1.SHUMEZ H.

2. Fritz Richard Schaudinn Paul Erich HoffmannZoologistDermatologist 3. Classification Kingdom Eubacteria PhylumSpirochaetes Class Spirochaetes Order Spirochaetales FamilyTreponemataceae Genus Treponema Species T. pallidum 4. The family Treponemataceae contains 3 genera Borrelia Treponema Leptospira 5. Subspecies There are at least four known subspecies of Treponemapallidum: Treponema pallidum pallidum Human Syphilis T. pallidum endemicum - Bejel T. pallidum pertenue - Yaws T. pallidum carateum Pinta Treponema cuniculi Rabbit syphilis 6. Avirulent strains Avirulent strains of Treponema pallidum: Reiter Nichols non pathogenic Noguchi Kazan Kroo 7. Microscopy Live treponemes are too slender to be seen byconventional light microscopy. Can be seen with : Negative staining with Indian Ink. Dieterle stain. Steiner stain. Warthin Starry stain. Can also be visualized by using dark-field microscopy. 8. Structure Helically coiled, corkscrew-shaped organisms. 6 to 15 long and 0.1 to 0.2 wide. Present 6-14 spirals. Ends are pointed with finely spiral terminal filaments. Exhibits characteristic motility. Consists of rapid rotation about its longitudinal axisand bending, flexing, and snapping about its fulllength. 9. Dark Field Microscopy 10. Electron Microscopy 11. Characteristics Fastidious organism that exhibits narrow optimalranges of : pH (7.2 to 7.4) Temperature (30 to 37C). It is rapidly inactivated by mild heat, cold,desiccation, and most disinfectants. Considered a strict anaerobe, but it is now known tobe microaerophilic. Treponemes multiply by binary transverse fission. 12. In Vivo generation time is about 30 hours. Not been successfully cultured In Vitro. Viable organisms can be maintained for 18 to 21 days incomplex media. Limited replication has been obtained by co-cultivation with tissue culture cells. 13. Ultra Structure Cytoplasmic membrane Cell wall (a thin peptidoglycan layer) Outer membrane layer Periplasmic flagella (also called endoflagella) Cytoplasmic filaments, also known as Cytoplasmicfibrils 14. The trilaminar Cytoplasmic membrane covers theprotoplasmic cylinder. The Murein layer overlies the cytoplasmic membrane. The organism has an Outer membrane which is lipidrich and contains low density of transmembraneproteins. This membrane contains the majority of thebacteriums integral membrane proteins andlipoproteins. 15. From each end of the cell, 3-4 Endoflagella wind roundthe axis of the cell. These Endoflagella do not protrude outside, butremain within the Outer membrane layer. The flagellar filament has a sheath and core structureand is composed of four major polypeptides. 16. Cryo Electron Tomography (CET) Image 17. Biochemical Structure Treponema is composed of approximately 70 %proteins, 20 % lipids, and 5 % carbohydrates. This lipid content is relatively high for bacteria. The lipid composition of T pallidum is complex,consisting of several phospholipids, includingcardiolipin, and a poorly characterized glycolipid. 18. Sample collection For direct examination, exudates from lesions ofprimary, secondary and early congenital syphilis arethe most useful. Clear, serous fluid free of erythrocytes, tissue debrisand other organisms is collected. Serum is the specimen of choice for bothnontreponemal and treponemal serological tests. Cerebrospinal fluid (CSF) testing is indicated incongenital and tertiary syphilis and when neurologicalsymptoms are present. 19. Laboratory Diagnosis Identification of Treponema pallidum in lesions Serologic tests Nontreponemal tests Treponemal tests20 20. Dark-field microscopy Simplest and most reliable method. Exudates and fluids from lesions are examined as a wetmount. Identification of T pallidum is based on thecharacteristic morphology and motility. This method is suitable when the lesions are moist. Examination should be done immediately afterspecimen collection. 21. This technique requires a trained, experiencedmicroscopist. Treatment with antibiotics may result in a false-negative finding. Dark-field microscopy has limited sensitivity. 22. Direct fluorescent antibody testfor T. pallidum It detects antigen and, thus, does not require thepresence of motile treponemes. Uses fluorescein isothiocyanate-labelled antibodyspecific to pathogenic treponemes Suitable for the examination of specimens from oraland rectal lesions. Does not differentiate between T pallidum and otherpathogenic treponemes. 23. Animal Inoculation Oldest method for detecting infection with T.pallidum. Rabbits were inoculated intratesticularly with T.Pallidum . 24. Nucleic acid amplification methods Highly sensitive Able to detect as low as one to 10 organisms perspecimen with high specificity. Used to monitor treatment . Used to differentiate new infections from oldinfections. May be available only through select laboratories. 25. Nontreponemal tests are rapid, simple andinexpensive. They are the only tests recommended to monitor thecourse of disease during and after treatment. Nontreponemal tests can also serve to detectreinfection. They are also used as screening tests. Limitations low specificity, low sensitivity in primaryand late latent syphilis, false-positive results 26. These include : Complement Fixation Tests Kahn Flocculation test VDRL Unheated Serum Reagin Test (USR) Rapid Plasma Reagin Test (RPR) Toluidine Red Unheated Serum Test (TRUST) 27. COMPLEMENT FIXATION TEST First developed by Wassermann in 1906. In this method, the patients serum containingantibodies is made to react with a standardisedantigen. Wassermann antigen extract of liver from newbornswho had died of congenital syphilis. Cholesterol and Lecithin were added to increasesensitivity of antigens. Complicated to perform, required many reagents and24 h to complete 28. KAHN FLOCCULATION TEST In 1922, Kahn introduced a occulation test withoutcomplement that could be read macroscopically in afew hours. Kahn antigen alcoholic extract of fresh beef heartwith cholesterol. On reaction with syphilitic serum, floccules areformed which can be seen with the naked eye. Standardization of the tests was difcult. 29. VDRL Slide micro flocculation test. Serum or CSF can be used. The basis of the test is that an antibody produced by apatient with syphilis reacts with an extract of ox heart Visualized through foaming of the test tube fluid, or"flocculation". It therefore detects anti-cardiolipin antibodies. Antigen - 0.03% cardiolipin, 0.21% lecithin and 0.9%cholesterol. 30. A reactive VDRL is seen in about 50-75% of patientswith primary syphilis and 100% in patients withsecondary syphilis. VDRL test can be quantitated by examining serialdilutions of serum and can be used to follow thecourse of illness, including the response to therapy. A dilution of > 1:8 is suggestive of syphilis. VDRL yields reproducible results, can be rapidlyperformed, acceptable levels of sensitivity andspecicity, valuable tool for mass screening 31. Biological false positiveSince the test employs a non-treponemal antigen, thereare many chances of biological false positive results. Pregnancy Menstruation Repeated blood loss Vaccination Severe trauma Antiphospholipid syndrome Drug addiction 32. SLE and other collagen vascular disorders Hepatitis or any other liver disease Malaria, Filariasis,Tuberculosis Malignancy Tropical eosinophilia Lepromatous leprosy Infectious mononucleosisProzone reactions are false-negative reactions that occur due to interference by high concentrations of target antibodies in a specimen. 33. UNHEATED SERUM REAGIN TEST Quantitative, microscopic, non treponemal,flocculation test similar to VDRL. The VDRL antigen is enhanced by the addition ofcholine chloride and EDTA. So the need for heating serum was eliminated. Plasma could also be used an acceptable samplesource. 34. RAPID PLASMA REAGIN TEST Rapid Plasma Reagin (RPR) test is a macroscopic NonTreponemal flocculation test , and is a simplifiedversion of the VDRL test. The RPR test uses a stabilized suspension of VDRLantigen to which charcoal particles are added to aidin the visualization of the test reaction. Serum or plasma can be used. RPR Teardrop card test and RPR 18mm circle card testare further refinements of this test which are usedcurrently for screening. 35. TRUST Toluidine Red Unheated Serum Test (TRUST) is amacroscopic Non Treponemal flocculation test. In the TRUST test, particles of toluidine red are usedin place of the charcoal particles of the RPR test as thevisualising agents. Serum or plasma can be used. Quantitative values allow evaluation of recentinfection and response to treatment. Used forscreening and follow up of therapy 36. Treponemal tests may remain reactive for years with orwithout treatment Treponemal test antibody titres correlate poorly withdisease activity. Therefore, treponemal tests should not be used toevaluate response to therapy, relapse or reinfection inpreviously treated patients. Treponemal tests do not differentiate venereal syphilisfrom endemic syphilis (yaws and pinta). Treponemal tests are used mainly as confirmatory teststo verify reactivity in nontreponemal tests. 37. These include : T. pallidum Immobilization (TPI) Reiters Antigen CFT Fluorescent Treponemal Antibody test (FTA) FTA Absorption test (FTA - ABS) T. pallidum haemagglutination assay (TPHA) T. pallidum Particle Agglutination Assay (TPPA) MHA TP PK TP 38. TPI T. pallidum immobilization (TPI) test Antigen - T. pallidum (Nichols strain) grown in rabbittestes. It is based on the ability of patients antibody andcomplement to immobilize living treponemes, asobserved by dark-eld microscopy. However, the TPI test was complicated, technicallydifcult, time-consuming, expensive to perform and isnot used much now. 39. REITERs ANTIGEN CFT Reiter protein complement xation test. Antigen - prepared from T. phagedenis, the Reitertreponeme, a nonpathogenic organism was used in acomplement xation test. High proportion of false-positive reactions. Less specic and sensitive than the TPI test. 40. Fluorescent treponemalantibody (FTA) test The FTA procedure uses a 1:5 dilution of the patientsserum in saline solution, reacted with a suspension ofReiter treponeme. FITC(uorescein isothiocyanate) was used as theconjugate, and the test was read under a microscopewith a UV light source. Nonspecic reactions were encountered inapproximately 25% of normal serum specimens. 41. To eliminate these false-positive reactions, the test wasmodied by diluting the patients serum 1:200, theFTA-200 test. The FTA 200 is highly specific but not very sensitive. 42. FTA absorption (FTA-ABS) test Generally regarded as the gold standard test forconfirming diagnosis. FTA-ABS is the most sensitive test in all stages ofsyphilis. The patients diluted serum (1:5) is added to the Reiterantigen and group treponemal antibodies areabsorbed leaving behind specific antibodies in theserum. 43. Results are reported as reactive, reactive minimal,nonreactive, or atypical uorescence It is a subjective test and difficult to standardise. Less than 1% false positives are due to HIV, SLE, RA orold-age. The FTA-ABS double staining test is a modification ofthe FTA-ABS test using a double staining procedurewith the addition of a contrasting counterstain. 44. T. pallidum haemagglutination test. The most appropriate test for confirming diagnosis. It is an indirect haemagglutination assay. Antigen formalinized, tanned, erythrocytessensitized with ultrasonicated material from T.pallidum (Nichols strain). The presence of treponemal antibody in the patientsserum is detected by the indirect agglutination of thesensitized erythrocytes and the subsequent formationof a mat of erythrocytes upon their settling. 45. Results are reported as reactive, nonreactive, orinconclusive. Specificity - 99% Biological False Positive in some cases of Leprosy. If gelatin particles are used instead of erythrocytes,test is called T. pallidum particle agglutination assay(TPPA). Microhaemagglutination assay for antibodies to T.pallidum (MHA-TP) uses reagents for a microvolumehaemagglutination test. Haemagglutination Treponemal test for Syphilis(HATTS) is another variant. 46. PK- T. pallidum (PK-TP) test PK-TP is a new haemagglutination test which hasachieved provisional status. The PK-TP reagent is composed of chickenerythrocytes which have been xed and thensensitized with components of sonicated T. pallidum. 47. Enzyme Immuno Assay A number of treponemal EIA tests are now available: Captia Syphilis M Captia Syphilis G Captia select Syph-G SpiroTek syphilis test Enzygnost Syphilis These are newer tests with provisional status which are being used now in a number of laboratories. 48. THANK YOU