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Syphilis: Diagnosis

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Syphilis: Diagnosis Syphilis: Diagnosis Lecture outline •Introduction •Diagnosis in the adult •Interpretation of serological tests •Syphilis/HIV interactions •Diagnosis in the infant Treponema pallidum Image: CDC PHIL Treponematoses (Differentiation based on clinical and epidemiological considerations) • Treponema carateum (pinta) – Central America; spread by close contact • skin only • Treponema pallidum subspecies endemicum (non-venereal endemic syphilis (“bejel”) – Middle East, SE Asia; spread by close contact • skin and bone only • Treponema pallidum subspecies pertenue (yaws) – Africa; spread by close contact • skin and bone only • Treponema pallidum subspecies pallidum (syphilis) – World-wide; spread by sexual intercourse • skin, bone, viscera, CNS, congenital infection Stages of syphilis Time after exposure Classification Early (infectious) syphilis 9-90 days Primary 6weeks - 6months Secondary ≤ 1 year (or ≤ 2 years) Early Latent Late (non-infectious) syphilis > 1 year (or > 2 years) Late Latent 3-20 years Tertiary Gummatous Cardiovascular Neurosyphilis Roles of syphilis testing • Diagnosis of active infection • Screen for infectious syphilis (early stage) • Screen for infection at any stage (early & late) • Confirmatory tests • Provide a guide to treatment status and monitor the efficacy of treatment • Detect neurological involvement (CSF) • Detect congenital infection Lab tests for Syphilis diagnosis • Direct detection of Treponema pallidum • NB Cannot be cultured in vitro • Dark ground microscopy (Sens 79-97%; Spec 77-100%) • Fluorescent antibody staining (Sens 73-100%; Spec 100%) • PCR (Sens 89-91%; Spec 99%) • Antibody detection • Detects antibodies against pathogenic treponemes •always reported as ‘treponemal’ serology • Incubation period 9 - 90 days • Natural history - many decades • Suspect neurosyphilis - test serum before CSF Methods of detecting T. pallidum in primary infection Dark ground Sensitivity Exudate; live treponemes; microscopy 79-97% morphology; dark ground microscope; experienced clinican and observer; genital lesions; 15 mins DFA-Tp Sensitivity Exudate; fixed treponemes; (MoAb to 73-?100% morphology; fluorescent 47KDa microscope; experienced antigen) observer; oral and rectal lesions; 30 mins; no kit PCR Sensitivity Exudate; specialised 75-95% equipment; objective; high specificity (T. pallidum subsp.); 2-4 hours; no kit EIA or TPPA Screening tests for syphilis What is available? • Non-treponemal tests • Cardiolipin antigen “Reagin” “Lipoidal” • VDRL slide test (read microscopically) • Rapid plasma reagin or carbon antigen test (RPR or VDRL/RPR) • Treponemal tests • Antigen from Nichols strain of T. pallidum • TPHA (erythrocytes as carrier) • TPPA (gelatin particles as carrier) • EIA (native and recombinant antigen) • Rapid immunochromatic strip tests VDRL slide test Positive Negative As seen through a microscope VDRL Carbon antigen test/RPR EIA (T. pallidum enzyme immunoassay) TPPA (or TPHA) test Two other tests for Syphilis antibodies • FTA-Abs (Fluorescent Treponemal Antibody- Absorbed) Long seen as the “Gold Standard”; now little used • Immunoblot (e.g. Inno-Lia) Reaction with protein fixed on nitrocellulose strips Developed as better confirmatory tests What we want in an ideal screening test ? • Sensitive (100%) • Specific (100%) • Simple to perform (automation) • Consistent quality of reagents • Objective reading • Reproducible • Cheap You don’t always get what you want! Screening with RPR/VDRL • Specificity > 99% • Problem of Biological False Positives (pregnancy, malaria, infectious mononucleosis, hepatitis, connective tissue disease, IV drug abuse) • Sensitivity varies by stage • 70-85% in primary • Prozone (false negatives) 1- 10% in early infection • ~100% in secondary • 60-80% in late stage infection • Usually negative after treatment • Cheap reagents and simple to perform • Labour intensive - not suited for automation •Subjective Screening with TPHA/TPPA TPHA • Specificity > 99.5% • Sensitivity • 70-80% in primary • 100% in all other stages (untreated and treated) • antibody persists after treatment (may become negative in HIV) TPPA • Specificity > 99.5% • Sensitivity – 90-95% in primary syphilis • Easier to perform and read than TPHA Neither test suited to automation Screening with EIA • Variety of EIAs • Native vs. recombinant T. pallidum antigens • Screening tests detect total IgG and IgM • Specificity > 99.5% • Sensitivity • 80-85% in primary and 100% in all other stages • Antibody persists after treatment (may become neg in HIV) • Objective reading • Suited to automated testing/ electronic reporting • Can test for other blood borne infections on same analyser • Not suitable for titration (staging/treatment monitoring) What to use as a primary screening test • EIA (first choice) • TPPA (second choice) • TPPA/TPHA plus VDRL (third choice) • Maximum detection of primary syphilis depends on high index of clinical suspicion • Window of 1-2 weeks when all serological screening tests may be negative • Perform a direct test if there is a lesion • Request EIA for specific IgM and/or • Repeat test 6 weeks later What to use as a confirmatory test? • Depends on • Resources and test volume • Screening test used • Confirmatory test should be • A treponemal test of a different type (i.e. using different antigens) • Equivalent sensitivity and specificity Predictive value as a measure of the utility of a diagnostic test • Predictive value is influenced by • Sensitivity; specificity; prevalence • Positive predictive value (PPV) • The probability that a positive result is a true result for the infection being tested for • Negative predictive value (NPV) • The probability that a negative result is a true result and excludes the infection being tested for Recommendation for confirmatory testing • TPPA (TPHA) when EIA is used to screen • EIA when TPPA (TPHA) is used to screen • The FTA-abs is no longer recommended as a first-line confirmatory test • Optimal profile to help stage disease; monitor treatment; and detect re-infection should include • Quantitative VDRL • (Quantitative) TPPA (TPHA) • EIA for anti-treponemal IgM Is there still a role for the FTA-abs as a confirmatory test ? • FTA-abs for many years considered "Gold standard" • Subjective; variation in performance of kit reagents • Poor reproducibility (within assay and between assay) • Reliable when testing VDRL positive sera • can score "Borderline reactions" negative and have • high sensitivity and specificity • Much less reliable with VDRL- TPHA/EIA + sera • False positives with some EIA's also false positive in FTA • False negative FTA-abs in HIV; falsely classified as BFPs • May have success with in-house tests, consistency of reagents and staff Can we use the immunoblot as a confirmatory test ? • Initially there were problems in defining a positive immunoblot result for tests using native T. pallidum antigen • Line immunoassays using recombinant antigens have overcome these problems • Hagedorn et al J Clin Microbiol 2002; 40: 973-8 • Sensitivity 100% • Specificity 99.3% • Can be useful in clarifying discrepancies Serological tests: active infection and staging disease • A VDRL/RPR titre of ≥16 and/or a positive IgM test indicate active disease and the need for treatment – Lower VDRL titres are found in untreated early infection – VDRL may exhibit prozone (false-negative due to v. high Ab levels), particularly in 2° stage, reinfection, HIV co-infection • The EIA IgM is often negative in late syphilis, and VDRL can be negative; this does not exclude the need for treatment Response to therapy VDRL / RPR Reactivity • Seroreversal rates vary depending on • Pre-treatment titre • Stage of disease • Previous episode of syphilis • Treatment regimen • Decrease in titre (primary and secondary) • Brown et al JAMA 1985; 253: 1296 - 9 • 4-fold at 3 months; 8-fold at 6 months • Romanowski Ann Intern Med 1991; 114:1005 - 9 • 4-fold at 6 months; 8-fold at 12 months • Early latent: 4-fold at 12 months • Patients may become ‘serofast’ at ≤4 (may be higher in HIV) Reinfection • A fourfold increase in titre (confirmed on a second specimen) suggests re-infection or relapse – Frequently reinfection produces a higher titre than first infection AND/OR • IgM becomes reactive again (confirmed on a second specimen) after it has become negative – Watch out for low positive indices which may indicate a ‘blip’ in test sensitivity – Not all reinfections result in a positive IgM test Syphilis serology in HIV infection • Very high levels of antibody often produced • Increased risk of prozone phenomenon • "Delayed seropositivity" rather than "Seronegative“ • Titres may not fall as expected after treatment • Conflicting reports, response dependent on: • Previous syphilis; stage; pre-Rx titre; regimen • Change in serological markers for syphilis •20-40% loss of reactivity to one treponemal test • False negative FTA tests Interpretation of serological tests Screening Confirmatory VDRL IgM Report test test Neg Not done Not done Not done Treponemal antibody not detected but advise repeat if at risk of recent infection. Neg Neg/Pos Neg/Pos Pos Suggests early primary infection. Advise repeat to confirm. Pos Pos Pos Pos Consistent with recent/active treponemal infection. Advise repeat to confirm Pos Pos Pos Neg Consistent with treponemal infection. Advise repeat to confirm Pos Pos Neg Neg Consistent with treponemal infection at some time. Advise repeat to confirm Pos Neg Neg Neg Treponemal antibody not detected. Pos Neg Pos Neg Treponemal antibody not detected. Congenital syphilis • Congenital
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