermal

cycling conditions.

Developed in 1983 by Kary Mullis ,[1] PCR is now a common and often indispensable technique used in

medical and biological research labs for a variety of applications.[2][3] These include DNA

cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary

diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the

detection and diagnosis of infectious diseases. In 1993, Mullis was awarded the Nobel Prize in

Chemistry for his work on PCR.[4]

PCR principles and procedure

Figure 1a: A thermal cycler for PCR

Figure 1b: An older model three-temperature thermal cycler for PCR

PCR is used to amplify a specific region of a DNA strand (the DNA target). Most PCR methods typically

amplify DNA fragments of up to ~10 kilo base pairs (kb), although some techniques allow for amplification

of fragments up to 40 kb in size.[5]

A basic PCR set up requires several components and reagents.[6] These components include:

DNA template that contains the DNA region (target) to be amplified.

Two primers that are complementary to the 3' (three prime) ends of each of the sense and anti-

sense strand of the DNA target.

Taq polymerase  or another DNA polymerase with a temperature optimum at around 70 °C.

Deoxynucleoside triphosphates  (dNTPs; also very commonly and erroneously called deoxynucleotide

triphosphates), the building blocks from which the DNA polymerases synthesizes a new DNA strand.

Buffer solution , providing a suitable chemical environment for optimum activity and stability of the DNA

polymerase.

Divalent  cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for

PCR-mediated DNA mutagenesis, as higher Mn2+concentration increases the error rate during DNA

synthesis[7]

Monovalent cation potassium ions.

The PCR is commonly carried out in a reaction volume of 10–200 μl in small reaction tubes (0.2–0.5 ml

volumes) in a thermal cycler. The thermal cycler heats and cools the reaction tubes to achieve the

temperatures required at each step of the reaction (see below). Many modern thermal cyclers make use of

thePeltier effect which permits both heating and cooling of the block holding the PCR tubes simply by

reversing the electric current. Thin-walled reaction tubes permit favorable thermal conductivity to allow for

rapid thermal equilibration. Most thermal cyclers have heated lids to prevent condensation at the top of the

reaction tube. Older thermocyclers lacking a heated lid require a layer of oil on top of the reaction mixture

or a ball of wax inside the tube.

[edit]Procedure

Figure 2: Schematic drawing of the PCR cycle. (1) Denaturing at 94–96 °C. (2) Annealing at ~65 °C (3) Elongation

at 72 °C. Four cycles are shown here. The blue lines represent the DNA template to which primers (red arrows) anneal

that are extended by the DNA polymerase (light green circles), to give shorter DNA products (green lines), which

themselves are used as templates as PCR progresses.

Typically, PCR consists of a series of 20-40 repeated temperature changes, called cycles, with each cycle

commonly consisting of 2-3 discrete temperature steps, usually three (Fig. 2). The cycling is often preceded

by a single temperature step (called hold) at a high temperature (>90°C), and followed by one hold at the

end for final product extension or brief storage. The temperatures used and the length of time they are

applied in each cycle depend on a variety of parameters. These include the enzyme used for DNA

synthesis, the concentration of divalent ions and dNTPs in the reaction, and the melting temperature (Tm)

of the primers.[8]

Initialization step: This step consists of heating the reaction to a temperature of 94–96 °C (or 98 °C if

extremely thermostable polymerases are used), which is held for 1–9 minutes. It is only required for

DNA polymerases that require heat activation by hot-start PCR.[9]

Denaturation step : This step is the first regular cycling event and consists of heating the reaction to 94–

98 °C for 20–30 seconds. It causes DNA melting of the DNA template by disrupting the hydrogen

bonds between complementary bases, yielding single-stranded DNA molecules.

Annealing step : The reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing

annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is

about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are

only formed when the primer sequence very closely matches the template sequence. The polymerase

binds to the primer-template hybrid and begins DNA synthesis.

Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq

polymerase has its optimum activitytemperature at 75–80 °C,[10][11] and commonly a temperature of

72 °C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand

complementary to the DNA template strand by adding dNTPs that are complementary to the template

in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the

end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase

used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum

temperature, the DNA polymerase will polymerize a thousand bases per minute. Under optimum

conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step,

the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific

DNA fragment.

Final elongation: This single step is occasionally performed at a temperature of 70–74 °C for 5–15

minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.

Final hold: This step at 4–15 °C for an indefinite time may be employed for short-term storage of the

reaction.

Figure 3: Ethidium bromide-stained PCR products after gel electrophoresis. Two sets of primers were used to amplify a

target sequence from three different tissue samples. No amplification is present in sample #1; DNA bands in sample #2

and #3 indicate successful amplification of the target sequence. The gel also shows a positive control, and a DNA

ladder containing DNA fragments of defined length for sizing the bands in the experimental PCRs.

To check whether the PCR generated the anticipated DNA fragment (also sometimes referred to as the

amplimer or amplicon), agarose gel electrophoresis  is employed for size separation of the PCR products.

The size(s) of PCR products is determined by comparison with a DNA ladder (a molecular weight marker),

which contains DNA fragments of known size, run on the gel alongside the PCR products (see Fig. 3).

PCR stages

The PCR process can be divided into three stages:

Exponential amplification: At every cycle, the amount of product is doubled (assuming 100% reaction

efficiency). The reaction is very sensitive: only minute quantities of DNA need to be present.[12]

Levelling off stage: The reaction slows as the DNA polymerase loses activity and as consumption of

reagents such as dNTPs and primers causes them to become limiting.

Plateau: No more product accumulates due to exhaustion of reagents and enzyme.

PCR optimization

In practice, PCR can fail for various reasons, in part due to its sensitivity to contamination causing

amplification of spurious DNA products. Because of this, a number of techniques and procedures have

been developed for optimizing PCR conditions.[13][14] Contamination with extraneous DNA is addressed with

lab protocols and procedures that separate pre-PCR mixtures from potential DNA contaminants. [6] This

usually involves spatial separation of PCR-setup areas from areas for analysis or purification of PCR

products, use of disposable plasticware, and thoroughly cleaning the work surface between reaction

setups. Primer-design techniques are important in improving PCR product yield and in avoiding the

formation of spurious products, and the usage of alternate buffer components or polymerase enzymes can

help with amplification of long or otherwise problematic regions of DNA.

Application of PCR

Selective DNA isolation

PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a specific region of

DNA. This use of PCR augments many methods, such as generating hybridization

probes for Southern or northern hybridization and DNA cloning, which require larger amounts of DNA,

representing a specific DNA region. PCR supplies these techniques with high amounts of pure DNA,

enabling analysis of DNA samples even from very small amounts of starting material.

Other applications of PCR include DNA sequencing to determine unknown PCR-amplified sequences in

which one of the amplification primers may be used in Sanger sequencing, isolation of a DNA sequence to

expedite recombinant DNA technologies involving the insertion of a DNA sequence into a plasmid or the

genetic material of another organism. Bacterial colonies (E. coli) can be rapidly screened by PCR for

correct DNA vector constructs.[15] PCR may also be used for genetic fingerprinting; a forensic technique

used to identify a person or organism by comparing experimental DNAs through different PCR-based

methods.

Some PCR 'fingerprints' methods have high discriminative power and can be used to identify genetic

relationships between individuals, such as parent-child or between siblings, and are used in paternity

testing (Fig. 4). This technique may also be used to determine evolutionary relationships among organisms.

Figure 4: Electrophoresis of PCR-amplified DNA fragments. (1) Father. (2) Child. (3) Mother. The child has inherited

some, but not all of the fingerprint of each of its parents, giving it a new, unique fingerprint.

Amplification and quantification of DNA

Because PCR amplifies the regions of DNA that it targets, PCR can be used to analyze extremely small

amounts of sample. This is often critical for forensic analysis, when only a trace amount of DNA is available

as evidence. PCR may also be used in the analysis of ancient DNAthat is tens of thousands of years old.

These PCR-based techniques have been successfully used on animals, such as a forty-thousand-year-

old mammoth, and also on human DNA, in applications ranging from the analysis of Egyptian mummies to

the identification of a Russian tsar .[16]

Quantitative PCR methods allow the estimation of the amount of a given sequence present in a sample—a

technique often applied to quantitatively determine levels of gene expression. Real-time PCR is an

established tool for DNA quantification that measures the accumulation of DNA product after each round of

PCR amplification.

PCR in diagnosis of diseases

PCR permits early diagnosis of malignant diseases such as leukemia and lymphomas, which is currently

the highest developed in cancer research and is already being used routinely. [citation needed] PCR assays can

be performed directly on genomic DNA samples to detect translocation-specific malignant cells at a

sensitivity which is at least 10,000 fold higher than other methods.[citation needed]

PCR also permits identification of non-cultivatable or slow-growing microorganisms such

as mycobacteria, anaerobic bacteria, or viruses from tissue cultureassays and animal models. The basis

for PCR diagnostic applications in microbiology is the detection of infectious agents and the discrimination

of non-pathogenic from pathogenic strains by virtue of specific genes.[citation needed]

Viral DNA can likewise be detected by PCR. The primers used need to be specific to the targeted

sequences in the DNA of a virus, and the PCR can be used for diagnostic analyses or DNA sequencing of

the viral genome. The high sensitivity of PCR permits virus detection soon after infection and even before

the onset of disease. Such early detection may give physicians a significant lead in treatment. The amount

of virus ("viral load") in a patient can also be quantified by PCR-based DNA quantitation techniques (see

below).

Variations on the basic PCR technique

Allele-specific PCR : a diagnostic or cloning technique which is based on single-nucleotide

polymorphisms (SNPs) (single-base differences in DNA). It requires prior knowledge of a DNA

sequence, including differences between alleles, and uses primers whose 3' ends encompass the

SNP. PCR amplification under stringent conditions is much less efficient in the presence of a mismatch

between template and primer, so successful amplification with an SNP-specific primer signals

presence of the specific SNP in a sequence.[17] See SNP genotyping for more information.

Assembly PCR  or Polymerase Cycling Assembly (PCA): artificial synthesis of long DNA sequences by

performing PCR on a pool of long oligonucleotides with short overlapping segments. The

oligonucleotides alternate between sense and antisense directions, and the overlapping segments

determine the order of the PCR fragments, thereby selectively producing the final long DNA product.[18]

Asymmetric PCR : preferentially amplifies one DNA strand in a double-stranded DNA template. It is

used in sequencing and hybridization probing where amplification of only one of the two

complementary strands is required. PCR is carried out as usual, but with a great excess of the primer

for the strand targeted for amplification. Because of the slow (arithmetic) amplification later in the

reaction after the limiting primer has been used up, extra cycles of PCR are required. [19] A recent

modification on this process, known as Linear-After-The-Exponential-PCR (LATE-PCR), uses a limiting

primer with a higher melting temperature (Tm) than the excess primer to maintain reaction efficiency as

the limiting primer concentration decreases mid-reaction.[20]

Helicase-dependent amplification : similar to traditional PCR, but uses a constant temperature rather

than cycling through denaturation and annealing/extension cycles. DNA helicase, an enzyme that

unwinds DNA, is used in place of thermal denaturation.[21]

Hot-start PCR : a technique that reduces non-specific amplification during the initial set up stages of the

PCR. It may be performed manually by heating the reaction components to the melting temperature

(e.g., 95°C) before adding the polymerase.[22] Specialized enzyme systems have been developed that

inhibit the polymerase's activity at ambient temperature, either by the binding of anantibody [9] [23]  or by

the presence of covalently bound inhibitors that only dissociate after a high-temperature activation

step. Hot-start/cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient

temperature and are instantly activated at elongation temperature.

Intersequence-specific PCR  (ISSR): a PCR method for DNA fingerprinting that amplifies regions

between simple sequence repeats to produce a unique fingerprint of amplified fragment lengths.[24]

Inverse PCR : is commonly used to identify the flanking sequences around genomic inserts. It involves

a series of DNA digestions and self ligation, resulting in known sequences at either end of the unknown

sequence.[25]

Ligation-mediated PCR : uses small DNA linkers ligated to the DNA of interest and multiple primers

annealing to the DNA linkers; it has been used for DNA sequencing, genome walking, and DNA

footprinting.[26]

Methylation-specific PCR  (MSP): developed by Stephen Baylin and Jim Herman at the Johns Hopkins

School of Medicine,[27] and is used to detect methylation of CpG islands in genomic DNA. DNA is first

treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is

recognized by PCR primers as thymine. Two PCRs are then carried out on the modified DNA, using

primer sets identical except at any CpG islands within the primer sequences. At these points, one

primer set recognizes DNA with cytosines to amplify methylated DNA, and one set recognizes DNA

with uracil or thymine to amplify unmethylated DNA. MSP using qPCR can also be performed to obtain

quantitative rather than qualitative information about methylation.

Miniprimer PCR : uses a thermostable polymerase (S-Tbr) that can extend from short primers

("smalligos") as short as 9 or 10 nucleotides. This method permits PCR targeting to smaller primer

binding regions, and is used to amplify conserved DNA sequences, such as the 16S (or eukaryotic

18S) rRNA gene.[28]

Multiplex Ligation-dependent Probe Amplification  (MLPA): permits multiple targets to be amplified with

only a single primer pair, thus avoiding the resolution limitations of multiplex PCR (see below).

Multiplex-PCR : consists of multiple primer sets within a single PCR mixture to produce amplicons of

varying sizes that are specific to different DNA sequences. By targeting multiple genes at once,

additional information may be gained from a single test run that otherwise would require several times

the reagents and more time to perform. Annealing temperatures for each of the primer sets must be

optimized to work correctly within a single reaction, and amplicon sizes, i.e., their base pair length,

should be different enough to form distinct bands when visualized by gel electrophoresis.

Nested PCR : increases the specificity of DNA amplification, by reducing background due to non-

specific amplification of DNA. Two sets of primers are used in two successive PCRs. In the first

reaction, one pair of primers is used to generate DNA products, which besides the intended target,

may still consist of non-specifically amplified DNA fragments. The product(s) are then used in a second

PCR with a set of primers whose binding sites are completely or partially different from and located 3'

of each of the primers used in the first reaction. Nested PCR is often more successful in specifically

amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the

target sequences.

Overlap-extension PCR : a genetic engineering technique allowing the construction of a DNA sequence

with an alteration inserted beyond the limit of the longest practical primer length.

Quantitative PCR  (Q-PCR): used to measure the quantity of a PCR product (commonly in real-time). It

quantitatively measures starting amounts of DNA, cDNA or RNA. Q-PCR is commonly used to

determine whether a DNA sequence is present in a sample and the number of its copies in the

sample. Quantitative real-time PCR has a very high degree of precision. QRT-PCR methods use

fluorescent dyes, such as Sybr Green, EvaGreen or fluorophore-containing DNA probes, such

as TaqMan, to measure the amount of amplified product in real time. It is also sometimes abbreviated

to RT-PCR (Real Time PCR) or RQ-PCR. QRT-PCR or RTQ-PCR are more appropriate contractions,

since RT-PCR commonly refers to reverse transcription PCR (see below), often used in conjunction

with Q-PCR.

Reverse Transcription PCR (RT-PCR): for amplifying DNA from RNA. Reverse transcriptase reverse

transcribes RNA into cDNA, which is then amplified by PCR. RT-PCR is widely used inexpression

profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript,

including transcription start and termination sites. If the genomic DNA sequence of a gene is known,

RT-PCR can be used to map the location of exons and introns in the gene. The 5' end of a gene

(corresponding to the transcription start site) is typically identified by RACE-PCR(Rapid Amplification of

cDNA Ends).

Solid Phase PCR : encompasses multiple meanings, including Polony Amplification  (where PCR

colonies are derived in a gel matrix, for example), Bridge PCR [29] (primers are covalently linked to a

solid-support surface), conventional Solid Phase PCR (where Asymmetric PCR is applied in the

presence of solid support bearing primer with sequence matching one of the aqueous primers) and

Enhanced Solid Phase PCR[30] (where conventional Solid Phase PCR can be improved by employing

high Tm and nested solid support primer with optional application of a thermal 'step' to favour solid

support priming).

Thermal asymmetric interlaced PCR (TAIL-PCR): for isolation of an unknown sequence flanking a

known sequence. Within the known sequence, TAIL-PCR uses a nested pair of primers with differing

annealing temperatures; a degenerate primer is used to amplify in the other direction from the

unknown sequence.[31]

Touchdown PCR  (Step-down PCR): a variant of PCR that aims to reduce nonspecific background by

gradually lowering the annealing temperature as PCR cycling progresses. The annealing temperature

at the initial cycles is usually a few degrees (3-5°C) above the Tm of the primers used, while at the later

cycles, it is a few degrees (3-5°C) below the primer Tm. The higher temperatures give greater

specificity for primer binding, and the lower temperatures permit more efficient amplification from the

specific products formed during the initial cycles.[32]

PAN-AC : uses isothermal conditions for amplification, and may be used in living cells.[33][34]

Universal Fast Walking : for genome walking and genetic fingerprinting using a more specific 'two-sided'

PCR than conventional 'one-sided' approaches (using only one gene-specific primer and one general

primer - which can lead to artefactual 'noise')[35] by virtue of a mechanism involving lariat structure

formation. Streamlined derivatives of UFW are LaNe RAGE (lariat-dependent nested PCR for rapid

amplification of genomic DNA ends),[36] 5'RACE LaNe[37] and 3'RACE LaNe.[38]

History

A 1971 paper in the Journal of Molecular Biology by Kleppe and co-workers first described a method using

an enzymatic assay to replicate a short DNA template with primers in vitro.[39] However, this early

manifestation of the basic PCR principle did not receive much attention, and the invention of the

polymerase chain reaction in 1983 is generally credited to Kary Mullis .[40]

At the core of the PCR method is the use of a suitable DNA polymerase able to withstand the high

temperatures of >90 °C (194 °F) required for separation of the two DNA strands in the DNA double

helix after each replication cycle. The DNA polymerases initially employed for in vitro experiments

presaging PCR were unable to withstand these high temperatures.[2] So the early procedures for DNA

replication were very inefficient, time consuming, and required large amounts of DNA polymerase and

continual handling throughout the process.

The discovery in 1976 of Taq polymerase  — a DNA polymerase purified from the thermophilic

bacterium, Thermus aquaticus , which naturally lives in hot (50 to 80 °C (122 to 176 °F))

environments[10]such as hot springs — paved the way for dramatic improvements of the PCR method. The

DNA polymerase isolated from T. aquaticus is stable at high temperatures remaining active even after DNA

denaturation,[11] thus obviating the need to add new DNA polymerase after each cycle.[3] This allowed an

automated thermocycler-based process for DNA amplification.

When Mullis developed the PCR in 1983, he was working in Emeryville, California for Cetus Corporation ,

one of the first biotechnology companies. There, he was responsible for synthesizing short chains of DNA.

Mullis has written that he conceived of PCR while cruising along the Pacific Coast Highway one night in his

car.[41] He was playing in his mind with a new way of analyzing changes (mutations) in DNA when he

realized that he had instead invented a method of amplifying any DNA region through repeated cycles of

duplication driven by DNA polymerase. In Scientific American, Mullis summarized the procedure:

"Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar

molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few

simple reagents, and a source of heat."[42] He was awarded the Nobel Prize in Chemistry in 1993 for his

invention,[4] seven years after he and his colleagues at Cetus first put his proposal to practice. However,

some controversies have remained about the intellectual and practical contributions of other scientists to

Mullis' work, and whether he had been the sole inventor of the PCR principle (see below).

Patent wars

The PCR technique was patented by Kary Mullis and assigned to Cetus Corporation , where Mullis worked

when he invented the technique in 1983. The Taq polymerase enzyme was also covered by patents. There

have been several high-profile lawsuits related to the technique, including an unsuccessful lawsuit brought

by DuPont. The pharmaceutical company Hoffmann-La Roche purchased the rights to the patents

in 1992 and currently holds those that are still protected.

A related patent battle over the Taq polymerase enzyme is still ongoing in several jurisdictions around the

world between Roche and Promega. The legal arguments have extended beyond the lives of the original

PCR and Taq polymerase patents, which expired on March 28, 2005.[43]

Nonlethal clinical techniques used in the diagnosis of diseases of fish.

Smith SA.

Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine,

Virginia Polytechnic Institute and State University, Blacksburg, 24061, USA.

AbstractNumerous nonlethal clinical techniques can be used on live fish to yield valuable diagnostic information. These

techniques include skin, fin, and gill biopsies; bacteriologic cultures of gill or skin lesions; tissue and fluid

aspiration; and radiography Most techniques can be performed on live fish without the use of anesthesia,

although light sedation of the fish often simplifies the procedure, making the procedure more easily accomplished

and less stressful on the fish. Because water conditions have a considerable effect on the health and well-being

of aquatic animals, an in-house evaluation of water quality (eg, temperature, pH, ammonia, nitrites, nitrates, and

salinity) is also paramount to any clinical diagnostic evaluation. As with domestic animals, a complete and

accurate history and thorough external examination are prerequisite to the selection of appropriate diagnostic

techniques as well as the formulation of any management or therapeutic plan. Through the correlation of clinical

history, water quality variables, and results of diagnostic testing, an informed plan of action can be devised to

correct acute or chronic problems in aquatic animals.

Strategies used by Doctors for DiagnosisBy manishrathi

[This blog is part of the series which I had been writing about various aspects of Doctor -

Patient Relationship. and how the function of diagnosis and treatment are at its core. In my

previous blog, I had talked about why disease name identification was a very important step

of the diagnosis process. This particular blog talks about what are the strategies which

doctors use to diagnose.]

In my previous set of blogs in this series I had started talking about what diagnosis means

from medical perspective.  One important realization I had was that

Diagnosis is about finding the name of the disease (and the dictionary definition of Diagnosis

–“process of determining the identity of (a disease, illness, etc.) by a medical examination”

– also conveys the same thing) and not really about treating a patient. As commonsensical as

this may sound – I have seen many do tend to miss out on this. According to me though –

understanding this subtle difference is important considering that it can have a direct impact

on how doctors and patients understand each other.

Now that I think I figured out what the real goal of Diagnosis process was i.e. the  ‘why’ part;

my next goal was to understand the ‘how’ part. Curiosity in my mind was how do doctor’s

diagnose and arrive at a conclusion. Was it through a logic or some magic or by simply

looking through a crystal mirror? The blog post from my brother – “What is in the Name?”

gave me some insights as to what goes in the doctor’s mind during the diagnosis. My next

step was to read and understand if there were any particular strategies using which doctors

typically came to a conclusion.

Towards this, I thought the four-strategy model suggested by D. L. Sackett et. all. was a good

starting point to start understanding how the typical clinical diagnosis happens (src – “The

diagnostic process in general practice: has it a two-phase structure?” by Anders Baerheim).

This blog is a summary of what those four strategies are.

Before I summarize them, I think it is important to keep in mind that these strategies are

categorized based on the characteristics of the approach used in it. I do not think doctors pre-

plan about the type of strategy they are going to use when they see a patient. Most of the

times it is a sub-conscious decision and also there is a good chance that hybrids of these

approaches are also used in real life practices. So beware – if you were to pop a question to

your doc about which technique he was going to use to diagnose you – don’t be surprised if

he gives you a blank stare!

So the four techniques described by Sackett et. all. are -

Pattern Recognition strategy -

Subconscious Recognition

While many can think about medical diagnosis as synonymous to any problem-solving

technique where some kind of scientific method is applied to come to a solution, this

technique of diagnosis is more instinctive especially when certain configuration of

symptoms/clues appears very classical. This results in a instant/unconscious generation of a

hypothesis. For example, diagnosing down syndrome after one look at the patient.

This technique of diagnosis for diseases/ailments is possibly the most common

strategy/technique used by the doctors – especially the ones who are the most seasoned.

More the experience of the doctor (probably clinical and not in terms of years) – better the

doctor gets at this. Majority of the time this technique is reflexive and possibly not reflective.

Interestingly this is not the technique which doctors are taught in their classrooms; but is

learnt on patients. This technique also forms the basis for ‘first’ diagnosis majority of the

time.

While Pattern Recognition strategy is the most popular strategy and majority of correct

diagnosis happen around using this technique – this technique of medical diagnosis also has

some inherent risks associated with it. First and foremost of course, the doctor need to be

very good at the skill of looking and sensing patterns. Second, there is always a good

possibility that the doctors could fail to look beyond the obvious patterns and hence there is a

increased risk of doctor’s tendency to close the diagnosis prematurely. Doctors are also

human beings. And like any human beings there is also the risk of introduction of self-biases

possibly for self-satisfaction. Unfortunately, this technique also seems to be quite prevalent in

over-stressed doctors.

Hypothetico-deductive strategy -

Probabilistic Diagnosis

Hypothetico-deductive strategy is a type of clinical reasoning model based on a combination

of both cognitive science and probabilistic theory. In this strategy, for diagnosing doctors first

do a restricted rule-outs i.e. possibilities or causes which they believe the patient is not

suffering from. Then they start with a short list of potential hypothesis based on the available

clues. This generation of hypotheses is followed byongoing analysis of patient information in

which further data/tests are collected and interpreted (typically in a cyclical manner).

Continued hypothesis creation and evaluation take place as various hypotheses are confirmed

or negated.

So in some sense in this technique the diagnosis moves from a generalization (multiple

hypotheses) to a specific conclusion. This technique is typically used in diagnosing uncommon

or rare diseases or where the doctor may not be experienced in a particular disease.

Pitfalls associated with this technique are that doctors require a very good understanding of

probability theory. They should have a good knack to work out the horses vs. zebra confusion

just based on hearing the hoof-beats. This technique can also turn out to be time and cost

consuming.

Algorithm strategy -

Sample Algorithm

This type of diagnosis process is based on Clinical Guidelines/Decision Rules which are

typically previously very well defined. When this approach is used, doctors typically refer to

the “step-by-step IF-THEN” logic or cause-effect logic well supplemented with bundles of

additional supporting information to arrive at a diagnosis. Click on the thumbnail on the right

to see a sample algorithm for diagnosis of asthma in older patients. Similar algorithms are

available for many such diseases where plenty of historical data is available. Today tools and

software are also available to assist doctors in such strategies.

While this method is typically suggested to reduce the diagnosis errors, unfortunately in

reality it is estimated that this approach is used in less than 10% of the diagnosis which takes

place out there.

Complete History strategy -

While the Hypothetico-deductive strategy described above can be categorized as ‘diagnosis

by probability’, Complete History strategy is typically exhaustion-based and can be

categorized as ‘diagnosis by possibility’. This is the approach where all the possibilities are

assumed. Then medical facts of the patients are collected and the assumed possibilities are

eliminated one by one till the time the diagnosis is reached.

This approach is typically used for diagnosing possibilities of a rare disease or possibly when

usage of any of the above listed strategy has not brought in the success of correct diagnosis.

In Doctor’s community – this approach is typically considered as the method of novice,

impractical, and inefficient.

Diagnosis of Diseases Caused by Protozoa and Helminths -  In order to supplement the conventional methods available for diagnosis, of different diseases, biotechnology has provided monoclonal antibodies and DNA probes as two very effective and most sensitive tools. Monoclonal antibodies and DNA probes are being prepared and made available for the diagnosis of a variety of diseases.

Monoclonal antibodies can be used through serological tests, which will take only minutes, while the conventional methods may sometimes take weeks, since they may require culturing of bacteria or viruses as in case of herpes virus or other viruses.

Similarly, DNA probes, which are even more sensitive than monoclonal antibodies, will, however, take hours (not minutes as in monoclonal antibodies) instead of weeks. Readymade DNA probes for herpes virus and other human, animal and plant viruses are being prepared. DNA probe kits are also available to help preparation of DNA probes and a market of DNA probes for several hundred million dollars (> $500,000,000) per year is now available. This market will expand and grow in future.

The diagnostic methods at the DNA level have the advantage over other methods, since in these methods genes of the parasite are examined and not the expressed product which changes in different stages of the life cycle of the parasite and also due to the environment.

Rapid progress in recent years has been made in the development of nucleic acid based assays for the diagnosis and epidemiological surveillance of human parasites. Probes are now available for a number of human parasites from the group Protozoa and Helminths (both Platyhelminths and Nematyhelminths).

In India also, recently (1986) a diagnostic probe for the detection of malaria has been constructed at the Astra Research Centre India (ARCI), which is established as a joint venture between India and AB Astra, Sweden, (inaugurated by Prime Minister, Rajiv Gandhi on Jan. 7, 1987). Methods for the detection of several other diseases are also being developed at this centre.

In the above use of DNA probes, DNA has to be radioactively labeled, which is not very safe in field study. Therefore, techniques for non radioactive labelling of probes, methods for quick preparation of target DNA and suitable protocols for hybridization are being developed. This in future will allow quick diagnosis of parasites in the field.

Overview of diagnostic methods  

In general, diagnostic tests can be grouped into 3 categories.: (1) direct detection, (2) indirect examination (virus isolation), and (3) serology. In direct examination, the clinical specimen is examined directly for the presence of virus particles, virus antigen or viral nucleic acids. In indirect examination, the specimen into cell culture, eggs or animals in an attempt to grow the virus: this is called virus isolation. Serology actually constitute by far the bulk of the work of any virology laboratory. A serological diagnosis can be made by the detection of rising titres of antibody between acute and convalescent stages of infection, or the detection of IgM. In general, the majority of common viral infections can be diagnosed by serology. The specimen used for direction detection and virus isolation is very important. A positive result from the site of disease would be of much greater diagnostic significance than those from other sites. For example, in the case of herpes simplex encephalitis, a positive result from the CSF or the brain would be much greater significance than a positive result from an oral ulcer, since reactivation of oral herpes is common during times of stress.  

1. Direct Examination of Specimen

1. Electron Microscopy morphology / immune electron microscopy2. Light microscopy histological appearance - e.g. inclusion bodies3. Antigen detection immunofluorescence, ELISA etc.4. Molecular techniques for the direct detection of viral genomes    

2. Indirect Examination

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

2. Eggs pocks on CAM - haemagglutination, inclusion bodies3. Animals disease or death confirmation by neutralization    

3. Serology

Detection of rising titres of antibody between acute and convalescent stages of infection, or the detection of IgM in primary infection.  

Classical Techniques Newer Techniques1. 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. Single Radial Haemolysis 5. Recombinant immunoblot assay (RIBA), line immunoassay

(Liatek) etc.   

1. Direct Examination

Direct examination methods are often also called rapid diagnostic methods because they can usually give a result either within the same or the next day. This is extremely useful in cases when the clinical management of the patient depends greatly on the rapid availability of laboratory results e.g. diagnosis of RSV infection in neonates, or severe CMV infections in immunocompromised patients. However, it is important to realize that not all direct examination methods are rapid, and conversely, virus isolation and serological methods may sometimes give a rapid result. With the advent of effective antiviral chemotherapy, rapid diagnostic methods are expected to play an increasingly important role in the diagnosis of viral infections.

1.1. Antigen Detection

Examples of antigen detection include immunofluorescence testing of nasopharyngeal aspirates for respiratory viruses e.g.. RSV, flu A, flu B, and adenoviruses, detection of rotavirus antigen in faeces, the pp65 CMV antigenaemia test, the detection of HSV and VZV in skin scrappings, and the detection of HBsAg in serum. (However, the latter is usually considered as a serological test). The main advantage of these assays is that they are rapid to perform with the result being available within a few hours. However, the technique is often tedious and time consuming, the result difficult to read and interpret, and the sensitivity and specificity poor. The quality of the specimen obtained is of utmost importance in order for the test to work properly.

(Virology Laboratory, Yale-New Haven Hospital)

1.2. Electron Microscopy (EM)

Virus particles are detected and identified on the basis of morphology. A magnification of around 50,000 is normally used. EM is now mainly used for the diagnosis of viral gastroenteritis by detecting viruses in faeces e.g. rotavirus, adenovirus, astrovirus, calicivirus and Norwalk-like viruses. Occasionally it may be used for the detection of viruses in vesicles and other skin lesions, such as herpesviruses and papillomaviruses. The sensitivity and specificity of EM may be enhanced by immune electron microscopy, whereby virus specific antibody is used to agglutinate virus particles together and thus making them easier to recognize, or to capture virus particles onto the EM grid. The main problem with EM is the expense involved in purchasing and maintaining the facility. In addition, the sensitivity of EM is often poor, with at least 105 to 106 virus particles per ml in the sample required for visualisation. Therefore the observer must be highly skilled. With the availability of reliable antigen detection and molecular methods for the detection of viruses associated with viral gastroenteritis, EM is becoming less and less widely used.

Electronmicrographs of viruses commonly found in stool specimens from patients suffering from gastroenteritis. From left to right: rotavirus, adenovirus, astroviruses, Norwalk-like viruses.  (Courtesy of Linda M. Stannard, University of Cape Town, http://www.uct.ac.za/depts/mmi/stannard/emimages.html)

1.3. Light Microscopy

Replicating virus often produce histological changes in infected cells. These changes may be characteristic or non-specific. Viral inclusion bodies are basically collections of replicating virus particles either in the nucleus or cytoplasm. Examples of inclusion bodies include the negri bodies and cytomegalic inclusion bodies found in rabies and CMV infections respectively. Although not sensitive or specific, histology nevertheless serves as a useful adjunct in the diagnosis of certain viral infections.

1.4.Viral Genome Detection

Methods based on the detection of viral genome are also commonly known as molecular methods. It is often said that molecular methods is the future direction of viral diagnosis. However in practice, although the use of these methods is indeed increasing, the role played by molecular methods in a routine diagnostic virus

laboratory is still small compared to conventional methods. It is certain though that the role of molecular methods will increase rapidly in the near future.Classical molecular techniques such as dot-blot and Southern-blot depend on the use of specific DNA/RNA probes for hybridization. The specificity of the reaction depends on the conditions used for hybridization. These techniques may allow for the quantification of DNA/RNA present in the specimen. However, it is often found that the sensitivity of these techniques is not better than conventional viral diagnostic methods.

Newer molecular techniques such as the polymerase chain reaction (PCR), ligase chain reaction (LCR), nucleic acid based amplification (NASBA), and branched DNA (bDNA) depend on some form of amplification, either the target nucleic acid, or the signal itself. bDNA is essentially a conventional hybridization technique with increased sensitivity. However, it is not as sensitive as PCR and other amplification techniques. PCR is the only amplification technique which is in common use. PCR is an extremely sensitive technique: it is possible to achieve a sensitivity of down to 1 DNA molecule in a clinical specimen. However, PCR has many problems, the chief among which is contamination, since only a minute amount of contamination is needed to give a false positive result. In addition, because PCR is so sensitive compared to other techniques, a positive PCR result is often very difficult to interpret as it does not necessarily indicate the presence of disease. This problem is particular great in the case of latent viruses such as CMV, since latent CMV genomes may be amplified from the blood of healthy individuals. Despite all this, PCR is being increasingly used for viral diagnosis, especially as the cost of the assay come down and the availability of closed automated systems that could also perform quantification (Quantitative PCR) e.g. real-time PCR and Cobas Amplicor.systems. Other amplification techniques such as LCR and NASBA are just as susceptible to contamination as PCR but that is ameliorated to a great extent by the use of propriatory closed systems. It is unlikely though that other amplification techniques will challenge the dominance of PCR since it is much easier to set up an house PCR assay than other assays. 

2. Virus Isolation

Cell cultures, eggs, and animals may be used for isolation. However eggs and animals are difficult to handle and most viral diagnostic laboratories depend on cell culture only. There are 3 types of cell cultures:

2.1. Types of cell cultures

1. Primary cells - e.g. Monkey Kidney. These are essentially normal cells obtained from freshly killed adult animals. These cells can only be passaged once or twice.

2. Semi-continuous cells - e.g. Human embryonic kidney and skin fibroblasts. These are cells taken from embryonic tissue, and may be passaged up to 50 times.

3. Continuous cells - e.g. HeLa, Vero, Hep2, LLC-MK2, BGM. These are immortalized cells i.e. tumour cell lines and may be passaged indefinitely.

Primary cell culture are widely acknowledged as the best cell culture systems available since they support the widest range of viruses. However, they are very expensive and it is often difficult to obtain a reliable supply. Continuous cells are the most easy to handle but the range of viruses supported is often limited.

2.2. Identification of growing virus

The presence of growing virus is usually detected by:

1. Cytopathic Effect (CPE) - may be specific or non-specific e.g. HSV and CMV produces a specific CPE, whereas enteroviruses do not.

2. Haemadsorption - cells acquire the ability to stick to mammalian red blood cells. Haemadsorption is mainly used for the detection of influenza and parainfluenzaviruses.

Confirmation of the identity of the virus may be carried out using neutralization, haemadsorption- inhibition, immunofluorescence, or molecular tests.

Left to Right: Cytopathic effect of HSV, enterovirus 71, and RSV in cell culture. Note the ballooning of cells in the cases of HSV and enterovirus 71. Note syncytia formation in the case of RSV. (Linda Stannard. University of Cape Town, Virology Laboratory, Yale-New Haven Hospital)

2.3 Problems with cell culture

The main problem with cell culture is the long period (up to 4 weeks) required for a result to be available. Also, the sensitivity is often poor and depends on many factors, such as the condition of the specimen, and the condition of the cell sheet. Cell cultures are also very susceptible to bacterial contamination and toxic substances in the specimen. Lastly, many viruses will not grow in cell culture at all e.g. Hepatitis B and C, Diarrhoeal viruses, parvovirus etc.

2.4 Rapid Culture Techniques

Rapid culture techniques are available whereby viral antigens are detected 2 to 4 days after inoculation. Examples of rapid culture techniques include shell vial cultures and the CMV DEAFF test. In the CMV DEAFF test, the cell sheet is grown on individual cover slips in a plastic bottle. After inoculation, the bottle then is spun at a low speed for one hour (to speed up the adsorption of the virus) and then incubated for 2 to 4 days. The cover slip is then taken out and examined for the presence of CMV early antigens by immunofluorescence. 

Left: Haemadsorption of red blood cells onto the surface of a cell sheet infected by mumps virus. Also note the presence of syncytia which is indistinguishable from that of RSV (Courtesy of Linda Stannard, University of Cape Town). Right: Positive CMV DEAFF test. (Virology Laboratory, Yale-New Haven Hospital)

The role of cell culture (both conventional and rapid techniques) in the diagnosis of viral infections is being increasingly challenged by rapid diagnostic methods i.e. antigen detection and molecular methods. Therefore, the role of cell culture is expected to decline in future and is likely to be restricted to large central laboratories.     

3. Serology

Serology forms the mainstay of viral diagnosis. This is what happens in a primary humoral immune response to antigen. Following exposure, the first antibody to appear is IgM, which is followed by a much higher titre of IgG. In cases of

reinfection, the level of specific IgM either remain the same or rises slightly. But IgG shoots up rapidly and far more earlier than in a primary infection. Many different types of serological tests are available. With some assays such as EIA and RIA, one can look specifically for IgM or IgG, whereas with other assays such as CFT and HAI, one can only detect total antibody, which comprises mainly IgG. Some of these tests are much more sensitive than others: EIAs and radioimmunoassays are the most sensitive tests available, whereas CFT and HAI tests are not so sensitive. Newer techniques such as EIAs offer better sensitivity, specificity and reproducibility than classical techniques such as CFT and HAI. The sensitivity and specificity of the assays depend greatly on the antigen used. Assays that use recombinant protein or synthetic peptide antigens tend to be more specific than those using whole or disrupted virus particles.

3.1. Criteria for diagnosing Primary Infection

1. A significant rise in titre of IgG/total antibody between acute and convalescent sera - however, a significant rise is very difficult to define and depends greatly on the assay used. In the case of CFT and HAI, it is normally taken as a four-fold or greater increase in titre. The main problem is that diagnosis is usually retrospective because by the time the convalescent serum is taken, the patient had probably recovered.

2. Presence of IgM - EIA, RIA, and IF may be are used for the detection of IgM. This offers a rapid means of diagnosis. However, there are many problems with IgM assays, such as interference by rheumatoid factor, re-infection by the virus, and unexplained persistence of IgM years after the primary infection.

3. Seroconversion - this is defined as changing from a previously antibody negative state to a positive state e.g. seroconversion against HIV following a needle-stick injury, or against rubella following contact with a known case.

4. A single high titre of IgG (or total antibody) - this is a very unreliable means of serological diagnosis since the cut-off is very difficult to define.

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

It is often very difficult to differentiate re-infection/re-activation from a primary infection. Under most circumstances, it is not important to differentiate between a primary infection and re-infection. However, it is very important under certain situations, such as rubella infection in the first trimester of pregnancy: primary infection is associated with a high risk of fetal damage whereas re-infection is not. In general, a sharp large rise in antibody titres is found in re-infection whereas IgM is usually low or absent in cases of re-infection/re-activation.

Serological events following primary infection and reinfection. Note that in reinfection, IgM may be absent or only present transiently at a low level.  

3.3. Limitations of serological diagnosis

How useful a serological result is depends on the individual virus.

1. For viruses such as rubella and hepatitis A, the onset of clinical symptoms coincide with the development of antibodies. The detection of IgM or rising titres of IgG in the serum of the patient would indicate active disease.

2. However, many viruses often produce clinical disease before the appearance of antibodies such as respiratory and diarrhoeal viruses. So in this case, any serological diagnosis would be retrospective and therefore will not be that useful.

3. There are also viruses which produce clinical disease months or years after seroconversion e.g. HIV and rabies. In the case of these viruses, the mere presence of antibody is sufficient to make a definitive diagnosis.

There are a number of problems associated with serology:-    

1. long length of time required for diagnosis for paired acute and convalescent sera

2. mild local infections such as HSV genitalis may not produce a detectable humoral immune response

3. Extensive antigenic cross-reactivity between related viruses e.g. HSV and VZV, Japanese B encephalitis and Dengue, may lead to false positive results

4. immunocompromised patients often give a reduced or absent humoral immune response.

5. Patients with infectious mononucleosis and those with connective tissue diseases such as SLE may react non-specifically giving a false positive result

6. Patients given blood or blood products may give a false positive result due to the transfer of antibody.

Complement Fixation Test in Microtiter Plate. Rows 1 and 2 exhibit complement fixation obtained with acute and convalescent phase serum specimens, respectively. (2-fold serum dilutions were used)  The observed 4-fold increase is significant and indicates infection.  

Microplate ELISA: coloured wells indicate reactivity. The darker the colour, the higher the reactivity    

3.4. Antibody in the CSF

In a healthy person, there should be little or no antibodies in the CSF. Where there is a viral meningitis or encephalitis, antibodies may be produced against the virus

by lymphocytes in the CSF. The finding of antibodies in the CSF is said to be significant when ratio between the titre of antibody in the serum and that in the CSF is less than 100. But this does depend on an intact blood-brain barrier. The problem is that in many cases of meningitis and encephalitis, the blood-brain barrier is damaged, so that antibodies in the serum can actually leak across into the CSF. This also happens where the lumbar puncture was traumatic in which case the spinal fluid would be bloodstained. So really, one should really check the integrity of the blood-brain barrier before making a definite diagnosis. One way to check the integrity of the blood brain barrier is to use a surrogate antibody that most individuals would have, such as measles virus, since most people would have been vaccinated. So the patient's serum and CSF for measles antibody. If the blood-brain barrier is intact, there should be little or no measles antibodies in the CSF.

Computer-Aided Differential Diagnosis of Diseases A...N Difficult to Differentiate

Mark D.Kats, doctor of science, Donetskaya St. 37/24, Severodonetsk, Lugansk region, 349940 Ukraine, tel.: (380-6452)-3-08-75, E-mail: [email protected] (Kats M.D.).

Attachment to the project:

"DEVELOPMENT OF INTELLIGENT SYSTEM FOR COMPUTER-AIDED DIFFERENTIAL DIAGNOSIS OF DISEASES A...N DIFFICULT TO DIFFERENTIATE".

We propose MEDICAL INSTITUTIONS to take part in development of intelligent systems which have no analogues worldwide enabling to carry out reliable differential diagnosis of each disease using mathematic models describing a great number of diseases under study which symptoms are similar.

PROGRAM OF WORKS ON DEVELOPMENT OF INTELLIGENT SYSTEM FOR COMPUTER-AIDED DIFFERENTIAL DIAGNOSIS WITHIN GROUP OF DISEASES A...N DIFFICULT TO DIFFERENTIATE.

1. Analysis of the problem status

Diagnosis is and will be the most important problem of medicine, and the accuracy of diagnosis achieved in certain historical periods determines mainly the state-of-the-art in medical science.

While examining a patient in modern diagnosis centers (anamnesis, physical examination, laboratory and instrument methods data, clinical data, etc.) a very large scope of initial data (more than 300 characteristics being measured mainly using numeral scales) is collected. If each of these characteristics is measured only using the most simple name-scales ("yes-no", "more-less") the quantity of initial data will make 2 bits to 300th power which is substantially higher than the number of elemental particles in all visible Universe.

As the human body is very complicated and it is characterized by practically infinite number of disease symptoms, symptoms and clinic of a disease are greatly influenced by the individual features of a patient and knowledge of specialists is limited, medical diagnosis, nowadays, is not a science but rather an art of a few highly qualified professionals.

After computers appeared and applied mathematics is developed, works related to attempts to formalize the diagnosis process using mathematical models boomed. The results of these works mainly did not come up to expectations and rare successes are connected either with relative simplicity of the problem (to differentiate diseases sufficiently remote from each other in the symptoms space) or with its inadequate simplification. As a result, at best models diagnosing a disease not worse than an average doctor appeared.

Principal difficulties in simulation of "large" systems (to which medical diagnosis systems also belong) made it necessary to look for roundabout ways. One of these ways being developed intensively at present is the creation of expert medical systems. An expert system is a computer system which incorporates formalized knowledge of specialists in a certain concrete subject and is able to take expert decisions within this subject (to solve problems in such a way as a man-expert would do it).

Efficiency of operation of the expert system depends in the first place on quantity and quality of the information available in its knowledge base. This is a weak point of expert systems because (1) knowledge base is formed on the basis of subjective ideas of experts whose knowledge is limited and (2) specialists are not able to formalize their knowledge as clear rules; moreover, many of them are not aware, on the whole , what rules they follow.

After much money has been spent for development of a great number of various medical expert systems and objective analysis of their efficiency has been carried out, it will appear the same as known a priori, from the definition:

- When solving relatively simple problems of differential diagnosis (those which are successfully solved by specialists using non-formalized approaches), accuracy of diagnosis achieved by means of expert systems and an expert will be close and sufficient.

- But when solving the most important and complicated problems of differential diagnosis, namely when differentiating diseases which are similar as to symptoms; prognosing progress of disease (for instance, acute myocardial infarction complications ), long-term consequences of surgical interference depending on selected type of operation; in case of early diagnosis (including the latent period) of chronic diseases with a prognosis unfavorable for the life (oncological diseases, chronic nephrism), etc., accuracy of diagnosis achievable by means of experts systems and expert will be close and substantially insufficient.

2. TAKEN DEFINITIONS AND FORMULATIONS OF DIFFERENTIAL DIAGNOSIS PROBLEMS

Quite a good progress in medical diagnosis and its conversion from intuitive art of a few talented professionals into a strict science with high level of formalization can be achieved only by transfer from the use of subjective diagnosis information provided by experts to objective information concerning dependence of diagnosis on individual characteristics of a patient and symptom values that are generated using methods provided by artificial intelligence.

There is a certain analogy between medical diagnosis and technical diagnosis. Notwithstanding that technical diagnosis problems are substantially less complex, their solution without use of mathematical models on the basis of paradigm adopted in the science at present is considered as not only incorrect but even indecent.

Under artificial intelligence we understand algorithm allowing to construct informative mathematical model of the object (system) under study carrying essential scope of new, untrivial information unknown before about relations between input and output variables of the object under study, on the basis of the table of experimental data describing behavior of the object (system) of any physical nature without additional a priori information about the structure of a model provided by an expert (specialist in this field) using only formalized procedures.

As applied to medical diagnosis, artificial intelligence allows to obtain for each of disease being differentiated a set of differential syndromes inherent only to a certain disease, many of them unknown before.

Under formal differential diagnosis we understand a formalized procedure (including also using computer) enabling to reliably differentiate each disease from the group of diseases similar as to symptoms using appropriate mathematical models.

On the basis of this definition differential diagnosis problems can be as follows:

- differential diagnosis within the group of diseases similar as to symptoms;

- prognosis of disease progress and outcome;

- early diagnosis of diseases dangerous for the life (for instance, cancerous disease in the latent period).

Taking into account the definitions taken the problem of differential diagnosis model construction can be presented as the following mathematical formulation.

GIVEN: Table of experimental data M=XxY (X={Xij}, i=1,m, j=1,n; Y={Yil}, i = 1, m, l=1,k) , each line of which contains information about symptoms values (Xij) and verified diagnosis Yil for the i-th patient. (Here m is a number of lines (patients) in table M, n is a number of columns (symptoms) in table M, K is a number of diseases to be differentiated.)

IT IS NECESSARY: to construct a mathematical model consisting of K of disjunctions of differential syndromes, each disjunction containing differential syndromes of only one of K of diseases to be differentiated on the basis of the table M by means of formalized procedures.

3. SOLUTION METHODS OF FORMAL DIFFERENTIAL DIAGNOSIS PROBLEMS

If a table, each line of which contains symptoms values and verified diagnosis for one patient, has been obtained on the basis of the examination results, and all observed patients belong to a set of diseases difficult to differentiate from each other, being studied, a mathematical model of differential diagnosis can be constructed using a new mathematical simulation method called mosaic portrait.

The essence of the method consists in realization of the following formalized procedures:

a) transformation of initial experimental data table M to table M' using formalized split of range of values of each symptom into subranges ( transfer from continual scales used for measurement of symptoms to discrete ones). In this case a specific code is applied to each subrange of values of each symptom;

b) search of codes combinations which can be found in table M' in lines belonging to one disease and not found in any line with other diseases.

These combinations are interpreted as differential syndromes of a corresponding disease. The mosaic model obtained in such a way consists of K of disjunctions, each containing differential syndromes of one of K of diseases to be differentiated. Number of differential syndromes is very large and most of them are new, untrivial, unknown before in the medical science.

Well-known methods of realization as per paragraph b require complete search for all possible combinations of subranges of values of all symptoms and belong to so called NP-problems. Computer time required for the solution of such problems depends exponentially on the number of input variables.

When the number of symptoms is more than 15 these problems are practically insoluble.

Using the mosaic portrait method, it is possible to solve problems with dimensionality of the order of 1000 input variables for acceptable time period.

4. NEW POSSIBILITIES WHEN USING MOSAIC PORTRAIT METHOD FOR SOLUTION OF MEDICAL PROBLEMS

Use of the mosaic portrait method allows to solve a number of critical problems of medicine solution of which by means of well-known methods caused serious and mostly irresoluble methodical and computing difficulties.

These problems include:

- formal differential diagnosis of diseases similar as to symptoms;

- formal prognosis of disease progress and outcome alternatives;

- formal selection of one case from a set of alternative surgical interferences on the basis of criteria of long-term consequences;

- early (or even in the latent period) diagnosis of chronic diseases with the prognosis dangerous for the life (oncological diseases, etc.);

- preliminary screening based on data of mass preventive examinations;

- construction of informative mathematical models using the table of experimental data resulting from any medical examination.

High self-descriptiveness of mosaic models allows to substantially increase the accuracy of differential diagnosis, to substantially reduce its cost and load on a patient by excluding invasive and low-informative diagnostic procedures, to formalize diagnosis procedure and to realize it by computer.

5. EXAMPLES OF PRACTICAL USE OF NEW METHODS OF DIFFERENTIAL DIAGNOSIS

At present there is a large experience in using the mosaic portrait method for construction of models of differential diagnosis of diseases difficult to differentiate and their practical use for formal diagnosis and disease outcome prognosis.

A prognosis method of myocardial infarction complications at the acute phase has been developed in collaboration with Military Medical Academy (Saint-Petersburg) [1,2].

Differential syndromes typical for each of consequences of myocardial infarction have been obtained on the basis of experimental data containing information about patients who died of infarction complications (cardiogenic shock, perfusion insufficiency, ventricular fibrillation, cardiac rupture) and had infarction without consequences. Namely these syndromes were used for diagnosis. Method of myocardial infarction consequences prognosis was adopted at Military Medical Academy (Saint-Petersburg), 23th hospital (Moscow), 20th hospital and 42nd polyclinic (Saint-Petersburg). 80-88% of prognosis have been verified by clinical data and in case of lethal outcome by results of pathologicoanatomic investigation. As a result of preventive treatment based on prognosis data rate of death of large-nidus infarction reduced by 36.8% and of small-nidus infarction by 45.1%.

Based on the results of this work, a software package for realization of an intelligent system and called "prognosis of myocardial infarction complications at acute phase" was developed.

Initial information about the state of a patient including anamnesis, examination data, electric cardiography data (total 39 indicants) is entered by keystroke in the question-answer mode.

The following problems are solved by this package:

- prognosis of one or several possible myocardial infarction complications according to the information about patient state collected at the first day of his stay in a hospital;

- in case of prognosis of several complications , the probability of realization of each of them;

- recommendations on preventive therapy of a complication (complications) being prognosticated and corresponding symptoms-eliminating therapy taking into account compatibility of medicines and treatments;

- every two days - a new prognosis corrected on the basis of treatment results and recommendations on preventive and symptoms-eliminating therapy corresponding to this prognosis;

- on inquiry, displaying of information concerning differential syndromes on which basis the prognosis was done;

- input of information about the patient, complications prognosis, syndromes, on which basis the prognosis was done, and recommended treatment into the data base.

A simple (without gastroscopy) and express method of differential diagnosis "gastric ulcer - gastric cancer" with accuracy of 96.4 % has been developed and put into practice in collaboration with Military and Medical Academy.

Based on the results of this work, a software package for realization of an intelligent system and called "DIFFERENTIAL DIAGNOSIS GASTRIC ULCER - GASTRIC CANCER" was developed.

Method of differential diagnosis of various pathogenesis ("shock lung", aspiration, atelectasis, toxic-septic, hypostatic and bronchial pneumonia ) for burnt patients which ensures early (1-2 days of progress) diagnosis, allows to differentiate the therapy and increase the efficiency of treatment was developed in cooperation with Kharkov Burn Centre [3,4].

On the basis of the results of this work, a software package for realization of intelligent system and called "DIFFERENTIAL DIAGNOSIS of pathogenesis of PNEUMONIA FOR burnT patients" was developed.

Reliable and express methods of differential diagnosis "2nd phase of hypertensive disease - chronic diffuse glomerulonephritis with hypertension" and chronic glomerulo-pyelonephritis" was developed and put into practice in collaboration with Military Medical Academy on the basis of biomicroscopy of bulbar conjunctiva.

6. MAIN TASKS AND FINAL GOAL OF THE WORK

The main task of the work is the creation of an efficient intelligent system having no analogues in the world practice, realized at computer and enabling to carry out correct computer-aided diagnosis within the group of diseases difficult to differentiate using formalized procedures.

The depth of the medical diagnosis system created will be constantly increasing: diagnosis accuracy will become higher due to feedback - model after-education based on reliably verified errors of formal diagnosis.

After development and patenting the intelligent differential diagnosis system will be represented as a final commercial product being PC software package.

As practically all EXPERT SYSTEMS AND INTELLIGENT SYSTEMS well-known as on today, allowing to obtain knowledge using the mosaic portrait method use the same language of algebra of logic, according to which any hypothesis is formulated as proposition "if..... then..." and the expert systems accumulate (or must do it) all well-known knowledge in this field, then in case of expert and intelligent systems created for the same field their intersection (common hypotheses) is an a priori known, trivial information; logic difference between propositions of expert and intelligent systems - misinformation (false information); logic difference between propositions of intelligent and expert systems - new, untrivial information unknown for the specialists in this field.

There is just the reason why intelligent medical systems can replace expert systems in the intelligent medical products market.

7. Program of works

Formal differential diagnosis models can be developed in collaboration with any medical institution (research institute, university, diagnostic center, hospital, etc.):

- specialized in studying (treating) of certain diseases;

- having qualified specialists and up-to-date diagnosis equipment for correct diagnosis verification;

- having archival materials or possibility to examine not less than 60 patient as to each disease of the group of diseases to be differentiated during acceptable time period (up to one year).

7.1 More precise definition of the problem. Required experimental data collection

7.1.1 Finalizing of the list of nosologic units to be differentiated.

7.1.2 Making of the list of input variables which can be used potentially to solve the problem. (This list must be surplus i.e. contain not only well-known variables selection of which is substantiated by positive experience of differential diagnosis of diseases being studied, but also other variables which are included on the basis of intuitive ideas of specialists.

7.1.3 Development of formalized history - unified form containing list of symptoms determined as per paragraph 7.1.2.

7.1.4 Determining of minimum required and sufficient scope of experimental studies to solve the problem of differential diagnosis and choose optimum treatment taking into account individual features of a patient.

7.1.5 Purposeful examination of patients suffering from diseases as per paragraph7.1.1; organizing of correct verification of diagnosis within the group of diseases under study; recording of results of examination using the unified form (see paragraph 7.1.3).

7.1.6 Collection of experimental data is finished by a table each line of which contains information about symptoms and verified diagnosis for one patient.

7.2 DEVELOPMENT OF INTELLIGENT COMPUTER SYSTEM PROPER CONSISTS OF THE FOLLOWING STAGES:

7.2.1 Construction of mathematical models of differential diagnosis on the basis of table of experimental data (see paragraph 7.1.6).

7.2.2 Minimization of the models as per paragraph 7.2.1.

7.2.3 Evaluation of adequacy of differential diagnosis models using new experimental data.

7.2.4 Repetition of paragraphs 7.2.1 and 7.2.2 using experimental data including the initial table as per paragraph 7.1.6 and the table used to evaluate adequacy of the model (paragraph 7.2.3).

7.2.5 Creation of intelligent medical system for development of software package providing the solution of the problem stated.

7.3 Constant after education of the system

As the errors are not excluded in the diagnosis on the basis of initial experimental data used for construction of a model the errors are also possible in case of formal differential diagnosis. It is found out experimentally that percentage of diagnosis errors in the initial data (accuracy of diagnosis achieved in a medical institution whose experimental data was used) and when using the model agree practically. After education of the model on the basis of additional experimental data recording actual errors of formal diagnosis will enable to construct the next version of the model ensuring diagnosis with less percentage of errors than made by specialists.

7.3.1 Development of formalized procedure of self-improvement of the system-after education of the models using reliably verified errors of formal diagnosis.

7.3.2 Collection of information about actual cases of inaccurate differential diagnosis at medical institutions participating in the development of the system.

7.3.3 Correction of the models and software package using information about actual cases of inaccurate diagnosis. (Sequential development of new, more precise versions of the system).

7.4 PATENTING OF INTELLIGENT MEDICAL SYSTEM

The models obtained by mosaic portrait methods contain a great number of new, unknown in the medical science differential syndromes for each of diseases to be differentiated which will allow to patent the corresponding system.

8. COMMERCIALIZATION OF THE SYSTEM

Arrangement of advertising campaign:

- development of demonstration disks;

- publications in medical journals, papers and presentations of demonstration versions at scientific conferences, delivery of advertising materials to practical medicine institutions, etc..

8.2 Commercialization of the system

References:

1. G.M.Yakovlev, V.N.Ardashev, M.D.Kats, T.A.Galkina. Mosaic portrait method in the myocardial infarction prognostication. Cardiology, 1981, No.6

2. Prognosis of outcome and complications of acute myocardial infarction (edited by V.P.Malygina). Moscow, Voyenizdat, 1987, p.128.

3. L.M.Tsogoeva, D.E.Pekarsky, S.F.Kudrya, M.D.Kats. Mosaic portrait method in differential diagnosis of pneumonia for burned patients. Clinic surgery, 1991, No.3.

4. L.M.Tsogoeva. Differential diagnosis and peculiarities of treatment of various forms of pneumonia for burned patients. Abstract of candidate thesis, Kharkov, 1991.

5. V.S.Zaitsev. Microcirculation state and rheologic properties of blood in case of hypertensive disease, chronic glomerulo- and pyelonephritis. Abstract of candidate thesis. Leningrad, 1984.

Information supplied by the Author December 1998.  Page last updated: January 19, 2004

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