y r a r Syphilis: Diagnosis b i L e r Lecture outline u t c e r L o •Introduction e h t n i l •Diagnosis in the adult au n y tests •Interpretation O of serological b D interactions •Syphilis/HIV I © M •Diagnosis in the infant C S E

y r a r Treponema pallidumib L e r u t c e r L o e h t n i u l a n O by D © I M

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Image: CDC PHIL

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y r a on Treponematoses (Differentiation based r b i clinical and epidemiological considerations) L e r u t c e r L o e h t n i u l a n O by D © I M C S

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

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y r a r Time after exposure Classification b i L Early (infectious) syphilis 9-90 days Primary re u t 6weeks - 6months Secondary c e r ≤ 1 year (or ≤ 2 years) LEarly Latent o e Late (non-infectious) syphilis th n i u l > 1 year (or > 2n years) Late Latent a y Tertiary 3-20 years O b D © Gummatous I M Cardiovascular C Neurosyphilis S Stages of syphilis

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Roles of syphilis testingary • Diagnosis of active infection

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r u t • Screen for infectious syphilis (early stage) c e (earlyr & late) • Screen for infection at any stage L o e h t n • Confirmatory tests li u a n • Provide a guideO to treatment status and monitor the y b D © efficacy of treatment I M • DetectCneurological involvement (CSF) S E congenital infection • Detect

Lab tests for Syphilis diagnosis y

r a r b • Direct detection of Treponema pallidum i L • NB Cannot be cultured in vitro e r • Dark ground microscopy u t •Fluorescent antibody staining c e r L •PCR o e h t n • Antibody detection li u a n • Detects antibodies against pathogenic treponemes y O b •always reported as ‘treponemal’ serology D I © • Incubation period 9 - 90 days M •C Natural history - many decades S E • Suspect neurosyphilis - test serum before CSF

y r Methods of detecting T. pallidum ain r b i primary infection L e r Dark ground Sensitivity u t c microscopy 79-97% e r L o e h t n i u l DFA-Tp Sensitivity a n y O73-?100% b D © I M C Sensitivity S PCR Exudate; live treponemes; morphology; dark ground microscope; experienced clinican and observer; genital lesions; 15 mins

Exudate; fixed treponemes; morphology; fluorescent microscope; experienced observer; oral and rectal lesions; 30 mins; no kit

(MoAb to 47KDa antigen)

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75-95%

Exudate; specialised equipment; objective; high specificity (T. pallidum subsp.); 2-4 hours; no kit

y r a r b i L e EIA or TPPA

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r u t c e r L o e h t n i u l a n O by D © I M

y r a r b i L e

Serological tests I • Non-treponemal tests

r u t c • VDRL slide test (read microscopically) e r L o • Rapid plasma reagin or carbon antigen test e h t n i (RPR or VDRL/RPR) u l a n • Wasserman (CFT) y O reaction b D © I M C S E • Cardiolipin antigen “Reagin” “Lipoidal”

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VDRL slide test

r u Negative t c e r L o e h t n i u l a n O by D © I M Positive

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As seen through a microscope

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r u t c e r L o e h t n i u l a n O by D © I M

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VDRL Carbon antigen test/RPR

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r u t c e r L o e h t n i u l a n O by D © I M

y r a r Serological Tests IIib L e r u • Treponemal tests t c • Antigen from Nichols strain of T. pallidum e r L • TPHA (erythrocytese as carrier) o h t n • TPPA (gelatin particles as u carrier) i l a n • TPLA (automated) y O b • EIA/CLIA (native and recombinant antigen) D I © •M Immunoblot (Western Blot or recombinant) C • FTA-Abs S E • Rapid immunochromatic strip tests

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EIA (T. pallidum enzyme immunoassay)

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r u t c e r L o e h t n i u l a n O by D © I M

TPPA (or TPHA) test

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r u t c e r L o e h t n i u l a n O by D © I M

y What we want in an ideal screening r a r test ? ib L e

r u t c e r • Specific (100%) L o e h • Simple to perform (automation) t n i u l a n ofy reagents • ConsistentO quality b D reading • Objective I © M • Reproducible C S •E Cheap

• Sensitive (100%)

You don’t always get what you want!

Screening with RPR/VDRL ry

a r b i • Specificity > 99% L • Problem of Biological False Positives (pregnancy, malaria, e r infectious mononucleosis, hepatitis, u connective tissue t disease, IV drug abuse) c e r • Sensitivity varies by stage L o e h • 70-85% in primary t n i u l • ~100% in secondary a n y stage infection • 60-80% in late O b D © • Prozone I M • Usually negative after treatment C • Cheap and simple S Egenerally automated (Mediace?) • Not • Subjective

TPHA

Screening with TPHA/TPPAry

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• 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)

r u t c e r L o e h t n i u l a n O by TPPA • SpecificityID > 99.5%© M • Sensitivity – 90-95% in primary syphilis C • Easier to perform and read than TPHA S E TPLA • Automated version (Sekisui Mediace)

y r a r b i • Variety of EIAs L • Native vs. recombinant T. pallidum antigens e r u t • Screening tests detect total IgG and IgM c e r L o e h • Specificity > 99.5% t n i u l a • Sensitivity n y in all other stages Oand 100% • 80-85% in primary b D after©treatment (may become neg in HIV) I • Antibody persists M C S • Objective reading E • Suited to automated testing/ electronic reporting Screening with EIA

• Can test for other blood borne infections on same analyser • Not suitable for titration (staging/treatment monitoring)

y r Window period a r b i L e r • Maximum detection of primary syphilis depends on high index of u t clinical suspicion c eall serological r • Window of 1-2 weeks when screening L o tests may be negative e h t n i • Perform a directltest if thereu is a lesion a n • Request EIA IgM y O for specific b and/orID © • Repeat test 6 weeks later M C S E

y r What to use as a confirmatoryratest? b i L e r • Depends on u t c • Resources and test volume e r L o • Screening test used e h t n i u l a n y be • Confirmatory O test should b D I © • A treponemal test of a different type (i.e. using M different antigens) C S E • Equivalent sensitivity and specificity

Predictive value as a measure ofy r the utility of a diagnostic testra

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r u t c e r • Sensitivity; specificity; prevalence L o e h t • Positive predictive value (PPV) n i u l a n • The probability that a positive result is a true y O b result forD the infection being tested for I © M • Negative predictive value (NPV) C S E• The probability that a negative result is a true • Predictive value is influenced by

result and excludes the infection being tested for

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y r a Recommendation for confirmatoryrtesting b i L e r TPPA (TPHA) when EIA is used touscreen t c EIA when TPPA (TPHA) is used to screen e r L o e h t n i u l stage disease; monitor Optimal profile to help a n y treatment; and O detect re-infection should include b D VDRL I • Quantitative © M C • (Quantitative) TPPA (TPHA) S E• EIA for anti-treponemal IgM

Can we use the immunoblot as ary a r confirmatory test ? ib

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r u t c e r L o e h t n i u l a n O by D © I M

• 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

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y r Serological tests: active infectionraand b i staging disease L e r • A VDRL/RPR titre of ≥16 and/ortu a positive IgM test c need for treatment indicate active disease ande the r L o – Lower VDRL titres are found in untreated early infection e h t n – VDRL may exhibit prozone (false-negative due to v. high Ab i u l levels), particularly in 2° stage, reinfection, HIV co-infection a n y in late syphilis, and • The EIA IgM O is often b negative Dbe negative; I VDRL can this does not exclude the © M need for treatment C S E

Response to therapy ry VDRL / RPR Reactivityra

b i • Seroreversal rates vary depending on L e • Pre-treatment titre r u t • Stage of disease c e r • Previous episode of syphilis L o e h • Treatment regimen t n i u l a (primary and secondary) • Decrease in titre n O by253: 1296 - 9 • Brown et al JAMA 1985; D I © 8-fold at 6 months • 4-fold at 3 months; M • Romanowski Ann Intern Med 1991; 114:1005 - 9 C S 4-fold at 6 months; 8-fold at 12 months E •• Early latent: 4-fold at 12 months

• Patients may become ‘serofast’ at ≤4 (may be higher in HIV)

y r a r Reinfection b i L e r u • A fourfold increase in titre (confirmed on a second t c specimen) suggests re-infection or relapse e r L o – Frequently reinfection produces a higher titre than first e h t infection n i u l a n AND/OR y O b • IgM becomes reactive again (confirmed on a second D I specimen) after it© has become negative M – C Watch out for low positive indices which may indicate a ‘blip’ S in test sensitivity E – Not all reinfections result in a positive IgM test

y r a r Syphilis serology in HIV infection b i L e • Very high levels of antibody often produced r u t • Increased risk of prozone phenomenon c e than "Seronegative“ • "Delayed seropositivity" rather r L o e h • Titres may not fall asn expected tafter treatment i u l a n • Conflicting reports, response dependent on: y O b • Previous syphilis; stage; pre-Rx titre; regimen D © I in serological markers for syphilis • ChangeM C •20-40% loss of reactivity to one treponemal test S E

• False negative FTA-abs tests

Interpretation of serological tests y

r a r Report b i L e r u t c e r L o e h t n i u l a n O by D © I M

Screening Confirmatory VDRL test test

Neg

Not done Not done Treponemal antibody not detected but advise repeat if at risk of recent infection. Suggests early primary infection. Neg/Pos Pos Advise repeat to confirm. Consistent with recent/active Pos Pos treponemal infection. Advise repeat to confirm Consistent with treponemal Pos Neg infection. Advise repeat to confirm Consistent with treponemal Neg Neg infection at some time. Advise repeat to confirm Treponemal antibody not detected. Neg Neg

Neg

Pos

Neg

Not done

Neg

Neg/Pos

Pos

Pos

Pos

Pos

Pos

Pos

Pos Pos

IgM

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Neg

Treponemal antibody not detected.

y r a r Congenital syphilis ib L e r u • Congenital syphilis is preventable t c epromptreffective – Antenatal screening and L o treatment e h t n i u l • Diagnosis complicated by the transplacental a n O bytreponemal IgG transfer of maternal D to the© fetus I antibodies M –C IgM antibodies do not cross the placenta S •ESerological testing of infant’s (not cord) blood and mother’s blood in parallel

y r a Congenital syphilis 2 (diagnosis) r b i L • Transplacental transfer of antibody e supported by: r u – Negative IgM EIA and reactive VDRL and/or TPPA titres t c