Serological Diagnosis of Syphilis

Serological Diagnosis of Syphilis Skill Based Learning:

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

Syphilis :

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

Syphilis was a Taboo:

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 3

SYPHILIS INTRODUCTION:

SYPHILIS INTRODUCTION 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 4

Introduction to Syphilis:

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 5

Other Related to Treponemes :

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

STAGES OF SYPHILIS:

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 7

Diagnosis of Syphilis:

The nontreponemal tests, VDRL and rapid plasma reagent (RPR), are antilipoidal antibodies 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 8 Diagnosis of Syphilis

Laboratory Diagnosis:

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

Nontreponemal Serologic Tests (continued):

10 Nontreponemal Serologic Tests (continued) 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

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 11 Diagnosis

Tests to Confirm :

S yphilis 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) Tests to Confirm Dr.T.V.Rao MD 12

The VDRL Testing Procedure :

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

VDRL - Background:

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 14

VDRL Test Basics:

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 15

VDRL – A Standard Test for Syphilis:

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 16

VDRL Serological Procedure Principles:

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 17

Preparation of Antigen:

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 18

Required Materials:

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 19

Specimen Collection and Preparation for Serum:

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 56 0 C for 30 minutes. Specimen must be at 23-29 0 C 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 2-8 0 C, maybe refrigerated up to 5 days, then frozen at <-20 0 C. Dr.T.V.Rao MD 20

Specimen Collection and Preparation for CSF:

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 21

Antigen Suspension Preparation:

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 22

Antigen Suspension Preparation:

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 23

Antigen Suspension:

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 24

Procedure: Step 1:

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

Procedure: Step 3:

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 26

Procedure: Step 4:

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 27

Procedure: Step 5:

Procedure: Step 5 Read test immediately after rotation with a 10x objective and Record the readings . Dr.T.V.Rao MD 28

Results for Serum Specimen:

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:

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 of CNS Infection with Syphilis:

31 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/mm 3 ) 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. Diagnosis

Specimen Collection and Preparation for CSF:

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

Testing CSF Samples :

Testing CSF Samples 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:

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:

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:

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:

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:

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:

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:

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

False Positive Reactions:

False Positive Reactions Lupus erythematosus Rheumatic fever Vaccinia and virus pneumonia Pneumococcal pneumonia Infectious mononucleosis Infectious hepatitis Leprosy Malaria Rheumatoid arthritis Pregnancy Aging individuals Dr.T.V.Rao MD 41

False Negative Reactions:

False Negative Reactions Technical error - unsatisfactory antigen or technique. Low antibody titers Presence of inhibitors in the patient’s serum Reduced ambient temperature (below 23 0 to 29 0 ) Prozone reaction Dr.T.V.Rao MD 42

RPR test :

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:

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:

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:

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:

Materials for RPR RPR Test Cards RPR Control Cards RPR Antigen Distilled Water Dispenstirs Rotator Dr.T.V.Rao MD 47

RPR Test Background:

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:

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

Specimen Processing :

Specimen Processing * 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 Dr.T.V.Rao MD 50

Prepare the Card:

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 51

Performing the Test:

Performing the Test Invert Dispenstir,and using the sealed end spread the specimen in the confines of the circle. Reconstitute the antigen bottle, by shaking. Holding the bottle in a straight vertical position drop one or two drops in the upper corner of each test circle, then place one “free falling” drop on each test area. Dr.T.V.Rao MD 52

Rotate card at Regulated Speed:

Rotate card at Regulated Speed Rotate card for 8 minutes on a mechanical rotator at 100 rpm. The test card she also be covered with a humidifier cover. After rotating mechanically, the test card should be rotated manually by hand 3 to four rotations and then read immediately macroscopically in the “wet” state under a high intensity lamp. Dr.T.V.Rao MD 53

Procedure for Controls:

Procedure for Controls A. Use Dispensator to draw up distilled water B. Drop 1 drop on the card test circle for each patient sample. C. Invert Dispensator and spread the water in the circle until the dried control is completely reconstituted. D. Add antigen as described for the patients E . Rotate for 8 minutes at 100 rpm Dr.T.V.Rao MD 54

Reactions of Controls:

Reactions of Controls The following reactions should be observed to compare against the test results: Reactive control - characteristic strong clumping. Reactive moderate control - moderate clumping. Non-reactive control - smooth, grayish appearance of unclumped particles Dr.T.V.Rao MD 55

Observe for Reactivity :

Observe for Reactivity Dr.T.V.Rao MD 56

A non reactive RPR sample:

A non reactive RPR sample Dr.T.V.Rao MD 57

Repeat all Positive Samples after Dilutions:

Repeat all Positive Samples after Dilutions Dr.T.V.Rao MD 58

Explanation of Results:

Explanation of Results A negative RPR test may indicate one of the following: 1. The patient does not have syphilis. 2. The infection is too recent for antibodies to be produced. (Repeated tests should be administered at 1 week, 1 month, and 3 month intervals to establish presence or absence of disease). 3. The syphilis is latent or inactive 4. Faulty immunodefense mechanism 5. Faulty lab techniques Dr.T.V.Rao MD 59

Explanation of Results:

Explanation of Results A positive reaction is not conclusive for syphilis. Several conditions produce biologic false positive results for syphilis. (False positive means that the test revealed a positive reaction when it was actually negative). False positives may reveal the presence of other serious diseases. Dr.T.V.Rao MD 60

Nontreponemal positive Tests Need Confirmation:

Nontreponemal positive Tests Need Confirmation Nontreponemal antigen tests are not entirely specific for syphilis and do not have satisfactory sensitivity in all stages of syphilis. Whenever the results of a nontreponemal antigen test disagree with the clinical impression, a treponemal antigen test such as the FTA-ABS should be performed . Dr.T.V.Rao MD 61

Non-syphilitic Conditions Giving Biologic False-Positive Results:

Non-syphilitic Conditions Giving Biologic False-Positive Results Malaria Leprosy Relapsing fever Infectious Mononucleosis Atypical pneumonia Viral pneumonia Lupus erythematous Measles pregnancy drug abuse Dr.T.V.Rao MD 62

Resolving False Positive RPR Tests:

Resolving False Positive RPR Tests False positive RPR tests may be resolved by testing the patient’s serum with a specific treponemal antigen tests . Dr.T.V.Rao MD 63

Treponema-Specific Tests for Syphilis :

Dr.T.V.Rao MD 64 Treponema-Specific Tests for Syphilis

Confirmatory Tests for Syphilis:

Treponemal tests are used to confirm reactive non –treponemal procedures. TPHA testing is now routinely done A positive FTA-ABS test almost always remains positive and therefore is not recommended for monitoring therapy. Dr.T.V.Rao MD 65 Confirmatory Tests for Syphilis

TREPONEMAL TESTS:

TREPONEMAL TESTS FTA-ABS Used as a confirmatory tests. Sensitivity and specificity high. 85% of patients with primary syphilis are reactive 99% with secondary syphilis > 95% with late syphilis (It may be the only test with a positive result for patients with cardiovascular or neurologic syphilis). Remains reactive for life in most, despite adequate therapy. Only 15-25 % of those treated for primary syphilis may turn negative by 2-3 yrs. False positive in other treponemal diseases (pinta, yaws..) and other spirochete diseases (Lyme, leptospirosis…) MHA-TP test (microhemagglutination assay for T. pallidum; a gglutination of RBCs to which T. pallidum antigens have been fixed is the basis).

TPHA and FTA-ABS Testing are commonly used Confirmatory Tests :

Dr.T.V.Rao MD 67 TPHA and FTA-ABS Testing are commonly used Confirmatory Tests

For Routine Testing a Combination of VDRL or RPR and TPHA is highly preferred :

Dr.T.V.Rao MD 68 For Routine Testing a Combination of VDRL or RPR and TPHA is highly preferred TPHA Test is a sensitive passive haemagglutination test, that detects specific Treponema pallidum antibodies in serum within one hour. Used in combination, the VDRL or RPR and TPHA Tests provide accurate and reliable confirmation of active syphilis infection. No specialized equipment is required and results are clearly visible and easily interpreted.

Microhemagglutination assay (MHA-TP)and TPHA (T. pallidum hemagglutination assay) :

T he MHA-TP was the earlier iteration of the TPHA. Occasionally, these tests are simply referred to as an indirect hemagglutination assay (IHA). The hemagglutination tests generally are simpler to perform than the fluorescent antibody tests and lend themselves to automation. The MHA-TP and TPHA tests are very rarely used currently. Both tests are quickly being replaced by newer and easier TP-PA and EIA-based tests (see below), including lateral flow strip test Dr.T.V.Rao MD 69 Microhemagglutination assay (MHA-TP)and TPHA (T. pallidum hemagglutination assay)

Microhemagglutination assay:

The MHA-TP and TPHA are used to confirm a syphilis infection after another method tests positive for the syphilis bacteria. The MHA-TP and TPHA tests detect antibodies to the bacteria that cause syphilis and can be used to detect syphilis in all stages, except during the first 3 to 4 weeks when antibody levels are too low. These tests are also suitable for use as a screening procedure. Neither of these tests is suitable for use on cerebrospinal fluid (CSF). Dr.T.V.Rao MD 70 Microhemagglutination assay

Principles of TPHA Test:

TPHA (Treponema Pallidum Hemagglutination) is an indirect hemagglutination assay carried out on micro plates for the qualitative and semi-qualitative detection of anti- Treponema pallidum specific antibodies in human serum. Avian blood cells stabilized and sensitized with a solution of T. pallidum antigen agglutinate in the presence of anti-T Pallidum antibodies, exhibiting a typical agglutination pattern. Dr.T.V.Rao MD 71 Principles of TPHA Test

Reading TPHA Results:

Dr.T.V.Rao MD 72 Reading TPHA Results The upper, left-hand well contains a positive control test. The red cells have had treponemal antigens attached and antibodies in the serum have caused these cells to agglutinate and form a mat across the bottom of the well. These antibodies can be presumed to be specific for Treponemes, since otherwise identical red cells that have not had the treponemal antigens attached do not cause haemagglutination, as seen in the bottom, left-hand well. A negative serum test is shown in the center and a patient's test is on the right. This result supports a diagnosis of syphilis.

EIA tests for Syphilis:

Enzyme immunoassay (EIA), also known as an enzyme linked immunosorbent assay (ELISA), for syphilis is a relatively new invention first appearing on the market in the mid-1990s. There are numerous benefits to the EIA platform over earlier technologies. Firstly, the majority of diseases that are considered to be of clinical and public health importance already exist in an EIA format, which is highly standardized even across international boundaries. This familiarity allows new EIAs to be readily accepted by clinicians and technicians with minimal difficulty. It also limits the need to purchase new capital equipment since most labs will already be equipped to handle EIAs. Dr.T.V.Rao MD 73 EIA tests for Syphilis

EIA tests Syphilis Gaining Importance:

EIA tests Syphilis Gaining Importance There have been several developments in particularly the advent of enzyme immunoassays (EIAs ) and, lately, the commercial availability of recombinant antigen-based tests Dr.T.V.Rao MD 74

Fluorescent Treponemal Antibody Absorption (FTA-abs):

The FTA-abs test detects antibodies to T. pallidum and can be used to detect syphilis infection at any stage except during the first 3 to 4 weeks after exposure (which is about the same time frame that the VDRL/RPR tests become effective) and in tertiary stages of the disease. In the secondary stage of syphilis, the FTA-abs test is most reliable and is reportedly positive in 100 percent of cases. It can be adapted to detect either IgG or IgM antibody. Dr.T.V.Rao MD 75 Fluorescent Treponemal Antibody Absorption (FTA-abs)

Gold Standard Confirmatory test:

Dr.T.V.Rao MD 76 Gold Standard Confirmatory test The FTA-abs is still generally regarded as the ‘ gold standard ’, but it has a number of limitations. It is a subjective test and difficult to standardize. It is sensitive , but the TPHA is more sensitive, except in the third and fourth weeks of infection; the TPHA is also more specific2.

Sensitivity of Serological Tests in Untreated Syphilis :

77 Sensitivity of Serological Tests in Untreated Syphilis Stage of Disease (Percent Positive [Range]) Test Primary Secondary Latent Tertiary VDRL 78 (74-87) 100 95 (88-100) 71 (37-94) RPR 86 (77-99) 100 98 (95-100) 73 FTA-ABS* 84 (70-100) 100 100 96 Treponemal Agglutination* 76 (69-90) 100 97 (97-100) 94 EIA 93 100 100 * FTA-ABS and TP-PA are generally considered equally sensitive in the primary stage of disease.

Causes of False-Positive Reactions in Serologic Tests for Syphilis:

78 Causes of False-Positive Reactions in Serologic Tests for Syphilis Disease RPR/VDRL FTA-ABS TP-PA Age Yes Autoimmune Diseases Yes Yes Cardiovascular Disease Yes Yes Dermatologic Diseases Yes Yes -- Drug Abuse Yes Yes Febrile Illness Yes Glucosamine/chondroitin sulfate Possibly Leprosy Yes No -- Lyme disease Yes Malaria Yes No Pinta, Yaws Yes Yes Yes Pregnancy Yes* Recent Immunizations Yes -- -- STD other than Syphilis Yes Source: Syphilis Reference Guide, CDC/National Center for Infectious Diseases, 2002 *May cause increase in titer in women previously successfully treated for syphilis Diagnosis

AIDS and Syphilis:

Dr.T.V.Rao MD 79 AIDS and Syphilis The Serological Tests in AIDS and HIV related infections should be interpreted with caution and expertise, need a better understanding of the progress of the Disease.

Nontreponemal Serologic Tests:

80 Nontreponemal Serologic Tests Principles Measure antibody directed against a cardiolipin-lecithin-cholesterol antigen Not specific for T. pallidum Titers usually correlate with disease activity and results are reported quantitatively May be reactive for life Nontreponemal tests include VDRL, RPR, TRUST,

References:

Thomas B. Wiggers, Associate Professor Clinical Laboratory Sciences, UMMC Additional photos:www.Kumc.EDU Center for disease control (1999). Guidelines for evaluation and acceptance of new syphilis serology tests for routine use. US department of health, education and welfare publication, Atlanta . Wasley G.D. (1988). Syphilis serology. Oxford press, New York. Abbot laboratories, Abbott Park, IL 60064. Dr.T.V.Rao MD 81 References

Slide 82:

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