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Serology is a division of Diagnostic Microbiology several diseases can be effectively diagnosed with use of Serological procedrues.

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  • Edmund Weil attended the German University of Prague. He obtained his doctorate in 1903 and then worked as 2nd assistant under the pathological anatomist Hans Chiari (1851-1916). In 1905 he entered the hygienical institute, at which he assumed leadership of the serological department in 1918. He was habilitated for hygiene in 1909, becoming ausserordentlicher professor in 1914. He died in Prague in 1922 from an infection of spotted fever he had attracted in a laboratory in Poland . n 1911, with the neuropsychologist Viktor Kafka, Weil described a previously unknown bacteriological reaction, now known as Weil-Kafka reaction. At the time, Kafka was an assistant in the psychiatric clinic of the German faculty in Prague.
  • Serology

    1. 1. Serology Principles and Interpretation in Infectious diseases Dr.T.V.Rao MD
    2. 2. Beginning of Serology <ul><li>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 . </li></ul>
    3. 3. Karl Landsteinar (1868-1943 ) <ul><li>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, </li></ul>
    4. 4. Specific antibodies establishes Blood grouping system
    5. 5. Purpose of Serological Tests <ul><li>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. </li></ul>
    6. 6. Serology <ul><li>The branch of laboratory medicine that studies blood serum for evidence of infection and other parameters by evaluating antigen-antibody reactions in vitro </li></ul>
    7. 7. Serology <ul><li>Serology is the scientific study of blood serum. In practice, the term usually refers to the diagnostic identification of antibodies in the serum </li></ul><ul><li>We can detect antigens too </li></ul>
    8. 8. Serology prerogative of Microbiology <ul><li>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. </li></ul>
    9. 9. Immunology/ Serology ? Precipitation Reactions <ul><li>Capillary tube precipitation (Ring Test) </li></ul><ul><li>Ouchterlony Double Diffusion (Immunodiffusion) </li></ul><ul><li>Radialimmunodiffusion (RID) </li></ul><ul><li>Immunoelectrophoresis (IEP) </li></ul><ul><li>Rocket Electroimmunodiffusion (EID) </li></ul><ul><li>Counterimmunoelectrophoresis (CIEP) </li></ul><ul><li>The above tests have moved to Biochemistry </li></ul>
    10. 10. Terms used in evaluating test methodology <ul><li>Sensitivity </li></ul><ul><ul><li>Analytical Sensitivity – ability of a test to detect very small amounts of a substance </li></ul></ul><ul><ul><li>Clinical Sensitivity – ability of test to give positive result if patient has the disease (no false negative results) </li></ul></ul>
    11. 11. Specificity <ul><li>Analytical Specificity – ability of test to detect substance without interference from cross-reacting substances </li></ul><ul><li>Clinical Specificity – ability of test to give negative result if patient does not have disease (no false positive results) </li></ul>
    12. 12. Affinity <ul><li>Affinity refers to the strength of binding between a single antigenic determinant and an individual antibody combining site. </li></ul><ul><li>Affinity is the equilibrium constant that describes the antigen-antibody reaction </li></ul>
    13. 13. Affinity <ul><li>Antibody affinity is the strength of the reaction between a single antigenic determinant and a single combining site on the antibody. </li></ul><ul><li>It is the sum of the attractive and repulsive forces operating between the antigenic determinant and the combining site . </li></ul>
    14. 14. Avidity <ul><li>Avidity is a measure of the overall strength of binding of an antigen with many antigenic determinants and multivalent antibodies </li></ul><ul><li>Avidity is influenced by both the valence of the antibody and the valence of the antigen. </li></ul><ul><li>Avidity is more than the sum of the individual affinities. </li></ul>
    15. 16. Dilution <ul><li>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 </li></ul>
    16. 17. Titer <ul><li>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 </li></ul>
    17. 18. Expression of Titers <ul><li>Expressed in term of the was in which they are made </li></ul><ul><li>Dilution 1 in 8 is a dilution made by mixing one volume of serum with seven volumes of diluents (Normal Saline ) </li></ul><ul><li>Incorrect to express dilution as 1/8 </li></ul>
    18. 19. Common methods in creating dilutions
    19. 20. Sero Conversion <ul><li>Seroconversion is the development of detectable specific antibodies to microorganisms in the blood serum as a result of infection or immunization. </li></ul>
    20. 21. Sero reversion <ul><li>S eroreversion is the opposite of seroconversion. This is when the tests can no longer detect antibodies or antigens in a patient’s serum </li></ul>
    21. 22. Testing paired Samples <ul><li>Testing for infectious diseases is performed on acute and convalescent specimens (about 2 weeks apart) Paired sample. </li></ul><ul><li>Must see 4-fold or 2-tube rise in titre to be clinically significant </li></ul>
    22. 23. Majority Diagnostic tests are Serological tests <ul><li>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. </li></ul>
    23. 24. Antigen and Antibody reactions can be identified by different methods
    24. 25. Precipitation <ul><li>Principle </li></ul><ul><ul><li>Soluble antigen + antibody (in proper proportions) –> visible precipitate </li></ul></ul><ul><ul><li>Lattice formation (antigen binds with Fab sites of 2 antibodies) </li></ul></ul><ul><li>Examples </li></ul><ul><ul><li>Double diffusion (Ouchterlony) </li></ul></ul><ul><ul><li>Single diffusion (radial immunodiffusion) </li></ul></ul><ul><ul><li>Imunoelectrphoresis </li></ul></ul><ul><ul><li>Immunofixation </li></ul></ul>
    25. 26. Agglutination <ul><li>Principle </li></ul><ul><ul><li>Particulate antigen + antibody –> clumping </li></ul></ul><ul><ul><li>Lattice formation (antigen binds with Fab sites of 2 antibodies forming bridges between antigens) </li></ul></ul><ul><li>Examples </li></ul><ul><ul><li>Direct agglutination (Blood Bank) </li></ul></ul><ul><ul><li>Passive Hemagglutination (treat RBC's with tannic acid to allow adsorption of protein antigens) </li></ul></ul><ul><ul><li>Passive latex agglutination (antigen attached to latex particle) </li></ul></ul>
    26. 27. Neutralization reactions <ul><li>Similar in principle and interpretation of results </li></ul><ul><li>Antibody-binding </li></ul><ul><li>Hemagglutination inhibition (serum antibody reacts with known nonparticulate antigen –> binding occurs) </li></ul><ul><li>Neutralization (antibody neutralizes toxin) </li></ul><ul><li>After binding, antibody is not available to react in indicator system </li></ul><ul><li>Results: </li></ul><ul><li>NO agglutination or NO hemolysis = positive reaction </li></ul><ul><li>Agglutination or hemolysis = negative reaction (antibody not bound in origin </li></ul>
    27. 28. <ul><li>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) </li></ul><ul><li>Example of inhibition: Hemagglutination inhibition test for rubella </li></ul><ul><li>Example of neutralization: antistreptolysin O test (ASO) </li></ul>Neutralization reactions
    28. 29. Complement fixation (CF) <ul><li>Antibody and antigen allowed to combine in presence of complement </li></ul><ul><li>If complement is fixed by specific antigen-antibody reaction, it will be unable to combine with indicator system </li></ul><ul><li>Precautions </li></ul><ul><li>Serum must be heat-activated </li></ul><ul><li>Stored serum becomes anti-complementary </li></ul><ul><li>Extensive QC/standardization required </li></ul><ul><li>Only use for IgM antibodies </li></ul>
    29. 30. Imunoelectrphoresis (IEP) Qualitative <ul><li>A serum sample is electrophoresed through an agar medium. </li></ul><ul><li>A trough is cut in the agar and filled with Ab. </li></ul><ul><li>A precipitin arc is then formed. </li></ul><ul><li>Because Ag diffuses radially and Ab from a trough diffuses, the reactants meet in optimal proportions for precipitation. </li></ul>
    30. 31. Serology can be done on various speciemns <ul><li>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. </li></ul><ul><li>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). </li></ul>
    31. 32. Enzyme immunoassay (EIA/ELISA ) <ul><li>Sandwich technique” </li></ul><ul><li>Monoclonal or polyclonal antibody adsorbed on solid surface (bead or microtiter plate) </li></ul><ul><li>Add patient serum; if antigen is present in serum, it binds to antibody coated bead or plate </li></ul><ul><li>Add excess labelled antibody (antibody conjugate); forms antigen-antibody-labelled antibody “sandwich” (antibody in conjugate is directed against another epitope of antigen being tested) </li></ul><ul><li>Add substrate, incubate, and read absorbance </li></ul><ul><li>Washing required between each step </li></ul><ul><li>Absorbance is directly proportional to antigen concentration </li></ul>
    32. 33. ELISA methods takes over <ul><li>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 </li></ul><ul><li>Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample </li></ul>
    33. 34. ELISA Most popular technological advance in Laboratory Medicine <ul><li>ELISA methods can detect any infectious disease provided if we have antibodies and antigen to any infection, enzyme or any substance </li></ul>
    34. 35. Serology applications in .. <ul><li>HIV testing </li></ul><ul><li>Serum HCG (pregnancy) </li></ul><ul><li>Tests for hepatitis antigens and antibodies </li></ul><ul><li>Antibodies to bacteria </li></ul><ul><li>Hepatitis Serology </li></ul>
    35. 36. Nephelometry <ul><li>Procedure </li></ul><ul><ul><li>Serum substance reacts with specific antisera and forms insoluble complexes </li></ul></ul><ul><ul><li>Light is passed through suspension </li></ul></ul><ul><ul><li>Scattered (reflected) light is proportional to number of insoluble complexes; compare to standards </li></ul></ul><ul><li>Examples </li></ul><ul><ul><li>Complement component concentration </li></ul></ul><ul><ul><li>Antibody concentration (IgG, IgM, IgA, etc.) </li></ul></ul><ul><li>Immunofluorescence </li></ul>
    36. 37. Immunofluorescence <ul><li>Direct – add fluorescein-labeled antibody to patient tissue, wash, and examine under fluorescent microscope </li></ul><ul><li>Indirect – add patient serum to tissue containing known antigen, wash, add labeled antiglobulin, wash, and examine under fluorescent microscope </li></ul><ul><li>Examples </li></ul><ul><ul><li>Testing for Antinuclear Antibodies (ANA) </li></ul></ul><ul><ul><li>Fluorescent Treponemal Antibody Test (FTA-Abs) </li></ul></ul>
    37. 38. Fluorescence polarization immunoassay (FPIA) <ul><li>Principle </li></ul><ul><li>Add reagent antibody and fluorescent-tagged antigen to patient serum </li></ul><ul><li>Positive test </li></ul><ul><ul><li>Antigen present in patient serum binds to reagent leaving most tagged antigen unbound </li></ul></ul><ul><ul><li>Unbound labeled antigens rotate quickly reducing amount of polarized light produced </li></ul></ul><ul><li>Negative test </li></ul><ul><ul><li>If no antigen present in patient serum, tagged antigen binds to reagent antibody </li></ul></ul><ul><ul><li>Tagged antigen-antibody complexes rotate slowly giving off increased polarized light </li></ul></ul>
    38. 39. Flow cytometry <ul><li>Method of choice for T- and B-cell analysis (lymphocyte phenotyping) </li></ul><ul><li>Principle </li></ul><ul><li>Incubate specimen with 1 or 2 monoclonal antibodies tagged with fluorochrome </li></ul><ul><li>Single cells pass through incident light of instrument (laser) which excites fluorochrome and results in emitted light of different wavelength </li></ul><ul><li>Intensity of fluorescence measured to detect cells possessing surface markers for the specific monoclonal antibodies that were employed </li></ul><ul><li>Forward light scatter indicates cell size or volume </li></ul><ul><li>90° side-scattered light indicates granula </li></ul>
    39. 40. Common uses Flow cytometry <ul><li>DNA analysis </li></ul><ul><li>Reticulocyte counts </li></ul><ul><li>Leukaemia/lymphoma classification </li></ul><ul><li>CD 4 cell estimations in AIDS/HIV patients. </li></ul>
    40. 41. Other Applications of agglutination tests in Serology <ul><li>i. Determination of blood types or antibodies to blood group antigens . </li></ul><ul><li>ii. To assess bacterial infections </li></ul><ul><li>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. </li></ul>
    41. 42. Georges-Fernand-Isidor Widal <ul><li>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 </li></ul>
    42. 43. Widal test a Popular test in diagnosis of Typhoid Fever <ul><li>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. </li></ul>
    43. 44. Widal test is century old , Is it loosing importance ? <ul><li>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. </li></ul>
    44. 45. Widal test – A standard tube agglutination test <ul><li>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. </li></ul>
    45. 46. Agglutination how it appear after reactivity <ul><li>O agglutination is granular </li></ul><ul><li>H agglutination is loose and floccular </li></ul>
    46. 48. Principle of the Test <ul><li>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. </li></ul>
    47. 49. Reading the Widal Test <ul><li>Read the results by viewing the tubes under good light against the dark background with x2 magnifying lens </li></ul><ul><li>Do not shake tubes before reading the results </li></ul><ul><li>Read titers as greatest dilutions giving visible agglutinations. </li></ul><ul><li>Limiting agglutination is 1in 200 the titer is 200 not to be reported as 1/200. </li></ul>
    48. 50. Interpretation of Widal test <ul><li>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. </li></ul>
    49. 51. Testing in Typhoid carriers <ul><li>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. </li></ul>
    50. 52. Importance of Vi antibodies <ul><li>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. </li></ul>
    51. 53. Prozone phenomenon in Agglutination tests <ul><li>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. </li></ul><ul><li>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 </li></ul>
    52. 54. Causes Of False-positive Widal Agglutination Tests <ul><li>Previous immunization with Salmonella antigen . </li></ul><ul><li>Cross - reaction with non – typhoidal Salmonella . </li></ul><ul><li>Variability and poorly standardized commercial antigen preparation . </li></ul><ul><li>Infection with malaria </li></ul><ul><li>other Enterobacteriaceae charring the same s - LPS . </li></ul>
    53. 55. Causes of Negative Widal Agglutination Test <ul><li>The carrier state </li></ul><ul><li>An inadequate inoculum of bacterial antigen in the host to induce antibody production </li></ul><ul><li>Technical difficulty or errors in the performance of the test. </li></ul><ul><li>Previous antibiotic treatment </li></ul><ul><li>Variability in the preparation of commercial antigens. </li></ul>
    54. 56. Declining importance of Widal test <ul><li>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. </li></ul>
    55. 57. Serology - Importance of repeated tests <ul><li>Criteria for diagnosing Primary Infection </li></ul><ul><ul><ul><li>4 fold or more increase in titre of IgG or total antibody between acute and convalescent sera </li></ul></ul></ul><ul><ul><ul><li>Presence of IgM </li></ul></ul></ul><ul><ul><ul><li>Seroconversion </li></ul></ul></ul><ul><ul><ul><li>A single high titre of IgG (or total antibody) - very unreliable </li></ul></ul></ul><ul><li>Criteria for diagnosing Reinfection </li></ul><ul><ul><ul><li>Four fold or more increase in titre of IgG or total antibody between acute and convalescent sera </li></ul></ul></ul><ul><ul><ul><li>Absence or slight increase in IgM </li></ul></ul></ul>
    56. 58. Typical Serological Profile After Acute Infection <ul><li>Note that during Reinfections, IgM may be absent or present at a low level transiently </li></ul>
    57. 59. Antigen – Antibody reactions presenting with precipitation
    58. 60. Precipitation Curve
    59. 61. Precipitation Curve
    60. 62. Measurement of Precipitation by Light <ul><li>Antigen-antibody complexes, when formed at a high rate, will precipitate out of a solution resulting in a turbid or cloudy appearance. </li></ul><ul><li>Turbidimetry measures the turbidity or cloudiness of a solution by measuring amount of light directly passing through a solution. </li></ul><ul><li>Nephelometry indirect measurement, measures amount of light scattered by the antigen-antibody complexes. </li></ul>
    61. 63. Screening Tests for Syphilis <ul><li>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. </li></ul>
    62. 64. Serological Diagnosis Of Syphilis <ul><li>Specific Anti- treponemal Antibody </li></ul><ul><li>Anti – treponemal Antibody </li></ul><ul><li>Reagin Antibody </li></ul><ul><li>(VDRL and RPR ) </li></ul><ul><li>Associated with higher false positives </li></ul>
    63. 65. Indication for testing for Syphilis <ul><li>Pregnant women </li></ul><ul><li>sexual contacts or partners of patients diagnosed with syphilis </li></ul><ul><li>children born to mothers with syphilis </li></ul><ul><li>patients with HIV infection </li></ul>
    64. 66. Tests For Reagin Antibody <ul><li>A large numbers of tests for Reagin : </li></ul><ul><ul><ul><li>VDRL ( Venereal Diseases Reference Laboratory ). </li></ul></ul></ul><ul><ul><ul><li>RPR ( Rapid Plasma Reagin ) </li></ul></ul></ul><ul><ul><ul><li>ART ( Automated Reagin Test ) </li></ul></ul></ul><ul><ul><ul><ul><li>Good sensitive screening </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Titre falls rapidly with treatment </li></ul></ul></ul></ul><ul><li>Reagin titre falls with treatment . </li></ul>
    65. 67. VDRL – A standard test for Syphilis <ul><li>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. </li></ul>
    66. 68. Screening tests should be reported with cautions <ul><li>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. </li></ul>
    67. 69. Combination of testes are desirable <ul><li>Syphilis serodiagnosis relies on a combination of nonspecific screening tests (antilipoidal antibodies) and Treponema pallidum -specific tests (anti- T . pallidum antibodies). </li></ul>
    68. 70. Measurement of Precipitation by Light <ul><li>Antigen-antibody complexes, when formed at a high rate, will precipitate out of a solution resulting in a turbid or cloudy appearance. </li></ul><ul><li>Turbidimetry measures the turbidity or cloudiness of a solution by measuring amount of light directly passing through a solution. </li></ul><ul><li>Nephelometry indirect measurement, measures amount of light scattered by the antigen-antibody complexes . </li></ul>
    69. 71. Confirmation is warranted <ul><li>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 </li></ul>
    70. 72. Non reactive and Reactive VDRL Tests
    71. 73. Rapid plasma reagin <ul><li>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. </li></ul>
    72. 74. Agglutination++   RPR         Agglutination+
    73. 75. Agglutination+: ve RPR Agglutination : -   
    74. 76. Biological false positives <ul><li>Biological False Positive Antibody ( BFP ) Reagin Antibody : associated with other diseases ( BFP ) </li></ul><ul><li>Acute : </li></ul><ul><ul><ul><li>Pneumonia </li></ul></ul></ul><ul><ul><ul><li>Vaccination with live attenuated viruses . </li></ul></ul></ul><ul><ul><ul><li>Malaria </li></ul></ul></ul><ul><ul><ul><li>Pregnancy </li></ul></ul></ul><ul><li>Chronic : </li></ul><ul><ul><ul><li>Leprosy – the only infection </li></ul></ul></ul><ul><li>Reagin titer falls rapidly with treatment </li></ul>
    75. 77. Serological Diagnosis Of Syphilis <ul><li>Test for specific Anti - treponemal Antibody </li></ul><ul><li>Absorbed fluorescent treponemal antibody ( FTA - ABs ) </li></ul><ul><li>Treponema Pallidum Immobilization Test ( TPI ) </li></ul><ul><ul><ul><li>Most sensitive </li></ul></ul></ul><ul><ul><ul><li>Utilize living Treponema maintained by passage in rabbits testes . </li></ul></ul></ul><ul><ul><ul><li>Expensive </li></ul></ul></ul><ul><ul><ul><li>Potentially hazardous . </li></ul></ul></ul><ul><ul><ul><li>Not done in the present contest as Technically demanding </li></ul></ul></ul>
    76. 78. Doing a quantization test RPR
    77. 79. Other Serological Methods in Diagnosis Of Syphilis <ul><li>Treponema pallidum haemagglutination ( TPHA ) test . </li></ul><ul><ul><ul><li>Sheep, chicken or turkey RBCs. Sensitized by attaching killed Treponema pallidum . </li></ul></ul></ul><ul><ul><ul><li>Agglutinate by presence of antibody </li></ul></ul></ul><ul><ul><ul><li>Less sensitive than FTA – Abs </li></ul></ul></ul><ul><ul><ul><li>Less reliable in the diagnosis of primary syphilis . </li></ul></ul></ul><ul><ul><ul><li>Sometimes false positive </li></ul></ul></ul>
    78. 80. Treponema Palladium Hemagglutination test TPHA
    79. 81. Other Serological Tests for Syphilis <ul><li>Anti – Treponemal Antibody </li></ul><ul><li>Anti-Treponemal ABs group detected by Reiter Protein Complement Fixation Test (RPCFT) </li></ul><ul><ul><li>Appears later than specific ABs </li></ul></ul><ul><ul><li>Some syphilis patient do not produce the form of ABs </li></ul></ul><ul><ul><li>Used is limited. </li></ul></ul>
    80. 82. Detection by FTA-ABS IgG and IgM <ul><li>In the FTA-ABS tests, the patient's blood serum is mixed with a preparation that prevents interference from antibodies to other treponemal infections. </li></ul>
    81. 83. FTA abs IgG and IgM detection continues to be a confirmatory test in diagnosis of Syphilis <ul><li>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.. </li></ul>
    82. 84. Principle of Fluorescent Method
    83. 85. Active Treponema Pallidum Infection <ul><li>Positive Specific Tests e . g . TPHA </li></ul><ul><li>Positive ( ≥1 / 8 ) of non - specific test ( VDRL ) </li></ul><ul><ul><li>TPI - T ( Treponema Pallidum Immobilization Test ) </li></ul></ul><ul><ul><li>FTA –T ( Fluorescent Treponema Test ) </li></ul></ul><ul><ul><li>Sometimes needed for confirmation . </li></ul></ul>
    84. 86. Emergin Methods in Diagnosis of Syphilis <ul><li>Currently, ELISA, Western blot, and PCR testing are being studied as additional diagnostic tests, particularly for congenital syphilis and neurosyphilis. </li></ul>
    85. 87. SPINAL FLUID TESTS in Syphilis. <ul><li>. 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. </li></ul>
    86. 88. CSF testing is indicated only in … <ul><li>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. </li></ul>
    87. 89. Biological false reactive VDRL test among the HIV infected patients <ul><li>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. </li></ul><ul><li>VirojWiwanitkit </li></ul>
    88. 90. Rickettsiae and Serology <ul><li>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 </li></ul><ul><li>Because of this, Rickettsiae cannot live in artificial nutrient environments and are grown either in tissue or embryo cultures (typically, chicken embryos are used). </li></ul><ul><li>Still we have to dependent on Weil Felix test </li></ul>
    89. 91. Weil and Felix contribute for testing <ul><li>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. </li></ul>
    90. 92. Weil-Felix reaction – A Heterophile agglutination Test <ul><li>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) </li></ul><ul><li>OX19, OX2 are strains of Proteus vulgaris. OXk is the strain of Proteus mirabilis. </li></ul>
    91. 93. Weil-Felix a Heterophile agglutination test <ul><li>The agglutination reactions, based on antigens common to both organisms, determine the presence and type of rickettsial infection </li></ul><ul><li>Because Rickettsiae are both fastidious and hazardous, few laboratories undertake their isolation and diagnostic identification </li></ul><ul><li>Weil-Felix test that is based on the cross-reactive antigens of OX-19 and OX-2 strains of Proteus vulgaris . </li></ul>
    92. 94. Interpretations in Weil-Felix reaction <ul><li>Sera from endemic typhus agglutinate OX19, OX2. Tick borne spotted fever agglutinate OX19, OX2. </li></ul><ul><li>Scrub Typhus agglutinate OXk strain </li></ul><ul><li>Test is negative in rickettsialpox, trench fever and Q-fever. False positive reaction may occur in urinary or other Proteus infections Test may be negative in 50 percent scrub typhus </li></ul>
    93. 95. Weil-Felix test indicated in when patients present with rashes <ul><li>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).. </li></ul>
    94. 96. Weil Felix test and Concentration Camps
    95. 97. Weil-Felix test positivity saves from Nazis <ul><li>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. </li></ul>
    96. 98. How they made Weil-Felix test Positive <ul><li>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 ) </li></ul>
    97. 99. Other Emerging Serological Tests
    98. 100. Co-agglutination <ul><li>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 </li></ul>
    99. 101. Co agglutination Test <ul><li>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. </li></ul>
    100. 102. Chemiluminescence <ul><li>Chemiluminescence is the emission of light with limited emission of heat (luminescence), as the result of a chemical reaction. </li></ul>
    101. 103. Chemiluminescent Immunoenzymatic Assay <ul><li>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. </li></ul>
    102. 104. Recent testing Advances <ul><li>The ToRC IgG kit simultaneously detects IgG class antibodies to Toxoplasmosis gondii, rubella and cytomegalovirus (CMV). </li></ul><ul><li>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. </li></ul>
    103. 105. False Positive Serological Tests <ul><li>Cross reacting antibody </li></ul><ul><li>Cross reactivation of latent organism ( Influenza Virus A infection activate CMV IgM – production </li></ul><ul><li>Presence of Rheumatoid factors </li></ul><ul><li>RF = IgM </li></ul><ul><li>RF + IgG = Complexed </li></ul><ul><li>= False positive organism - specific IgM Antibody </li></ul>
    104. 106. False Negative Serologic Test <ul><li>Immune system not intact </li></ul><ul><li>Delay in Antibody response ( Lyme disease - Legionnaire’s Disease ) </li></ul><ul><li>Competition for Antigen binding site of antibody ) </li></ul><ul><li>IgM binds to the Antigen IgG site </li></ul><ul><li>IgG binds to the Antigen IgM site </li></ul><ul><li>Prozone Phenomena </li></ul>
    105. 107. Usefulness of Serological Results <ul><li>How useful a serological result is depends on the individual virus. </li></ul><ul><li>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. </li></ul>
    106. 108. Rota Virus - wether serology useful ? <ul><li>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. </li></ul><ul><li>Acute presence of Antigen is much useful in Diagnosis </li></ul>
    107. 109. Antibody detection is definitive Diagnosis <ul><li>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. </li></ul>
    108. 110. Problems with Serology <ul><li>Long period of time required for diagnosis for paired acute and convalescent sera. </li></ul><ul><li>Mild local infections such as HSV genitalis may not produce a detectable humoral immune response. </li></ul><ul><li>Extensive antigenic cross-reactivity between related viruses e.g. HSV and VZV, Japanese B encephalitis and Dengue, may lead to false positive results. </li></ul>
    109. 111. Problems with Serology Other Health condtions interfere <ul><li>Immunocompromised patients often give a reduced or absent humoral immune response. </li></ul><ul><li>Patients with infectious mononucleosis and those with connective tissue diseases such as SLE may react non-specifically giving a false positive result. </li></ul><ul><li>Patients given blood or blood products may give a false positive result due to the transfer of antibody </li></ul>
    110. 112. Automations <ul><li>One of the first successful attempts to automate </li></ul><ul><li>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. </li></ul>
    111. 113. Created as part of Dr.T.V.Rao MD’s ‘e’ learning Programme Email [email_address]