Lecture outline - ESCMID

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 syphilisTime after exposure ClassificationEarly (infectious) syphilis9-90 days Primary6weeks - 6months Secondary≤ 1 year (or ≤ 2 years) Early LatentLate (non-infectious) syphilis> 1 year (or > 2 years) Late Latent3-20 years Tertiary

GummatousCardiovascularNeurosyphilis

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 microscopy

Sensitivity79-97%

Exudate; live treponemes; morphology; dark ground microscope; experienced clinican and observer; genital lesions; 15 mins

DFA-Tp (MoAb to 47KDa antigen)

Sensitivity73-?100%

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

PCR Sensitivity 75-95%

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

EIA or TPPA

Screening tests for syphilisWhat 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 stripsDeveloped 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/TPPATPHA

• 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 therapyVDRL / 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 testsScreening test

Confirmatory test

VDRL IgM Report

Neg Not done Not done Not done

Neg Neg/Pos Neg/Pos Pos

Pos Pos Pos Pos

Pos Pos Pos Neg

Pos Pos Neg Neg

Pos Neg Neg Neg

Pos Neg Pos Neg

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

Treponemal antibody not detected.

Congenital syphilis

• Congenital syphilis is preventable– Antenatal screening and prompt effective

treatment• Diagnosis complicated by the transplacental

transfer of maternal treponemal IgG antibodies to the fetus– IgM antibodies do not cross the placenta

• Serological testing of infant’s (not cord) blood and mother’s blood in parallel

Congenital syphilis 2 (diagnosis)

• Transplacental transfer of antibody supported by:– Negative IgM EIA and reactive VDRL and/or TPPA titres

<fourfold higher that those of the mother• Congenital infection supported by:

– Positive IgM EIA– Fourfold or greater increase in VDRL or TPPA titre above

that of the mother (and confirmed on a second specimen)• Definitive diagnosis provided by:

– Demonstration of T. pallidum in tissues or secretions (umbilical cord, placenta, nasal discharge, skin lesion)

• See also case definitions: http://www.ecdc.europa.eu/en/activities/surveillance/ESSTI/Pages/Case%20definition.aspx#congenital

Congenital syphilis 3 (follow-up)

• If congenital syphilis is indicated by physical signs or laboratory tests, perform CSF serology

• IgM may be negative at birth and should be repeated up to 12 weeks

• IgG (including VDRL and TPPA) tests should be repeated until negative