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)
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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
microscopy 79-97%
Exudate; live treponemes;
morphology; dark ground
microscope; experienced
clinican and observer;
genital lesions; 15 mins
DFA-Tp
Sensitivity
73-?100%
Exudate; fixed treponemes;
morphology; fluorescent
microscope; experienced
observer; oral and rectal
lesions; 30 mins; no kit
Sensitivity
75-95%
Exudate; specialised
equipment; objective; high
specificity (T. pallidum
subsp.); 2-4 hours; no kit
(MoAb to
47KDa
antigen)
PCR
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 AntibodyAbsorbed)
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)
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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
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EIA (first choice)
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TPPA (second choice)
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TPPA/TPHA plus VDRL (third choice)
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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?
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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 ?
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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
test
test
IgM
Report
Neg
Not done
Neg
Neg/Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
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
Pos
Neg
Pos
Neg
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