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  1. Serological Diagnosis of SyphilisSkill Based Learning Dr.T.V.Rao MD Dr.T.V.Rao MD

  2. Syphilis "He who knows syphilis, knows medicine" Sir William Osler Dr.T.V.Rao MD

  3. Syphilis was a Taboo • Poster for testing of syphilis, showing a man and a woman bowing their heads in shame (ca. 1936). Dr.T.V.Rao MD

  4. SYPHILISINTRODUCTION • Caused by Treponema pallidum. • Transmission: sexual; maternal-fetal, and rarely by other means. • Primary and secondary syphilis in the US dropped by ~ 90 %t from 1990 to 2000, the number of cases have gone up since then. • A dramatic increase in cases in men from 2000 to 2002 reflected syphilis in MSM. • Syphilis increases the risk of both transmitting and getting infected with HIV. Perform HIV testing in all patients with syphilis. Dr.T.V.Rao MD

  5. Introduction to Syphilis • Syphilis is one of a group of diseases caused by spirochete organisms of the genus Treponema. Sexually acquired syphilis occurs worldwide and is caused by T. pallidum subspecies pallidum. Dr.T.V.Rao MD

  6. Other Related to Treponemes • Related Treponemes cause the non-venereal treponematosesbejel, or endemic syphilis (T. pallidum endemicum), yaws (T. pallidum pertenue), and pinta (T. carateum). Dr.T.V.Rao MD

  7. 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) Dr.T.V.Rao MD

  8. Diagnosis of Syphilis • The nontreponemal tests, VDRL and rapid plasma reagent (RPR), are antilipoidalantibodies seen in other disease states, pregnancy, and occasionally after vaccination. They are nonspecific and cannot rule in disease. These tests have sensitivities approaching 80% in patients with symptomatic primary syphilis and virtually 100% in patients with secondary syphilis. • – A positive VDRL/RPR should be quantified and titers followed at regular intervals after treatment. As such, its value is in response to treatment. However, it does not correlate with symptom resolution. • – Most patients have nonreactive nontreponemal tests within several years after successful treatment for syphilis, but a significant number have persistently positive tests, the so-called serofast reaction. Dr.T.V.Rao MD

  9. Diagnosis Laboratory Diagnosis • Identification of Treponema pallidum in lesions • Darkfield microscopy • Direct fluorescent antibody - T. pallidum (DFA-TP) • Serologic tests • Nontreponemal tests • Treponemal tests

  10. Advantages: Rapid and inexpensive Easy to perform and can be done in clinic or office Quantitative Used to follow response to therapy Can be used to evaluate possible reinfection Disadvantages: May be insensitive in certain stages False-positive reactions may occur Prozone effect may cause a false-negative reaction (rare) Diagnosis Nontreponemal Serologic Tests (continued)

  11. Diagnosis • Patients with a reactive VDRL or RPR should have the result confirmed by specific treponemal testing. FTA-ABS and or EIA. • • Tertiary syphilis Serology is used in the diagnosis. Evaluation of neurosyphilis requires a lumbar puncture (LP) and evaluation of the CSF. • – The CDC currently recommends LP only if the patient is seroreactive and HIV positive, has symptoms of neurosyphilis Dr.T.V.Rao MD

  12. Tests to Confirm • Syphilis may be confirmed either via blood tests or direct visualization using microscopy. Typical diagnosis is with blood tests using nontreponemal and/or treponemal tests. Nontreponemal test are used initially and include venereal disease research laboratory (VDRL) and rapid plasma regain however as these test occasionally are falsely positive confirmation is required with a treponemal test such as treponemal pallidum particle agglutination (TPHA) or fluorescent treponemal antibody absorption test (FTA-Abs) Dr.T.V.Rao MD

  13. The VDRL Testing Procedure Dr.T.V.Rao MD

  14. VDRL - Background • The Venereal Disease Research Laboratory (VDRL) test is one of two variations of flocculation procedures used for serological testing of syphilis, the other being the Rapid Plasma Reagin (RPR). Flocculation testing is based on antibody detection with the interaction of soluble antigen with an antibody that results in a precipitate formation of fine particles. Dr.T.V.Rao MD

  15. VDRL Test Basics • The VDRL is a confirmatory serological micro flocculation slide test used for the detection of syphilis antibodies. In a VDRL procedure, the patient’s serum is heat-inactivated and mixed with a buffered saline suspension of VDRL Antigen containing cardiolipin, lecithin and cholesterol that binds with Reagin, an antibody-like protein. A combination of Reagin and VDRL Antigen form microscopic clumping called flocculation. Dr.T.V.Rao MD

  16. VDRL – A Standard Test for Syphilis • The VDRL can be used for qualitative and quantitative measurements and is recommended when a patient suspected of having syphilis has a negative dark field microscopy result or when atypical lesions are present. Dr.T.V.Rao MD

  17. VDRL Serological Procedure Principles • VDRL Antigen is a nontreponemal antigen composed of cardiolipin cholesterol and lecithin. The nontreponemal tests measures anti-lipid antibodies, which are formed by the host in response to lipids released from damaged host cells early in infection with T. pallidum, and lipid-like material form the treponemal cell surface. During syphilis infection, an antibody-like substance called reagin can be detected in the patient’s serum or CSF. Dr.T.V.Rao MD

  18. Preparation of Antigen • Prepare a fresh antigen suspension each testing day. Once prepared, it should be used within 8 hours. • Store prepared suspension at 23-29)C. • Test antigen suspension reactivity with control sera (Reactive, Weakly reactive and Nonreactive). Test serum dilutions within 1 hour after heat inactivation. • Use antigen suspension only if it produces the expected reactivity with the control sera comparable to results obtained with the reference antigen. Dr.T.V.Rao MD

  19. Required Materials • VDRL Antigen with buffered saline solution containing 1% sodium chloride, pH 6.0+/-0.1 with 0.05% formaldehyde preservative • Reactive, weakly reactive and nonreactive serum • 0.9% saline, non-disposable 1cc glass syringe and calibrated needles without bevel-18 gauge(serum) or 21-22 gauge(CSF), slide cards(serum) or concavity slides(CSF) • Stirrers • Rotator Dr.T.V.Rao MD

  20. Specimen Collection and Preparation for Serum • Collect 5-8 ml of blood by aseptic venipuncture in a red top tube. • Allow blood to clot at room temperature then centrifuge to obtain serum. • Heat the test sera at 560C for 30 minutes. • Specimen must be at 23-290C when tested. • Specimen must be clear of hemolysis and show no visible evidence of bacteria contamination. • Store at room temperature for 4 hours, after which store at 2-80C, maybe refrigerated up to 5 days, then frozen at <-200C. Dr.T.V.Rao MD

  21. Specimen Collection and Preparation for CSF • Centrifuge and decant the specimen • Specimens do not require heat inactivation before testing. • Spinal fluids that are visibly contaminated or that contain gross blood are unsatisfactory Dr.T.V.Rao MD

  22. Antigen Suspension Preparation • Pipette 0.4ml of VDRL buffered saline to the bottom of a round 30 ml glass stoppered bottle with a flat inner-bottom surface. Gently tilt bottle so that VDRL buffered saline will cover the entire inner-bottom surface of the bottle. • Add 0.5 ml of VDRL Antigen directly into the saline while continuously but gently rotating the bottle on a flat surface from the lower half of a 1.0 ml pipette graduated cylinder to the tip. Add antigen drop by drop at a rate that allows about 6 sec for 0.5 ml of antigen. Keep pipette tip in the upper third of the bottle and do not splash saline unto the pipette. Dr.T.V.Rao MD

  23. Antigen Suspension Preparation • Expel the last drop of antigen without touching pipette to the saline and continue rotation of the bottle for 10 sec. • Add 4.1 ml of buffered saline from a 5 ml pipette. Do not drop saline directly on antigen; allow it to flow down the side of the bottle. • Cap the bottle and mix by gentle inversion. Allow to stand for 5 minutes but no more than 2 hours. The suspension is ready for use. • Remix suspension by swirling only Dr.T.V.Rao MD

  24. Antigen Suspension • Cap the bottle and mix by gentle inversion. Allow to stand for 5 minutes but no more than 2 hours. The suspension is ready for use. • Remix suspension by swirling only Dr.T.V.Rao MD

  25. Procedure: Step 1 Wells should be labeled as reactive ®, weakly reactive (WR), and nonreactive (NR), Dr.T.V.Rao MD

  26. Procedure: Step 3 Add one drop (.01 ml) of sensitized antigen suspension to each specimen with a 21 or 22 gauge needle. Dr.T.V.Rao MD

  27. Procedure: Step 4 • Rotate slides for 8 minutes on a mechanical rotator at 180 rpm. Note: when the tests are performed in a dry climate, the slides may be covered with a box lid to prevent evaporation. Dr.T.V.Rao MD

  28. Results for Serum Specimen • Qualitative Testing - Medium to large clumps (Reactive); Small clumps (Weakly Reactive); No clumping or very slight roughness (Nonreactive). • Verify control sera results for expectation. If reactions are not as expected, the test is invalid and results can not be reported. Dr.T.V.Rao MD

  29. Reporting the Results • Perform a quantitative test to endpoint on all serum samples that produce reactive, weakly reactive or “rough” nonreactive results in the qualitative slide test. • Quantitative Testing - Report the titer as the highest dilution that produces a Reactive (not weakly reactive) results Dr.T.V.Rao MD

  30. Diagnosis Diagnosis of CNS Infection with Syphilis No test can be used alone to diagnose neurosyphilis. • VDRL-CSF: highly specific but insensitive • Diagnosis usually depends on the following factors: • Reactive serologic test results, • Abnormalities of CSF cell count or protein, or • A reactive VDRL-CSF with or without clinical manifestations. • CSF leukocyte count usually is elevated (>5 WBCs/mm3) in patients with Neurosyphilis. • The VDRL-CSF is the standard serologic test for CSF, and when reactive in the absence of contamination of the CSF with blood, it is considered diagnostic of Neurosyphilis.

  31. Specimen Collection and Preparation for CSF • Centrifuge and decant the specimen • Specimens do not require heat inactivation before testing. • Spinal fluids that are visibly contaminated or that contain gross blood are unsatisfactory Dr.T.V.Rao MD

  32. TestingCSFSamples Quantitative tests are run on all spinal fluids found to be reactive in the qualitative test. Prepare fluid as follows: A. Pipette 0.2 ml of 0.9% saline into each of 5 or more tubes. Dr.T.V.Rao MD

  33. Testing of CSF Samples Add 0.2ml of unheated spinal fluid to tube 1, mix well and transfer 0.2 ml to tube 2 . Dr.T.V.Rao MD

  34. Testing of CSF Samples Continue mixing and transferring 0.2 ml from one tube to the next until the last tube is reached. The respective dilutions are 1:2, 1:4, 1:8, 1:16. Etc., Dr.T.V.Rao MD

  35. Reporting CSF Samples 2. Test each spinal fluid dilution and undiluted spinal fluid as described under “VDRL slide qualitative on spinal fluid.” 3. Report results in terms of the greatest spinal fluid dilution (dils) that produces a reactive result. Dr.T.V.Rao MD

  36. All Positive Samples tested by Quantitative Method • In Quantitative Testing - Report the titer in terms of the highest dilution that produces a reactive (not weakly reactive) result. Dr.T.V.Rao MD

  37. Quantitative Testing and Reporting • In Quantitative Testing- Report the titer in terms of the highest dilution that produces a reactive (not weakly reactive) result. Dr.T.V.Rao MD

  38. Interpretation • Nonreactive VDRL - with clinical evidence may indicate early primary syphilis, a prozone reaction in secondary or late syphilis. • Nonreactive VDRL- with no clinical evidence may indicate no current infection or an effectively treated infection. • Quantitative VDRL- detects changes in reagin titer. Serum samples displaying a fourfold increase in titer on a repeated sample may indicate an infection, reinfection or treatment failure. A fourfold decrease during treatment indicates adequate therapy. Dr.T.V.Rao MD

  39. Sources of Error • False positive reactions - occur in 10% to 30% of positive serological tests for syphilis and consist of nonsyphilitic positive VDRL. reactions with cardiolipin type antigens. • False negative reactions - consist of conditions and a variety of situations. • Weakly reactive- caused by very early infection, lessening of the activity of the disease after treatment and improper technique or questionable reagents. Dr.T.V.Rao MD

  40. Lupus erythematosus Rheumatic fever Vaccinia and virus pneumonia Pneumococcal pneumonia Infectious mononucleosis Infectious hepatitis Leprosy Malaria Rheumatoid arthritis Pregnancy Aging individuals False Positive Reactions Dr.T.V.Rao MD

  41. False Negative Reactions • Technical error - unsatisfactory antigen or technique. • Low antibody titers • Presence of inhibitors in the patient’s serum • Reduced ambient temperature (below 230 to 290) • Prozone reaction Dr.T.V.Rao MD

  42. RPR test The RPR test is a nontreponemal testing procedure for the serologic detection of syphilis. Dr.T.V.Rao MD

  43. Principle of RPR Test • The RPR Card antigen suspension is a carbon particle cardiolipin antigen that detects reagin. • Reagin is an antibody like substance present in serum or plasma from individuals with syphilis. • The reagin binds to the test antigen which consists of cardiolipin-lecithin coated particles that cause macroscopic flocculation. Dr.T.V.Rao MD

  44. Principle of RPR • When a specimen such as serum or plasma contains antibody, flocculation occurs with the resulting aggregation of the carbon particles. • The flocculation appears as black clumps against the white background of the plastic coated card. Dr.T.V.Rao MD

  45. Principle of RPR • Antibodies associated with syphilis begin to appear in the blood 4 to 6 weeks after infection. Nontreponemal tests determine the presence of reagin. Reagin is a nontreponemal autoantibody directed against cardiolipin antigens. Dr.T.V.Rao MD

  46. Materials for RPR • RPR Test Cards • RPR Control Cards • RPR Antigen • Distilled Water • Dispenstirs • Rotator Dr.T.V.Rao MD

  47. RPR Test Background • The RPR test uses a white plastic coated card that consist of several circles that are 18 mm in diameter. • The controls which are strongly reactive, moderately reactive, and non-reactive are contained on the control card in a dried form. Dr.T.V.Rao MD

  48. Specimen Collection • Unheated Plasma - specimen should be collected with an anticoagulant such as EDTA or heparin, plasma must be stored at 2°C to 8°C. Plasma must be tested within in 24 hrs. of collection Dr.T.V.Rao MD

  49. *The addition of choline chloride, which inactivates complement enables the serum to be tested without prior heating. Unheated serum- centrifuge for sedimentation of cellular elements, serum may be frozen until time of testing. Heated Serum- transfer serum to clean tube and place in 56°C water bath for 30 minutes Specimen Processing Dr.T.V.Rao MD

  50. Prepare the Card • Label rings on test card with numbers of samples to be tested • Use Dispenstir to draw up serum sample. • Hold Dispenstir in a perpendicular position directly over the test circle to which the specimen is to be delivered. • Squeeze Dispenstir to allow 1 drop to fall on to each circle Dr.T.V.Rao MD

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