syphilis serology ppt, syphilis, laboratory diagnosis of syphilis, VDRL, FTA-ABS

syphilis serology ppt, syphilis, laboratory diagnosis of syphilis, VDRL, FTA-ABS

1. Dr. Sanjay singh AIIMS Diagnostic Evaluation of Syphilis

2. Syphilis "He who knows syphilis, knows medicine" Sir William Osler 2

3. Caused by Treponema pallidum . Motile spiral-shaped gram – ve bacteria Characteristic cock-screw motility Inability to survive outside in an animal host Cannot be cultured in vitro Size : approx 10–14 μm in length and 0.1–0.2 μm in diameter, 10 regular spirals at interval of about 1 μm Transmission : sexual; maternal-fetal, and rarely by other means INTRODUCTION

4. Dr.T.V.Rao MD 4 Ultra Structure Contains many strongly antigenic protein

5. 5

6. STAGES OF SYPHILIS Primary Secondary Latent Early latent Late latent Late or tertiary May involve any organ, but main parts are: Neurosyphilis Cardiovascular syphilis Late benign ( gumma )

7. Direct detection of Treponema Pallidum Nontreponemal S erological Tests Treponemal Serological Tests Diagnosis of Syphilis

8. TESTS FOR DIRECT DETECTION OF T PALLIDUM Animal Inoculation Dark Field microscopy Direct fluorescent antibody test Direct tests for T pallidum in tissue sections Nucleic acid amplification methods

9. NONTREPONEMAL SEROLOGICAL TESTS Microscopic nontreponemal tests VDRL (Venereal disease research laboratory test) USR (Unheated serum reagin Test) Macroscopic nontreponemal tests RPR (Rapid plasma reagin test) TRUST (Toluidine red unheated serum test)

10. TREPONEMAL SEROLOGICAL TESTS FTA-ABS (Fluorescent treponemal antibody absorption test) FTA-ABS double-staining (Fluorescent treponemal antibody absorption double staining test) TP-PA test ( Treponema pallidum particle agglutination test) Western blots EIAs (Enzyme immunoassays)/Rapid tests

11. Animal inoculation Oldest method for detecting infection Most sensitive method for detecting infectious treponemes and is used as the gold standard for measuring the sensitivity of methods such as the PCR Rabbit is most commonly used Any source of specimen can be used as long as the material is less than 1 h old or was frozen immediately after collection Inoculation of sample : Intratesticular or I ntradermal

12. Incubation period : I nversely proportional to the size of inoculum. S ensitivity of RIT approaches 100 % if the number of organisms exceeds 23 and patient has not received antibiotic treatment.

13. Dark Field Microscopy One of simplest and most reliable for the direct detection of T pallidum Exudates and fluids from lesions are examined as a wet mount Examination should be done immediately M ost productive during 1˚, 2˚, early relapsing, and early congenital syphilis when lesions contains large numbers of treponemes (chancres , condylomata latum , or mucous patches)

14. Comparison of Light Pathways of bright field and dark field Microscopy

15. Procedure Clean the lesion with a saline soaked gauze and squeeze it between index finger and thumb to produce a serous exudate (avoid contamination with blood) E xudate is then transferred onto a glass slide by directly pressing it on the lesion Normal saline can be added to the exudate to make the material homogenous Specimen should immediately be examined as delay in examination reduces the motility of the treponemes

16. Results T.pallidum is identified by its typical morphology and characteristic movements T.pallidum is differentiated from the other treponemes by the tightness of spirals and characteristic cork screw movements

17. Organism Location Coils Length (μm) Width (μm) Rotation T. pallidum subsp. pallidum Skin and mucosal lesions Spiral shape, 10–13 coils Medium , 10 ( 6–20) Very thin, 0.13–0.15 Slow to rapid; like a cork-screw , may rotate without changing place T. refringens Normal genital flora Spiral shape, 2–3 coils Short,5 - 8 Thick, 0.20–0.30 Very rapid; active serpentine-like , rotates sometimes so rapidly that it looks straight T . phagedenis , Reiter treponeme Normal genital flora Spiral shape, 10–12 coils (10–30) Medium long , 10–12 (10–30 ) Thick , 0.20–0.25 (0.20–0.40) Slow to rapid; rotates without changing place T. denticola Normal oral flora Spiral shape, 6–8 coils (2–8 ) Medium , 8 ( 6–16) Very thin, 0.15–0.20 Slow to rapid, often jerky

18. Demonstration of spirochetes

19. It is a practical alternative to dark field examination Specimen collection is same as that of dark field microscopy Slide is air dried and fixed with either acetone for 10 min or 100% methanol for 10 sec Smear is stained with fluorescein- labeled anti T . pallidum globulin and examined under fluorescent microscope Direct fluorescent antibody - T.pallidum (DFA-TP)

20. Advantages More sensitive and specific than dark field microscopy Samples from oral mucosa can also be examined Slides need not be examined immediately Disadvantages Can’t differentiate T. pallidum subsp from eachother

21. T pallidum in tissue sections 1˚ Syphilis P /V & P/J infiltrate of lymphocytes, plasma cells, and macrophages. Capillary endothelial proliferation and subsequent obliteration of small blood vessels may be appreciable. Focal erosion or ulceration is common.

22. 2˚ Syphilis Histologically similar to that of the primary chancre but infiltrate is less intense “ Lichenoid- psoriasiform ” configuration with a perijunctional infiltrate of lymphocytes, histiocytes , and plasma cells Sometimes histiocytic component of the infiltrate is prominent, and thus the biopsy may assume a “ lichenoid-granulomatous ” configuration

23. L ichenoid infiltrate

24. Traditionally Warthin starry method has been used for staining of tissue section Organisms can also be identified by PCR and a polyclonal antibody against T. pallidum is available for IHC Both PCR and IHC are much more specific than histochemistry in diagnosis of syphilis

25. Secondary syphilis: numerous spirochetes are present (Warthin-Starry stain)

26. Immunoperoxidase staining

27. Immunoperoxidase Conventional silver stain  Serology N = 10 9 6 7 Immunoperoxidase technique for detecting spirochetes in tissue sections : comparison with other methods Phelps RG, Knispel J, Tu ES et al. Int J Dermatol . 2000 Aug;39(8):609-13.

28. Treponema pallidum distribution patterns in mucocutaneous lesions of primary and secondary syphilis: an immunohistochemical and ultrastructural study . Martín- Ezquerra G, Fernandez- Casado A, Barco D et al. Hum Pathol . 2009 ;40(5 ):624-30. No. of Patient Warthin-Starry stain IHC p value Primary Syphilis (N = 8) 4 8 < 0.05 Secondary Syphilis (N = 26) 13 21 < 0.05

29. PCR Increasingly becoming the investigation of choice for identifying T.pallidum from the early lesions of syphilis A number of well-preserved DNA sequences have been identified that are specific for T.pallidum and do not appear to be found in other treponemes Assays based on these primers have been shown to be sensitive and specific in the diagnosis of early syphilis H ighly sensitive, able to detect as low as 1 to 10 organisms per specimen with high specificity.

30. Sample size : 12 patients of 2˚ Syphilis P olyclonal antibody directed against T. pallidum was positive in 90 % of samples Bacteria were located in epidermis and upper dermis 47-kDa surface protein gene could be amplified by PCR in 75 % samples When combining both techniques, T. pallidum was detected in 92% of the samples Diagnosing Treponema pallidum in secondary syphilis by PCR and immunohistochemistry. Buffet M, Grange PA, Gerhardt P et al. J Invest Dermatol . 2007;127(10):2345-50.

31. Antitreponemal antibody response IgM antibodies are produced ∼ 2 weeks after exposure, followed by IgG antibodies 2 weeks after IgM production T.pallidum infection produces antibodies to more than 20 different polypeptide antigens. Antibodies are of two types : 1) Non specific antibodies ( reagins ) : directed against lipoidal antigen of T . pallidum as well as mitochondrial & nuclear membranes of human cells 2) Specific anti-treponemal antibodies : directed against T.pallidum Early responses are against TpN47 and some of the flagellar proteins, followed by TpN15 and TpN17 In 2˚ syphilis, there is a disproportionate increase in antitreponemal IgG3-specific responses

32. Early latent syphilis : Faint – to moderate IgM and strong IgG reactivity are evident Late Latent syphilis : Faint IgM & variable IgG IgM antibodies decrease rapidly, becoming undetectable within 6–12 months after treatment Several studies suggest that decreasing IgM levels indicate adequacy of treatment . In contrast, IgG1 and IgG3 antitreponemal antibodies can persist for years despite therapy

33. NONTREPONEMAL SEROLOGICAL TESTS Four nontreponemal tests are currently considered standard tests: All these non treponemal tests measure anti lipoidal IgM and IgG antibodies These tests are used for initial screening and for follow up after treatment Microscopic tests Macroscopic tests VDRL RPR USR TRUST

34. Non Treponemal Tests They can be performed as a : Qualitative test (to check for presence or absence of antibodies) Quantitative test (to check the amount of antibodies present in the serum) Except for VDRL & RPR tests, most lipoidal antigen tests are not used

35. These t ests use basic antigen formula containing standardized amounts of cardiolipin , cholesterol and lecithin. Only tests recommended to monitor the course of disease during and after treatment. Nontreponemal tests can also serve to detect reinfection Limitations : R educed sensitivity in primary syphilis and late latent syphilis F alse-positive results False negative results

36. False-positive reactions Acute False positive reaction < 6 months Chronic False positive reaction > 6 months Viral infections Malaria Immunizations Pregnancy Laboratory errors Connective tissue diseases IV drug abusers Narcotic addiction Ageing Leprosy Malignancy

37. False Negative Reaction : Prozone Phenomenon O ccur due to interference by high concentrations of target antibodies in a specimen. Such specimens gives a clearly positive reaction when diluted and retested, a process that brings the antibody-to-antigen ratio within the optimal range . Prozone reactions occur in 1 to 2% of patients with secondary syphilis.

38. VDRL Venereal Disease Research Laboratory (VDRL) Test is a slide flocculation test employed in the diagnosis of syphilis Since the antigen used in this test is cardiolipin , which is a lipoidal extracted from beef heart , it is not a specific test. Antibodies reacting with cardiolipin antibodies have been traditionally termed “ reagin ” Antigen : lipid component of T pallidum or as a result of tissue injury following infection

39. VDRL- Test Requirements Patient’s serum, water bath, freshly prepared cardiolipin antigen, VDRL slide, mechanical rotator, pipettes, hypodermic syringe with unbeveled needle and microscope. Reactive and non-reactive serum controls are also required VDRL antigen : 0.03 % cardiolipin 0.21 % lecithin 0.9 % cholesterol Cardiolipin antigen must be freshly constituted each day of test. The working antigen is a buffered saline suspension of cardiolipin .

40. VDRL slide : This is a glass slide measuring 2 X 3 inch with 12 concave depressions, each measuring 16 mm in diameter and 1.75 mm deep . Patients’ serum is inactivated by heating at 56˚C for 30 minutes in a water bath to remove non-specific inhibitors (such as complement).

41. Qualitative test : 0.05 ml of inactivated serum is taken into one well . 1/60th ml (or 1 drop from 18 gauge needle) of cardiolipin antigen is added with help of a syringe to the well and rotated at 180 rpm for 4 minutes . Slide is then viewed under low power objective of a microscope for flocculation. Depending on size, the results are graded as weakly reactive (W) or reactive (R ). Reactive samples are then subjected to quantitative test.

42. QUANTITATIVE TEST : This is performed to determine the antibody titers Serum is doubly diluted in saline from 1 in 2 to 1:256 or more Reported as the highest dilution giving a reactive (not weakly reactive) result

43. Reporting of results Results of the test are reported as: REACTIVE : P ast / present infection with a pathogenic T.pallidum, which is either treated or untreated (or) a false positive reaction WEAKLY REACTIVE : Past/present infection, false positive reaction, Serofast NON REACTIVE : N o current infection (or) an effectively treated infection , but it does not rule out syphilis in incubation period

44. A four fold rise in titer  I nfection Reinfection Treatment failure A four fold decrease in titer  Effective therapy When a non treponemal test shows a persistent reactivity with no signs of decline in titer after 6 months of adequate therapy or Fails to show a four fold decrease of an initial high titer within 1 year SERORESISTANCE (SEROFAST )

45. Unheated Serum Reagin test USR antigen is VDRL antigen stabilized by addition of EDTA , so need for daily preparation of an antigen suspension is eliminated Choline chloride is added to eliminate the need to heat inactivate the serum. Addition of choline chloride also enhances the reactivity of the antigen USR test is performed and reported in a manner similar to the VDRL slide test on serum

46. RPR & TRUST Both tests are based on USR antigen TRUST and RPR card test antigens differ only in the visualization agent added to antigen For the RPR card test, sized charcoal particles are added to the antigen For the TRUST paint pigment particles are added Particles of both tests become entrapped in antigen-antibody lattice formed with a reactive serum.

47. Slides are read macroscopically to determine the presence of clumping (flocculation) Results of card tests are reported as either reactive, regardless of the size of the clumps , or nonreactive. All serum samples exhibiting any degree of reactivity or roughness should be quantitated to an endpoint titer

48. Treponemal Tests In these tests, entire T.pallidum or its fragments are used as the antigen to detect antibodies directed against treponemal cellular components These tests are used for confirmation of the disease either in past/present Treponemal tests become reactive before non treponemal tests but unlike non treponemal tests they remain positive for many years even after adequate therapy Treponemal tests are technically more difficult and costly to perform than nontreponemal tests and cannot be used to monitor treatment

49. Commonly used Treponemal tests Fluorescent Treponemal Antibody Absorption (FTA-Abs) test Fluorescent Treponemal Antibody Absorption double staining (FTA-Abs-DS) test Treponema pallidum Haemagglutination Assay (TPHA) Treponemal Enzyme Immunoassay (EIA)

50. Fluorescent Treponemal Antibody Absorption (FTA-Abs) test Serum for testing is diluted in sorbent (containing extract of Reiter treponemal culture) to absorb non specific antibodies Serum is placed on a microscopic slide to which the antigen (a suspension of T . pallidum organism) is fixed Conjugated fluorescein labeled antihuman globulin is added

51. The intensity of fluorescence is reported as REACTIVE, BORDERLINE, NON REACTIVE. False positives and negatives may occur It is most sensitive serological test in early stages of syphilis at present ADVANTAGES High specificity & sensitivity Can detect recent infection 1-2 weeks before other assays DISADVANTAGES Expensive Time consuming Well trained personnel is required

52. Fluorescent Treponemal Antibody Absorption double staining (FTA-Abs-DS) test Serum for testing is diluted in sorbent (containing extract of Reiter treponemal culture) to absorb non specific antibodies Serum is placed on a microscopic slide to which the antigen (a suspension of T . pallidum organism) is fixed Class - specific tetramethylrhodamine isothiocyanate – labeled antihuman immunoglobulin G is added Counterstain , fluorescein isothiocyanate (FITC)- labeled anti-treponemal globulin, is added to locate T. pallidum when the slide is examined with the FITC filter.

53. Treponema pallidum Haemagglutination Assay (TPHA ) A qualitative haemagglutination test using tanned formalinised sheep RBC’s as carrier for T.pallidum antigen (sensitized cells ) TPPA : Treponema pallidum particle agglutination B ased on agglutination of coloured particle carriers sensitized with T pallidum antigen Uses gelatin particles instead of erythrocytes, thus eliminating nonspecific reactions with plasma samples

54. Treponemal Enzyme Immunoassay ( EIA) In this test, serum is added to microwells coated with a treponemal antigen An enzyme labeled anti human Ig conjugate & enzyme substrate are added to detect antigen-antibody reaction after incubation It has advantages of higher specificity than FTA-Abs and automated or semi automated processing and objective reading of results

55. Western Blot

56. Performance of serological tests for syphilis   Percentage of sensitivity by stage of untreated syphilis Test Primary Secondary Latent Late Specificity VDRL 78 (74–87) 100 96 (88–100) 71 (34–94) 98 (96–99) RPR card 86 (77–99) 100 98 (95–100) 73 98 (93–99) USR 80 (72–88) 100 95 (88–100)   99 TRUST 85 (77–86) 100 98 (95–100)   99(98–99) FTA-ABS 84 (70–100) 100 100 96 97 (84–100) FTA-ABS DS 80 (70–100) 100 100   98 (97–100) TP-PA 88 (86–100) 100 100   96 (95–100) IgM EIA 93 85 64     Immune capture EIA 77 100 100 100 99 Chemiluminescence assay 98 100 100 100 99

57. RPR TPHA IgM EIA _ _ _ No syphilis or incubating syphilis _ _ + Early primary syphilis + + + Primary or secondary + _ + Early infection + + _ Late secondary or latent + _ _ Biologic false positive, late syphilis _ + _ Late infection, treated syphilis or false positive treponemal test Increasing + Increasing Re- infection, relapse Interpretation

58. Diagnosis according to stages Early syphilis Dark field microscopic examination : - Most specific and sensitive Non treponemal tests: - Positive in 80% cases Treponemal tests: - Positive in 80 - 90 % cases

59. 2. Secondary syphilis Dark field microscopic examination: - fluid from moist wet lesions and lymph node aspirate Non treponemal tests: - Always positive, usually at a high dilution Treponemal tests: - Always positive

60. Early Latent syphilis Non treponemal tests: - positive in 95-98% cases Treponemal tests: - positive in 97-100% cases Diagnosis  based on reactive serological tests- treponemal and non treponemal in absence of any apparent signs of disease

61. Late Latent syphilis Non treponemal tests : - positive in 34 - 94% cases Treponemal tests : - positive 94 - 96 % cases

62. Diagnosis of Neurosyphilis CSF examination is done in : Patients with neurosyphilis In patients with syphilis of more than 2 years duration to exclude asymptomatic neurosyphilis Before retreatment of patients who have had relapses after any form of treatment As a follow up procedure for patients who have been treated for neurosyphilis In all infants suspected of prenatal syphilis

63. CSF sample is taken and a cell count is made It is further checked for protein abnormalities and subjected to VDRL test Diagnosis of neurosyphilis is indicated by 1. I ncreased cell count (> 10 lymphocytes per mm 3 of CSF) 2. Increased proteins (> 40 mg% in the CSF) 3. REACTIVE VDRL test Serum VDRL test is reactive in about 2/3 rd of the cases

64. Cardiovascular syphilis - Serological tests - usually reactive, esp. if extensive involvement Negative reaction may accompany a localized lesion Congenital syphilis : Demonstration of T. pallidum by direct examination from nasal discharge or from early lesions Positive treponemal test in a titre, higher than mother or serially rising FTA- IgM test is more specific with infection

65. Diagnostic Algorithm

66. Diagnosis of Syphilis in HIV Unusual serologic responses have been observed among HIV-infected persons who have syphilis 1. Serologic titers higher than expected 2. False negative serologic test results 3. Delayed appearance of seroreactivity Both treponemal and nontreponemal serologic tests should be interpreted in usual manner for majority of patients who are coinfected with T. pallidum and HIV.

67. When clinical findings are s/o syphilis but serologic tests are nonreactive, alternative tests (e.g., biopsy of a lesion, DGM, or DFA staining of lesion material) might be useful for diagnosis Neurosyphilis should be considered in the differential diagnosis of neurologic disease in HIV- infected persons.