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Serology in Infectious Diseases

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Serology in Infectious Diseases

Serology in Infectious Diseases

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  • 1. SerologyPrinciples and Interpretation in Infectious diseases Dr.T.V.Rao MD Dr.T.V.Rao MD 1
  • 2. 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. Dr.T.V.Rao MD 2
  • 3. Karl Landsteiner (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, Dr.T.V.Rao MD 3
  • 4. 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 individuals blood type. Serology blood tests help to diagnose patients with certain immune deficiencies associated with the lack of antibodies, such as X-linked agammaglobulinemia. Dr.T.V.Rao MD 4
  • 5. Serology• The branch of laboratory medicine that studies blood serum for evidence of infection and other parameters by evaluating antigen- antibody reactions in vitro Dr.T.V.Rao MD 5
  • 6. 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 Dr.T.V.Rao MD 6
  • 7. 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. Dr.T.V.Rao MD 7
  • 8. 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 Dr.T.V.Rao MD 8
  • 9. 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) Dr.T.V.Rao MD 9
  • 10. 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) Dr.T.V.Rao MD 10
  • 11. 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 Dr.T.V.Rao MD 11
  • 12. 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 . Dr.T.V.Rao MD 12
  • 13. 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. Dr.T.V.Rao MD 13
  • 14. Dr.T.V.Rao MD 14
  • 15. 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 Dr.T.V.Rao MD 15
  • 16. 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 TiterDr.T.V.Rao MD 16
  • 17. 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 Dr.T.V.Rao MD 17
  • 18. Common methods in creating dilutions Dr.T.V.Rao MD 18
  • 19. Sero Conversion• Seroconversion is the development of detectable specific antibodies to microorganisms in the blood serum as a result of infection or immunization. Dr.T.V.Rao MD 19
  • 20. 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 Dr.T.V.Rao MD 20
  • 21. 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 Dr.T.V.Rao MD 21
  • 22. 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 Dr.T.V.Rao MD 22 antibodies.
  • 23. Antigen and Antibodyreactions can be identified by different methods Dr.T.V.Rao MD 23
  • 24. 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 Dr.T.V.Rao MD 24
  • 25. 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 RBCs with tannic acid to allow adsorption of protein antigens) – Passive latex agglutination (antigen attached to latex particle) Dr.T.V.Rao MD 25
  • 26. 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 haemolysis = positive reaction• Agglutination or haemolysis = negative reaction (antibody not bound in origin Dr.T.V.Rao MD 26
  • 27. 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) Dr.T.V.Rao MD 27
  • 28. 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 Dr.T.V.Rao MD 28
  • 29. 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. Dr.T.V.Rao MD 29
  • 30. Serology can be done on various specimens• 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). Dr.T.V.Rao MD 30
  • 31. Enzyme immunoassay (EIA/ELISA)• Sandwich technique”• Monoclonal or polyclonal antibody adsorbed on solid surface (bead or microliter 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 Dr.T.V.Rao MD 31
  • 32. 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 Dr.T.V.Rao MD 32
  • 33. 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 Dr.T.V.Rao MD 33
  • 34. Serology applications in..• HIV testing• Serum HCG (pregnancy)• Tests for hepatitis antigens and antibodies• Antibodies to bacteria• Hepatitis Serology Dr.T.V.Rao MD 34
  • 35. 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 Dr.T.V.Rao MD 35
  • 36. Immunofluorescence• Direct – add fluorescein-labelled antibody to patient tissue, wash, and examine under fluorescent microscope• Indirect – add patient serum to tissue containing known antigen, wash, add labelled antiglobulin, wash, and examine under fluorescent microscope• Examples – Testing for Antinuclear Antibodies (ANA) – Fluorescent Treponemal Antibody Test (FTA-Abs) Dr.T.V.Rao MD 36
  • 37. 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 labelled 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 Dr.T.V.Rao MD 37
  • 38. 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 fluororochrome 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 Dr.T.V.Rao MD 38
  • 39. Common uses Flow cytometry• DNA analysis• Reticulocyte counts• Leukaemia/lymphoma classification• CD 4 cell estimations in AIDS/HIV patients. Dr.T.V.Rao MD 39
  • 40. Other Applications of agglutination tests in Serology i. Determination of blood types or antibodies to blood group antigens.ii. To assess bacterial infectionse.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. Dr.T.V.Rao MD 40
  • 41. 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 Dr.T.V.Rao MD 41
  • 42. Widal test a Popular test indiagnosis 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 Dr.T.V.Rao MD 42 H-flagellar in the blood.
  • 43. 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. Dr.T.V.Rao MD 43
  • 44. 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. Dr.T.V.Rao MD 44
  • 45. Agglutination how it appear after reactivity•O agglutination is granular•H agglutination is loose and floccular Dr.T.V.Rao MD 45
  • 46. Dr.T.V.Rao MD 46
  • 47. 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 patients serum, is measured by adding a constant amount of antigen (Salmonella typhi) to the serially diluted serum. Dr.T.V.Rao MD 47
  • 48. 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. Dr.T.V.Rao MD 48
  • 49. 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. Dr.T.V.Rao MD 49
  • 50. 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. Dr.T.V.Rao MD 50
  • 51. 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. Dr.T.V.Rao MD 51
  • 52. Prozone phenomenon in Agglutination testsProzone 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 Dr.T.V.Rao MD 52
  • 53. 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 . Dr.T.V.Rao MD 53
  • 54. 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. Dr.T.V.Rao MD 54
  • 55. 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. Dr.T.V.Rao MD 55
  • 56. Serology - Importance of repeated testsCriteria for diagnosing Primary Infection • 4 fold or more increase in titer of IgG or total antibody between acute and convalescent sera • Presence of IgM • Seroconversion • A single high titer of IgG (or total antibody) - very unreliableCriteria for diagnosing Reinfection • Four fold or more increase in titer of IgG or total antibody between acute and convalescent sera Dr.T.V.Rao MD 56 • Absence or slight increase in IgM
  • 57. Typical Serological Profile After Acute InfectionNote that during Reinfections, IgM may be absent or present at a low level transiently Dr.T.V.Rao MD 57
  • 58. Antigen – Antibody reactionspresenting with precipitation Dr.T.V.Rao MD 58
  • 59. Precipitation Curve Dr.T.V.Rao MD 59
  • 60. Precipitation Curve Dr.T.V.Rao MD 60
  • 61. 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 theDr.T.V.Rao MD antigen-antibody complexes. 61
  • 62. 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. Dr.T.V.Rao MD 62
  • 63. Serological Diagnosis Of SyphilisI. Specific Anti- Treponemal AntibodyII. Anti – Treponemal AntibodyIII. Reagin Antibody (VDRL and RPR)Associated with higher false positives Dr.T.V.Rao MD 63
  • 64. 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 Dr.T.V.Rao MD 64
  • 65. 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 Titer falls rapidly with treatment• Reagin titer falls with treatment. Dr.T.V.Rao MD 65
  • 66. 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 patients 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 patients blood. Dr.T.V.Rao MD 66
  • 67. 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. Dr.T.V.Rao MD 67
  • 68. 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). Dr.T.V.Rao MD 68
  • 69. 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. Dr.T.V.Rao MD 69
  • 70. 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 Dr.T.V.Rao MD 70
  • 71. Non reactive and Reactive VDRL Tests Dr.T.V.Rao MD 71
  • 72. Rapid plasma reagin• The rapid plasma reagin (RPR) test works on the same principle as the VDRL. It is available as a kit. The patients serum is mixed with cardiolipin on a plastic-coated card that can be examined with the naked eye. Dr.T.V.Rao MD 72
  • 73. Agglutination++ RPR Agglutination+ Dr.T.V.Rao MD 73
  • 74. Agglutination+: ve RPR Agglutination :- Dr.T.V.Rao MD 74
  • 75. Biological false positives• Biological False Positive Antibody (BFP) Reagin Antibody: associated with other diseases (BFP)A. Acute: • Pneumonia • Vaccination with live attenuated viruses. • Malaria • PregnancyB. Chronic: • Leprosy – the only infection• Reagin titer falls rapidly with treatment Dr.T.V.Rao MD 75
  • 76. Serological Diagnosis Of SyphilisTest for specific Anti - Treponemal Antibody1. Absorbed fluorescent Treponemal antibody (FTA - ABs)2. Treponema Pallidum Immobilization Test (TPI) A. Most sensitive B. Utilize living Treponema maintained by passage in rabbits testes. C. Expensive D. Potentially hazardous.MD Dr.T.V.Rao 76 E. Not done in the present contest as Technically demanding
  • 77. Doing a quantization test RPR Dr.T.V.Rao MD 77
  • 78. Other Serological Methods in Diagnosis Of SyphilisTreponema pallidum haemagglutination (TPHA) test. A. Sheep, chicken or turkey RBCs. Sensitized by attaching killed Treponema pallidum. B. Agglutinate by presence of antibody C. Less sensitive than FTA – Abs D. Less reliable in the diagnosis of primary syphilis. E. Sometimes false positive Dr.T.V.Rao MD 78
  • 79. Treponema PalladiumHaemagglutination test TPHA Dr.T.V.Rao MD 79
  • 80. Other Serological Tests for Syphilis• Anti – Treponemal Antibody• Anti-Treponemal ABs group detected by Reiter Protein Complement Fixation Test (RPCFT) A. Appears later than specific ABs B. Some syphilis patient do not produce the form of ABs C. Used is limited. Dr.T.V.Rao MD 80
  • 81. Detection by FTA-ABS IgG and IgM• In the FTA-ABS tests, the patients blood serum is mixed with a preparation that prevents interference from antibodies to other Treponemal infections. Dr.T.V.Rao MD 81
  • 82. 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.. Dr.T.V.Rao MD 82
  • 83. Principle of Fluorescent Method Dr.T.V.Rao MD 83
  • 84. Active Treponema Pallidum Infection1. Positive Specific Tests e.g. TPHA2. Positive ( ≥1/ 8) of non-specific test (VDRL) • TPI-T (Treponema Pallidum Immobilization Test) • FTA –T (Fluorescent Treponema Test) • Sometimes needed for confirmation. Dr.T.V.Rao MD 84
  • 85. Emerging 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. Dr.T.V.Rao MD 85
  • 86. 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. Dr.T.V.Rao MD 86
  • 87. 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. Dr.T.V.Rao MD 87
  • 88. 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.• Dr.T.V.Rao MD 88
  • 89. Rickettsia and Serology• Rickettsia 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, Rickettsia 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 Dr.T.V.Rao MD 89
  • 90. 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. Dr.T.V.Rao MD 90
  • 91. 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. Dr.T.V.Rao MD 91
  • 92. 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. Dr.T.V.Rao MD 92
  • 93. 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 infections Test may be negative in 50 percent scrub typhus Dr.T.V.Rao MD 93
  • 94. Weil-Felix test indicated 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).. Dr.T.V.Rao MD
  • 95. Weil Felix test and Concentration Camps Dr.T.V.Rao MD 95
  • 96. 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. Dr.T.V.Rao MD 96
  • 97. 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 ) Dr.T.V.Rao MD 97
  • 98. Other EmergingSerological Tests Dr.T.V.Rao MD 98
  • 99. 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 Dr.T.V.Rao MD 99
  • 100. Co agglutination TestAgglutination test inwhich inert particles(latex beads or heat-killed S aureusCowan 1 strain withprotein A) are coatedwith antibody to anyof a variety ofantigens and thenused to detect theantigen in specimensor in isolated bacteria. Dr.T.V.Rao MD 100
  • 101. Chemiluminescence• Chemiluminescen ce is the emission of light with limited emission of heat (luminescence), as the result of a chemical reaction. Dr.T.V.Rao MD 101
  • 102. Chemiluminescent Immunoenzymatic Assay• Process for the quantitative and qualitative determination of antigens, antibodies and their complexes by means of a chemiluminescent labelling substance activated or excited to chemiluminescence by an analytical reagent. By means of a serological reaction, initially an antigen/antibody complex is formed which is treated with a chemiluminescent conjugate containing chemiluminescent triphenylmethane dyes and the chemiluminescence of the chemiluminescent complex formed is measured. Dr.T.V.Rao MD 102
  • 103. 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. Dr.T.V.Rao MD 103
  • 104. False Positive Serological Tests1. Cross reacting antibody2. Cross reactivation of latent organism (Influenza Virus A infection activate CMV IgM – production3. Presence of Rheumatoid factors RF = IgM RF + IgG = Complexed = False positive organism- specific IgM Antibody Dr.T.V.Rao MD 104
  • 105. False Negative Serologic Test1. Immune system not intact2. Delay in Antibody response (Lyme disease - Legionnaire’s Disease)3. Competition for Antigen binding site of antibody) IgM binds to the Antigen IgG site IgG binds to the Antigen IgM site4. Prozone Phenomena Dr.T.V.Rao MD 105
  • 106. 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. Dr.T.V.Rao MD 106
  • 107. Rota Virus - whether 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 Dr.T.V.Rao MD 107
  • 108. 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. Dr.T.V.Rao MD 108
  • 109. 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. Dr.T.V.Rao MD 109
  • 110. Problems with Serology Other Health conditions 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 Dr.T.V.Rao MD 110
  • 111. If we are a busy lab…. Dr.T.V.Rao MD 111
  • 112. Why Automate?• Reduce variability and improve quality• Reduce labor and test costs• Improve workflow in the laboratory• Avoid potential ergonomic issues Dr.T.V.Rao MD 112
  • 113. 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. Dr.T.V.Rao MD 113
  • 114. Definitions (1)• Quality Control - QC refers to the measures that must be included during each assay run to verify that the test is working properly.• Quality Assurance - QA is defined as the overall program that ensures that the final results reported by the laboratory are correct.• “The aim of quality control is simply to ensure that the results generated by the test are correct. However, quality assurance is concerned with much more: that the right test is carried out on the right specimen, and that the right result and right interpretation is delivered to the right person at the right time” Dr.T.V.Rao MD 114
  • 115. Definitions (2)• Quality Assessment - quality assessment (also known as proficiency testing) is a means to determine the quality of the results generated by the laboratory. Quality assessment is a challenge to the effectiveness of the QA and QC programs.• Quality Assessment may be external or internal, examples of external programs include NEQAS, HKMTA, and Q- probes. Dr.T.V.Rao MD 115
  • 116. Variables that affect the quality of results • The educational background and training of the laboratory personnel • The condition of the specimens • The controls used in the test runs • Reagents • Equipment • The interpretation of the results • The transcription of results • The reporting of results Dr.T.V.Rao MD 116
  • 117. Errors in measurement• True value - this is an ideal concept which cannot be achieved.• Accepted true value - the value approximating the true value, the difference between the two values is negligible.• Error - the discrepancy between the result of a measurement and the true (or accepted true value). Dr.T.V.Rao MD 117
  • 118. Random Error• An error which varies in an unpredictable manner, in magnitude and sign, when a large number of measurements of the same quantity are made under effectively identical conditions.• Random errors create a characteristic spread of results for any test method and cannot be accounted for by applying corrections. Random errors are difficult to eliminate but repetition reduces the influences of random errors.• Examples of random errors include errors in pipetting and changes in incubation period. Random errors can be minimized by training, supervision and adherence to standard operating procedures. Dr.T.V.Rao MD 118
  • 119. Random Errors x x x x xTrue x x x xValue x x x x x x x x x Dr.T.V.Rao MD 119
  • 120. Systematic Error• An error which, in the course of a number of measurements of the same value of a given quantity, remains constant when measurements are made under the same conditions, or varies according to a definite law when conditions change.• Systematic errors create a characteristic bias in the test results and can be accounted for by applying a correction.• Systematic errors may be induced by factors such as variations in incubation temperature, blockage of plate washer, change in the reagent batch or modifications in testing method. Dr.T.V.Rao MD 120
  • 121. Systematic Errors x x x x x x x xTrue xValue Dr.T.V.Rao MD 121
  • 122. Internal Quality Control Program for Serological TestingAn internal quality control program depend on the use ofinternal quality control (IQC) specimens, Shewhart ControlCharts, and the use of statistical methods for interpretation.Internal Quality Control SpecimensIQC specimens comprises either (1) in-house patient sera(single or pooled clinical samples), or (2) international serumstandards with values within each clinically significant ranges. Dr.T.V.Rao MD 122
  • 123. For Articles of Interest onAntibiotics follow me on Dr.T.V.Rao MD 123
  • 124. • Created by Dr.T.V.Rao MD for ‘e’ learning resources forMicrobiologists in Developing World • Email • doctortvrao@gmail.com Dr.T.V.Rao MD 124