Serology

Serology Principles and Interpretation in Infectious diseases Dr.T.V.Rao MD : Serology Principles and Interpretation in Infectious diseases Dr.T.V.Rao MD

Beginning of Serology : Beginning of Serology Serology as a science began in 1901. Austrian American immunologist Karl Landsteiner (1868-1943) identified groups of red blood cells as A, B, and O. From that discovery came the recognition that cells of all types, including blood cells, cells of the body, and microorganisms carry proteins and other molecules on their surface that are recognized by cells of the immune system.

Karl Landsteinar (1868-1943) : Karl Landsteinar (1868-1943) An Austrian physician by training, Landsteiner played an integral part in the identification of blood groups. He demonstrated the catastrophic effect of transfusing with the wrong type of blood,

Specific antibodies establishes Blood grouping system : Specific antibodies establishes Blood grouping system

Purpose of Serological Tests : Purpose of Serological Tests Serological tests may be performed for diagnostic purposes when an infection is suspected, in rheumatic illnesses, and in many other situations, such as checking an individual's blood type. Serology blood tests help to diagnose patients with certain immune deficiencies associated with the lack of antibodies, such as X-linked agammaglobulinemia.

Serology : Serology The branch of laboratory medicine that studies blood serum for evidence of infection and other parameters by evaluating antigen-antibody reactions in vitro

Serology : Serology Serology is the scientific study of blood serum. In practice, the term usually refers to the diagnostic identification of antibodies in the serum We can detect antigens too

Serology prerogative of Microbiology : Serology prerogative of Microbiology It is rather curious that, although serum for a multitude of constituents in biochemistry and haematological laboratories, the term serology has come to imply almost exclusively the detection of antibodies in serum for antibodies in infectious diseases, and terminology has become prerogative of microbiologists.

Immunology/ Serology? Precipitation Reactions : Immunology/ Serology? Precipitation Reactions Capillary tube precipitation (Ring Test) Ouchterlony Double Diffusion (Immunodiffusion) Radialimmunodiffusion (RID) Immunoelectrophoresis (IEP) Rocket Electroimmunodiffusion (EID) Counterimmunoelectrophoresis (CIEP) The above tests have moved to Biochemistry

Terms used in evaluating test methodology : Terms used in evaluating test methodology Sensitivity Analytical Sensitivity – ability of a test to detect very small amounts of a substance Clinical Sensitivity – ability of test to give positive result if patient has the disease (no false negative results)

Specificity : Specificity Analytical Specificity – ability of test to detect substance without interference from cross-reacting substances Clinical Specificity – ability of test to give negative result if patient does not have disease (no false positive results)

Affinity : Affinity Affinity refers to the strength of binding between a single antigenic determinant and an individual antibody combining site. Affinity is the equilibrium constant that describes the antigen-antibody reaction

Affinity : Affinity Antibody affinity is the strength of the reaction between a single antigenic determinant and a single combining site on the antibody. It is the sum of the attractive and repulsive forces operating between the antigenic determinant and the combining site .

Avidity : Avidity Avidity is a measure of the overall strength of binding of an antigen with many antigenic determinants and multivalent antibodies Avidity is influenced by both the valence of the antibody and the valence of the antigen. Avidity is more than the sum of the individual affinities.

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Dilution : Dilution Estimating the antibody by determining the greatest degree to which the serum may be diluted without losing the power to given an observable effect in a mixture with specific antigen

Titer : Titer Different dilutions of serum are tested in mixture with a constant amount of antigen and greatest reacting dilution is taken as the measure or Titer

Expression of Titers : Expression of Titers Expressed in term of the was in which they are made Dilution 1 in 8 is a dilution made by mixing one volume of serum with seven volumes of diluents (Normal Saline ) Incorrect to express dilution as 1/8

Common methods in creating dilutions : Common methods in creating dilutions

Sero Conversion : Sero Conversion Seroconversion is the development of detectable specific antibodies to microorganisms in the blood serum as a result of infection or immunization.

Sero reversion : Sero reversion Seroreversion is the opposite of seroconversion. This is when the tests can no longer detect antibodies or antigens in a patient’s serum

Testing paired Samples : Testing paired Samples Testing for infectious diseases is performed on acute and convalescent specimens (about 2 weeks apart) Paired sample. Must see 4-fold or 2-tube rise in titre to be clinically significant

Majority Diagnostic tests are Serological tests : Majority Diagnostic tests are Serological tests There are several serology techniques that can be used depending on the antibodies being studied. These include: ELISA, agglutination, precipitation, complement-fixation, and fluorescent antibodies.

Antigen and Antibody reactions can be identified by different methods : Antigen and Antibody reactions can be identified by different methods

Precipitation : Precipitation Principle Soluble antigen + antibody (in proper proportions) –> visible precipitate Lattice formation (antigen binds with Fab sites of 2 antibodies) Examples Double diffusion (Ouchterlony) Single diffusion (radial immunodiffusion) Imunoelectrphoresis Immunofixation

Agglutination : Agglutination Principle Particulate antigen + antibody –> clumping Lattice formation (antigen binds with Fab sites of 2 antibodies forming bridges between antigens) Examples Direct agglutination (Blood Bank) Passive Hemagglutination (treat RBC's with tannic acid to allow adsorption of protein antigens) Passive latex agglutination (antigen attached to latex particle)

Neutralization reactions : Neutralization reactions Similar in principle and interpretation of results Antibody-binding Hemagglutination inhibition (serum antibody reacts with known nonparticulate antigen –> binding occurs) Neutralization (antibody neutralizes toxin) After binding, antibody is not available to react in indicator system Results: NO agglutination or NO hemolysis = positive reaction Agglutination or hemolysis = negative reaction (antibody not bound in origin

Neutralization reactions : Generally, positive control samples used in inhibition or neutralization tests show no reaction and negative control samples show a reaction (opposite of results in direct agglutination testing) Example of inhibition: Hemagglutination inhibition test for rubella Example of neutralization: antistreptolysin O test (ASO) Neutralization reactions

Complement fixation (CF) : Complement fixation (CF) Antibody and antigen allowed to combine in presence of complement If complement is fixed by specific antigen-antibody reaction, it will be unable to combine with indicator system Precautions Serum must be heat-activated Stored serum becomes anti-complementary Extensive QC/standardization required Only use for IgM antibodies

Imunoelectrphoresis (IEP)Qualitative : Imunoelectrphoresis (IEP)Qualitative A serum sample is electrophoresed through an agar medium. A trough is cut in the agar and filled with Ab. A precipitin arc is then formed. Because Ag diffuses radially and Ab from a trough diffuses, the reactants meet in optimal proportions for precipitation.

Serology can be done on various speciemns : Serology can be done on various speciemns Some serological tests are not limited to blood serum, but can also be performed on other bodily fluids such as semen and saliva, which have (roughly) similar properties to serum. Serological tests may also be used forensically, generally to link a perpetrator to a piece of evidence (e.g., linking a rapist to a semen sample).

Enzyme immunoassay (EIA/ELISA) : Enzyme immunoassay (EIA/ELISA) Sandwich technique” Monoclonal or polyclonal antibody adsorbed on solid surface (bead or microtiter plate) Add patient serum; if antigen is present in serum, it binds to antibody coated bead or plate Add excess labelled antibody (antibody conjugate); forms antigen-antibody-labelled antibody “sandwich” (antibody in conjugate is directed against another epitope of antigen being tested) Add substrate, incubate, and read absorbance Washing required between each step Absorbance is directly proportional to antigen concentration

ELISA methods takes over : ELISA methods takes over Enzyme-linked immunosorbent assay, also called ELISA, enzyme immunoassay or EIA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample

ELISA Most popular technological advance in Laboratory Medicine : ELISA Most popular technological advance in Laboratory Medicine ELISA methods can detect any infectious disease provided if we have antibodies and antigen to any infection, enzyme or any substance

Serology applications in.. : Serology applications in.. HIV testing Serum HCG (pregnancy) Tests for hepatitis antigens and antibodies Antibodies to bacteria Hepatitis Serology

Nephelometry : Nephelometry Procedure Serum substance reacts with specific antisera and forms insoluble complexes Light is passed through suspension Scattered (reflected) light is proportional to number of insoluble complexes; compare to standards Examples Complement component concentration Antibody concentration (IgG, IgM, IgA, etc.) Immunofluorescence

Immunofluorescence : Immunofluorescence Direct – add fluorescein-labeled antibody to patient tissue, wash, and examine under fluorescent microscope Indirect – add patient serum to tissue containing known antigen, wash, add labeled antiglobulin, wash, and examine under fluorescent microscope Examples Testing for Antinuclear Antibodies (ANA) Fluorescent Treponemal Antibody Test (FTA-Abs)

Fluorescence polarization immunoassay (FPIA) : Fluorescence polarization immunoassay (FPIA) Principle Add reagent antibody and fluorescent-tagged antigen to patient serum Positive test Antigen present in patient serum binds to reagent leaving most tagged antigen unbound Unbound labeled antigens rotate quickly reducing amount of polarized light produced Negative test If no antigen present in patient serum, tagged antigen binds to reagent antibody Tagged antigen-antibody complexes rotate slowly giving off increased polarized light

Flow cytometry : Flow cytometry Method of choice for T- and B-cell analysis (lymphocyte phenotyping) Principle Incubate specimen with 1 or 2 monoclonal antibodies tagged with fluorochrome Single cells pass through incident light of instrument (laser) which excites fluorochrome and results in emitted light of different wavelength Intensity of fluorescence measured to detect cells possessing surface markers for the specific monoclonal antibodies that were employed Forward light scatter indicates cell size or volume 90° side-scattered light indicates granula

Common uses Flow cytometry : Common uses Flow cytometry DNA analysis Reticulocyte counts Leukaemia/lymphoma classification CD 4 cell estimations in AIDS/HIV patients.

Other Applications of agglutination tests in Serology : Other Applications of agglutination tests in Serology i. Determination of blood types or antibodies to blood group antigens. ii. To assess bacterial infections e.g. A rise in titer of an antibody to a particular bacterium indicates an infection with that bacterial type. N.B. a fourfold rise in titer is generally taken as a significant rise in antibody titer.

Georges-Fernand-Isidor Widal : Georges-Fernand-Isidor Widal Widal in 1896, and Widal & Sicard in 1896 described the Widal reaction, and this test has proved of value in cases where positive cultures have been unobtainable

Widal test a Popular test in diagnosis of Typhoid Fever : Widal test a Popular test in diagnosis of Typhoid Fever The Widal test is a presumptive serological test for Enteric fever or Undulant fever. In case of Salmonella infections, it is a demonstration of agglutinating antibodies against antigens O-somatic and H-flagellar in the blood.

Widal test is century old ,Is it loosing importance ? : Widal test is century old ,Is it loosing importance ? In this reaction antibodies react with antigens on the surface of particulate objects and cause the objects to clump together, or agglutinate. These reactions were the earliest to be adapted to diagnostic laboratory. Widal test is used for diagnosis of typhoid fever. This test, developed by Georges Fernand I. Widal (French physician) in 1896, is now supplemented by more sophisticated procedures.

Widal test – A standard tube agglutination test : Widal test – A standard tube agglutination test Test can be performed by the tube dilution technique which permits, the assay of antibody titre. In this, a constant amount of the antigen is added to a  series of tubes containing serum dilutions. After mixing, the tubes are incubated at a particular temperature and the highest dilution of serum showing visible agglutination is determined.

Agglutination how it appear after reactivity : Agglutination how it appear after reactivity O agglutination is granular H agglutination is loose and floccular

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Principle of the Test : Principle of the Test A classic example of the agglutination reaction is seen in the widal test for diagnosis of typhoid fever. In this test the antibody content of the patient's serum, is measured by adding a constant amount of antigen (Salmonella typhi) to the serially diluted serum.

Reading the Widal Test : Reading the Widal Test Read the results by viewing the tubes under good light against the dark background with x2 magnifying lens Do not shake tubes before reading the results Read titers as greatest dilutions giving visible agglutinations. Limiting agglutination is 1in 200 the titer is 200 not to be reported as 1/200.

Interpretation of Widal test : Interpretation of Widal test Test results need to be interpreted carefully in the light of past history of enteric fever, typhoid vaccination, general level of antibodies in the populations in endemic areas of the world.

Testing in Typhoid carriers : Testing in Typhoid carriers Many known carriers of typhoid bacilli possess antibody against the Vi (virulence) antigen of S. typhi. This is a surface antigen easily lost during cultivation(Vi tires seem to correlate better with the carrier state than do O or H titres). For this reason, Felix et al. suggested the use of Vi agglutination for detection of carriers.

Importance of Vi antibodies : Importance of Vi antibodies Many known carriers of typhoid bacilli possess antibody against the Vi (virulence) antigen of S. typhi. This is a surface antigen easily lost during cultivation(Vi tires seem to correlate better with the carrier state than do O or H titres). For this reason, Felix et al. suggested the use of Vi agglutination for detection of carriers.

Prozone phenomenon in Agglutination tests : Prozone phenomenon in Agglutination tests Prozone effect - Occasionally, it is observed that when the concentration of antibody is high (i.e. lower dilutions), there is no agglutination and then, as the sample is diluted, agglutination occurs. The lack of agglutination at high concentrations of antibodies is called the prozone effect. Lack of agglutination in the prozone is due to antibody excess resulting in very small complexes that do not clump to form visible agglutination

Causes Of False-positive Widal Agglutination Tests : Causes Of False-positive Widal Agglutination Tests Previous immunization with Salmonella antigen. Cross-reaction with non – typhoidal Salmonella. Variability and poorly standardized commercial antigen preparation. Infection with malaria other Enterobacteriaceae charring the same s-LPS .

Causes of Negative Widal Agglutination Test : Causes of Negative Widal Agglutination Test The carrier state An inadequate inoculum of bacterial antigen in the host to induce antibody production Technical difficulty or errors in the performance of the test. Previous antibiotic treatment Variability in the preparation of commercial antigens.

Declining importance of Widal test : Declining importance of Widal test The value of the salmonella agglutination tests has declined as the incidence of typhoid fever has decreased, at least in the developed world, the general use of vaccines has increased, and ever increasing -numbers of antigenically related serotypes of Salmonella have been recognised.

Serology - Importance of repeated tests : Serology - Importance of repeated tests Criteria for diagnosing Primary Infection 4 fold or more increase in titre of IgG or total antibody between acute and convalescent sera Presence of IgM Seroconversion A single high titre of IgG (or total antibody) - very unreliable Criteria for diagnosing Reinfection Four fold or more increase in titre of IgG or total antibody between acute and convalescent sera Absence or slight increase in IgM

Typical Serological Profile After Acute Infection : Typical Serological Profile After Acute Infection Note that during Reinfections, IgM may be absent or present at a low level transiently

Antigen – Antibody reactions presenting with precipitation : Antigen – Antibody reactions presenting with precipitation

Precipitation Curve : Precipitation Curve

Precipitation Curve : Precipitation Curve

Measurement of Precipitation by Light : Measurement of Precipitation by Light Antigen-antibody complexes, when formed at a high rate, will precipitate out of a solution resulting in a turbid or cloudy appearance. Turbidimetry measures the turbidity or cloudiness of a solution by measuring amount of light directly passing through a solution. Nephelometry indirect measurement, measures amount of light scattered by the antigen-antibody complexes.

Screening Tests for Syphilis : Screening Tests for Syphilis Serologic methods are divided into two classes. One class, the nontreponemal tests, detects antibodies to lipoidal antigens present in either the host or T. pallidum; examples are the Venereal Disease Research Laboratory and rapid plasma reagin and tests.

Serological Diagnosis Of Syphilis : Serological Diagnosis Of Syphilis Specific Anti- treponemal Antibody Anti – treponemal Antibody Reagin Antibody (VDRL and RPR) Associated with higher false positives

Indication for testing for Syphilis : Indication for testing for Syphilis Pregnant women sexual contacts or partners of patients diagnosed with syphilis children born to mothers with syphilis patients with HIV infection

Tests For Reagin Antibody : Tests For Reagin Antibody A large numbers of tests for Reagin: VDRL (Venereal Diseases Reference Laboratory). RPR (Rapid Plasma Reagin) ART (Automated Reagin Test) Good sensitive screening Titre falls rapidly with treatment Reagin titre falls with treatment.

VDRL – A standard test for Syphilis : VDRL – A standard test for Syphilis NONTREPONEMAL ANTIGEN TESTS. Nontreponemal antigen tests are used as screeners. They measure the presence of reagin, which is an antibody formed in reaction to syphilis. In the venereal disease research laboratory (VDRL) test, a sample of the patient's blood is mixed with cardiolipin and cholesterol. If the mixture forms clumps or masses of matter, the test is considered reactive or positive. The serum sample can be diluted several times to determine the concentration of reagin in the patient's blood.

Screening tests should be reported with cautions : Screening tests should be reported with cautions Reactivity in these tests generally indicates host tissue damage that may not be specific for syphilis. Because these tests are easy and inexpensive to perform, they are commonly used for screening, and with proper clinical signs they are suggestive of syphilis. The other class of test, the Treponemal tests, uses specific Treponemal antigens.

Combination of testes are desirable : Combination of testes are desirable Syphilis serodiagnosis relies on a combination of nonspecific screening tests (antilipoidal antibodies) and Treponema pallidum-specific tests (anti-T. pallidum antibodies).

Measurement of Precipitation by Light : Measurement of Precipitation by Light Antigen-antibody complexes, when formed at a high rate, will precipitate out of a solution resulting in a turbid or cloudy appearance. Turbidimetry measures the turbidity or cloudiness of a solution by measuring amount of light directly passing through a solution. Nephelometry indirect measurement, measures amount of light scattered by the antigen-antibody complexes.

Confirmation is warranted : Confirmation is warranted Confirmation of infection requires a reactive Treponemal test. Examples of the Treponemal tests are the microhemagglutination assay for antibodies to T. pallidum and the fluorescent treponemal antibody absorption test. These tests are more expensive and complicated to perform than the nontreponemal tests. On the horizon are a number of direct antigen, enzyme-linked immunosorbent assay, and PCR technique

Non reactive and Reactive VDRL Tests : Non reactive and Reactive VDRL Tests

Rapid plasma reagin : Rapid plasma reagin The rapid plasma reagin (RPR) test works on the same principle as the VDRL. It is available as a kit. The patient's serum is mixed with cardiolipin on a plastic-coated card that can be examined with the naked eye.

Agglutination++  RPR       Agglutination+ : Agglutination++  RPR       Agglutination+

Agglutination+: ve RPR Agglutination : - : Agglutination+: ve RPR Agglutination : -

Biological false positives : Biological false positives Biological False Positive Antibody (BFP) Reagin Antibody: associated with other diseases (BFP) Acute: Pneumonia Vaccination with live attenuated viruses. Malaria Pregnancy Chronic: Leprosy – the only infection Reagin titer falls rapidly with treatment

Serological Diagnosis Of Syphilis : Serological Diagnosis Of Syphilis Test for specific Anti - treponemal Antibody Absorbed fluorescent treponemal antibody (FTA - ABs) Treponema Pallidum Immobilization Test (TPI) Most sensitive Utilize living Treponema maintained by passage in rabbits testes. Expensive Potentially hazardous. Not done in the present contest as Technically demanding

Doing a quantization test RPR : Doing a quantization test RPR

Other Serological Methods in Diagnosis Of Syphilis : Other Serological Methods in Diagnosis Of Syphilis Treponema pallidum haemagglutination (TPHA) test. Sheep, chicken or turkey RBCs. Sensitized by attaching killed Treponema pallidum. Agglutinate by presence of antibody Less sensitive than FTA – Abs Less reliable in the diagnosis of primary syphilis. Sometimes false positive

Treponema Palladium Hemagglutination testTPHA : Treponema Palladium Hemagglutination testTPHA

Other Serological Tests for Syphilis : Other Serological Tests for Syphilis Anti – Treponemal Antibody Anti-Treponemal ABs group detected by Reiter Protein Complement Fixation Test (RPCFT) Appears later than specific ABs Some syphilis patient do not produce the form of ABs Used is limited.

Detection by FTA-ABS IgG and IgM : Detection by FTA-ABS IgG and IgM In the FTA-ABS tests, the patient's blood serum is mixed with a preparation that prevents interference from antibodies to other treponemal infections.

FTA abs IgG and IgM detection continues to be a confirmatory test in diagnosis of Syphilis : FTA abs IgG and IgM detection continues to be a confirmatory test in diagnosis of Syphilis The test serum is added to a slide containing T. pallidum. In a positive reaction, syphilitic antibodies in the blood coat the spirochetes on the slide. The slide is then stained with fluorescein, which causes the coated spirochetes to fluoresce when the slide is viewed under ultraviolet (UV) light..

Principle of Fluorescent Method : Principle of Fluorescent Method

Active Treponema Pallidum Infection : Active Treponema Pallidum Infection Positive Specific Tests e.g. TPHA Positive ( =1/ 8) of non-specific test (VDRL) TPI-T (Treponema Pallidum Immobilization Test) FTA –T (Fluorescent Treponema Test) Sometimes needed for confirmation.

Emergin Methods in Diagnosis of Syphilis : Emergin Methods in Diagnosis of Syphilis Currently, ELISA, Western blot, and PCR testing are being studied as additional diagnostic tests, particularly for congenital syphilis and neurosyphilis.

SPINAL FLUID TESTS in Syphilis. : SPINAL FLUID TESTS in Syphilis. . Testing of cerebrospinal fluid (CSF) is an important part of patient monitoring as well as a diagnostic test. The VDRL and FTA-ABS tests can be performed on CSF as well as on blood. An abnormally high white cell count and elevated protein levels in the CSF, together with positive VDRL results, suggest a possible diagnosis of neurosyphilis.

CSF testing is indicated only in… : CSF testing is indicated only in… CSF testing is not used for routine screening. It is used most frequently for infants with congenital syphilis, HIV-positive patients, and patients of any age who are not responding to penicillin treatment.

Biological false reactive VDRL test among the HIV infected patients : Biological false reactive VDRL test among the HIV infected patients Fewer reports on the biological false positive VDRL in HIV individuals are documented. In this work, the author studied the rate of biological false reactive VDRL among the HIV-infected patients. Of interest, in this study, the rate is significantly lower (by Fishers exact test) than a recent previous report among prostitutes in India (10/94, about 10.6 %). In the general population, the biological false positive VDRL generally returns to negative within 14 weeks, without other clinical significance. VirojWiwanitkit

Rickettsiae and Serology : Rickettsiae and Serology Rickettsiae is a genus of motile, Gram-negative, non-spore forming, highly pleomorphic bacteria that can present as cocci (0.1 µm in diameter), rods (1–4 µm long) or thread-like (10 µm long). Obligate intracellular parasites Because of this, Rickettsiae cannot live in artificial nutrient environments and are grown either in tissue or embryo cultures (typically, chicken embryos are used). Still we have to dependent on Weil Felix test

Weil and Felix contribute for testing : Weil and Felix contribute for testing In 1915, Weil and Felix showed that serum of patients infected with any member of the typhus group of diseases contains agglutinins for one or more strains of O X Proteus. In cases of typhus fever the reaction usually appears before the sixth day and reaches its height in the second week.

Weil-Felix reaction – A Heterophile agglutination Test : Weil-Felix reaction – A Heterophile agglutination Test A Weil-Felix reaction is a type of agglutination test in which patients serum is tested for agglutinins to O antigen of certain non-motile Proteus and rickettsial strains(OX19, OX2, OXk) OX19, OX2 are strains of Proteus vulgaris.OXk is the strain of Proteus mirabilis.

Weil-Felix a Heterophile agglutination test : Weil-Felix a Heterophile agglutination test The agglutination reactions, based on antigens common to both organisms, determine the presence and type of rickettsial infection Because Rickettsiae are both fastidious and hazardous, few laboratories undertake their isolation and diagnostic identification Weil-Felix test that is based on the cross-reactive antigens of OX-19 and OX-2 strains of Proteus vulgaris.

Interpretations in Weil-Felix reaction : Interpretations in Weil-Felix reaction Sera from endemic typhus agglutinate OX19, OX2.Tick borne spotted fever agglutinate OX19, OX2. Scrub Typhus agglutinate OXk strain Test is negative in rickettsialpox, trench fever and Q-fever.False positive reaction may occur in urinary or other Proteus infectionsTest may be negative in 50 percent scrub typhus

Weil-Felix testindicated in when patients present with rashes : Weil-Felix testindicated in when patients present with rashes Test for diagnosis of typhus and certain other rickettsial diseases. The blood serum of a patient with suspected rickettsial disease is tested against certain strains of (OX-2, OX-19, OX-K)..

Weil Felix test and Concentration Camps : Weil Felix test and Concentration Camps

Weil-Felix test positivity saves from Nazis : Weil-Felix test positivity saves from Nazis In Poland, during World War II, where a pair of quick-thinking doctors used a little-known organism to keep the Nazis at bay.The microorganisms is Proteus OX19. . Its one remarkable feature is that human antibodies for Proteus OX19 cross-react with the antibodies for Ricksettia – the bacterium responsible for the deadly disease typhus. Blood from a patient infected with Proteus Ox19 will give a false-positive in the most common typhus screening method, the Weil-Felix test.

How they made Weil-Felix test Positive : How they made Weil-Felix test Positive While the Polish doctors could, and did, inject a number of other people with Proteus to induce positive Weil-Felix results, an on-site Nazi medical team could well have proved their undoing. Fortunately, ingenuity and a good dose of hospitality and alcohol prevented them from being uncovered. ( From the British Medical Journal )

Other Emerging Serological Tests : Other Emerging Serological Tests

Co-agglutination : Co-agglutination Co agglutination is similar to the latex agglutination technique for detecting antigen (described above). Protein A, a uniformly distributed cell wall component of Staphylococcus aureus, is able to bind to the Fc region of most IgG isotype antibodies leaving the Fab region free to interact with antigens present in the applied specimens. The visible agglutination of the S. Aureus particles indicates the antigen-antibody reactions

Co agglutination Test : Co agglutination Test Agglutination test in which inert particles (latex beads or heat-killed S aureus Cowan 1 strain with protein A) are coated with antibody to any of a variety of antigens and then used to detect the antigen in specimens or in isolated bacteria.

Chemiluminescence : Chemiluminescence Chemiluminescence is the emission of light with limited emission of heat (luminescence), as the result of a chemical reaction.

Chemiluminescent Immunoenzymatic Assay : Chemiluminescent Immunoenzymatic Assay Process for the quantitative and qualitative determination of antigens, antibodies and their complexes by means of a chemiluminescing labelling substance activated or excited to chemiluminescence's by an analytical reagent. By means of a serological reaction, initially an antigen/antibody complex is formed which is treated with a chemiluminescing conjugate containing chemiluminescing triphenylmethane dyes and the chemiluminescence of the chemiluminescing complex formed is measured.

Recent testing Advances : Recent testing Advances The ToRC IgG kit simultaneously detects IgG class antibodies to Toxoplasmosis gondii, rubella and cytomegalovirus (CMV). The HSV-1 and HSV-2 IgG kit utilises type-specific proteins to simultaneously detect and differentiate IgG class antibodies to the two most common herpes subtypes, HSV-1 and HSV-2.

False Positive Serological Tests : False Positive Serological Tests Cross reacting antibody Cross reactivation of latent organism (Influenza Virus A infection activate CMV IgM – production Presence of Rheumatoid factors RF = IgM RF + IgG = Complexed = False positive organism-specific IgM Antibody

False Negative Serologic Test : False Negative Serologic Test Immune system not intact Delay in Antibody response (Lyme disease - Legionnaire’s Disease) Competition for Antigen binding site of antibody) IgM binds to the Antigen IgG site IgG binds to the Antigen IgM site Prozone Phenomena

Usefulness of Serological Results : Usefulness of Serological Results How useful a serological result is depends on the individual virus. For example, 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 titers of IgG in the serum of the patient would indicate active disease.

Rota Virus - wether serology useful ? : Rota Virus - wether serology useful ? However, many viruses often produce clinical disease before the appearance of antibodies such as respiratory and diarrheal viruses. So in this case, any serological diagnosis would be retrospective and therefore will not be that useful. Acute presence of Antigen is much useful in Diagnosis

Antibody detection is definitive Diagnosis : Antibody detection is definitive Diagnosis 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.

Problems with Serology : Problems with Serology Long period of time required for diagnosis for paired acute and convalescent sera. Mild local infections such as HSV genitalis may not produce a detectable humoral immune response. Extensive antigenic cross-reactivity between related viruses e.g. HSV and VZV, Japanese B encephalitis and Dengue, may lead to false positive results.

Problems with SerologyOther Health condtions interfere : Problems with SerologyOther Health condtions interfere Immunocompromised patients often give a reduced or absent humoral immune response. Patients with infectious mononucleosis and those with connective tissue diseases such as SLE may react non-specifically giving a false positive result. Patients given blood or blood products may give a false positive result due to the transfer of antibody

Automations : Automations One of the first successful attempts to automate antibody tests was made by Weitz (1967) at the Lister Institute, London. The apparatus developed by Weitz (Fig. 3) allowed the performance of up to 12 titrations in a single operation, with even less manipulation than that required for a single test done by a more conventional technique.

Created as part of Dr.T.V.Rao MD’s ‘e’ learning Programme : Created as part of Dr.T.V.Rao MD’s ‘e’ learning Programme Email doctortvrao@gmail.com