Laboratory diagnosis of syphilis

Simple presentation of syphilis lab diagnosis

1. Laboratory Diagnosis of Syphilis Patricia Antonette E. Pomar, RMT NRL-SLH/SACCL

2. Objectives Short introduction of Syphilis Stages of Syphilis Infection Laboratory Diagnosis of Syphilis Traditional vs. Reverse Algorithm for Syphilis Screening Algorithm used in NRL-SLH / SACCL Interpretation of result and follow up

3. Genus Treponema Etiologic agent: Treponema pallidum helically coiled, cork-screw shaped Cannot be cultured in-vitro Transmission Sexual Trans-placental Percutaneous following contact with infectious lesions Blood Transfusion Treponema pallidum on Darkfield Microscopy

4. Genus Treponema pallidum subspecies causes Treponema pallidum subsp. pallidum Venereal syphilis Treponema pallidum subsp. pertenue Yaws Treponema pallidum subsp. endemicum Endemic syphilis / Bejel Treponema pallidum subsp. carateum Pinta

5. Stages of Syphilis Infection Primary Secondary Latent Tertiary Development of primary lesion or ‘CHANCRE” at the site of inoculation Secondary lesions, most commonly mucocutaneous Host suppresses infection, but no lesions are clinically apparent Approximately 30% of untreated patients progress to the tertiary stage within 1 to 20 years Painless May persist from weeks to months Only evidence is a positive serologic test Rare because of the widespread availability and use of antibiotics Highly infectious Clinical manifestations: -Rash (75%–100%) -Lymphadenopathy (50%–86%) -Malaise -Mucous patches (6%–30%) - Condylomata lata (10%–20%) -Alopecia (5%) -Liver and kidney involvement can occur -Splenomegaly is occasionally present May occur between primary and secondary stages, between secondary relapses, and after secondary stage Manifestations - Gummatous lesions -Cardiovascular syphilis Heals within 3-6 weeks Categories: Early latent: <1 year duration Late latent:  1 year duration Serologic test may not be positive in early primary syphilis Serologic test are usually highest in titer

6. Primary Syphilis Source : CDC/ NCHSTP/ Division of STD Prevention /STD Clinical Slides

7. Secondary Syphilis Generalized rash Condylomata lata

8. Secondary Syphilis Nickel / Dime Lesions Alopecia

9. Tertiary Syphilis Serpiginous Gummata of Forearm Ulcerating Gumma

10. Late Syphilis—Cardiovascular 11 Source : CDC/ NCHSTP/ Division of STD Prevention, STD Clinical Slides

11. Neurosyphilis Occurs when T. pallidum invades the central nervous system (CNS) May occur at any stage of syphilis Can be asymptomatic Early neurosyphilis occurs a few months to a few years after infection Clinical manifestations can include acute syphilitic meningitis, meningovascular syphilis, and ocular involvement Neurologic involvement can occur decades after infection and is rarely seen Clinical manifestations can include general paresis, tabes dorsalis, and ocular involvement Ocular involvement can occur in early or late neurosyphilis. Spirochetes in Neural Tissue

12. Congenital Syphilis Occurs when T. pallidum is transmitted from a pregnant woman to her fetus May lead to stillbirth, neonatal death, and infant disorders such as deafness, neurologic impairment, and bone deformities Transmission can occur during any stage of syphilis; risk is much higher during primary and secondary syphilis Fetal infection can occur during any trimester of pregnancy Wide spectrum of severity exists; only severe cases are clinically apparent at birth Early lesions (most common): Infants <2 years old; usually inflammatory Late lesions: Children >2 years old; tend to be immunologic and destructive

13. Congenital Syphilis Mucous Patches Perforation of Palate

14. Laboratory Diagnosis

15. NON TREPONEMAL TREPONEMAL VDRL (Venereal Disease Research Laboratory) RPR (Rapid Plasma Reagin) TRUST (Toluidine Red Unheated Serum Test) USR (Unheated Serum Reagin) TP-PA (Treponema Pallidum Particle Agglutination) FTA-abs (Fluorescent Treponemal Antibody -Absorbed) EIA (Enzyme Immunoassay)

16. NON TREPONEMAL TESTS (Screening) Principles T. pallidum infection leads to the production of reagin Reagin – Antibodies to substances released from cells damaged by T. pallidum Reagin reacts with cardiolipin Cardiolipin – a phospholipid component of certain eukaryotic and prokaryotic membranes Not specific for T. pallidum Titers usually correlate with disease activity and results are reported quantitatively (can be tittered) May be reactive for life, referred to as “ serofast ”

17. NON TREPONEMAL Advantages Disadvantages 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 May be insensitive in certain stages False-positive reactions may occur Prozone effect may cause a false-negative reaction (rare)

18. NON TREPONEMAL TESTS RAPID PLASMA REAGIN (RPR) SERUM VENEREAL DISEASE RESEARCH LABORATORY (VDRL) CEREBROSPINAL FLUID Cardiolipin Charcoal Reagin Serum or CSF

19. NON TREPONEMAL TESTS

20. NON TREPONEMAL TESTS

21. TREPONEMAL TESTS Specific for T. pallidum Measure antibody (IgM & IgG) directed against T. pallidum antigens by particulate agglutination (TP-PA) or immunofluorescence (FTA-abs) May remain positive after treatment More sensitive and specific than non- trep . tests More expensive and labor intensive Can not quantitate…not useful for following response to treatment

22. TREPONEMAL TESTS ADVANTAGES DISADVANTAGES High Specificity Possibly higher sensitivity during early and late syphilis stages compared to non-treponemal tests Newer Methods Objective result interpretation Automation option High throughput High reproducibility/ precision Remain positive despite treatment Cannot be used to monitor response to therapy Conventional Methods Subjective interpretation requiring technician expertise to read Newer Methods Expensive instrumentation Higher cost/test

23. Direct Fluorescence Antibody-TP

24. Darkfield Microscopy

25. Enzyme Immunoassay (EIA)

26. TP – Particle Agglutination (TPPA) TP – Hemagglutination (TPHA)

27. Interpretation of Results Possible Explanation Treponemal Tests (TP-PA/ FTA-ADS) Non- Treponemal tests (RPR/ VDRL) Syphilis - recent or previous Yaws or pinta + + No syphilis False positive – + Consistent with previously treated or untreated Syphilis Yaws, Pinta , Bejel + – No s yphilis Syphilis in incubation period – –

28. Syphilis Screening Algorithms: Traditional versus Reverse Screening

29. Traditional Algorithm Non-treponemal test (e.g., RPR) Treponemal test (e.g., FTA) Negative for syphilis Non-reactive Non-reactive Syphilis Negative for syphilis Reactive Reactive

30. Traditional Algorithm ADVANTAGES DISADVANTAGES Results show good correlation with disease status Rapid, inexpensive screening method Excellent option for laboratory with small throughput Recommended by the CDC Manual (RPR) and subjective interpretation Screening method is non-specific and may lead to false-positive results Not suitable for high throughput laboratories Potentially lower sensitivity for detecting early syphilis and late/latent disease

31. Reverse Algorithm Treponemal test ( eg , EIA) Non-Treponemal test (eg, RPR) Negative for syphilis Non-reactive Non-reactive Syphilis Second Treponemal Test (e.g., TP-PA) Reactive Reactive Non-reactive Reactive Evaluation Required* Negative for syphilis

32. Reverse Algorithm ADVANTAGES DISADVANTAGES Objective interpretation of results Results from EIA or MFI can be interfaced with LIS Specific screening test for anti- T. pallidum antibodies Potentially increased detection of patients with early syphilis Higher cost/sample Higher assay complexity Increased detection of patients with screen (+), RPR (-) results

33. Follow Up Primary or secondary syphilis Reexamine at 6 and 12 months. Follow-up titers should be compared to the maximum or baseline nontreponemal titer obtained on day of treatment. Latent syphilis Reexamine at 6, 12, and 24 months. HIV-infected patients 3, 6, 9, 12 and 24 months for primary or secondary syphilis 6, 12, 18, and 24 months for latent syphilis Neurosyphilis Serologic testing as above Repeat CSF examination at 6-month intervals until normal