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Syphilis: Diagnosis
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Lecture outline
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•Introduction
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•Diagnosis in the
adult au
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y tests
•Interpretation
O of serological
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•Syphilis/HIV
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•Diagnosis
in the infant
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Treponema pallidumib
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Image: CDC PHIL
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Treponematoses (Differentiation based
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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
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Time after exposure
Classification
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Early (infectious) syphilis
9-90 days
Primary re
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6weeks - 6months
Secondary
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≤ 1 year (or ≤ 2 years) LEarly Latent
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e
Late (non-infectious) syphilis th
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> 1 year (or > 2n
years)
Late
Latent
a
y Tertiary
3-20 years O
b
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Cardiovascular
C
Neurosyphilis
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Stages of syphilis
E
Roles of syphilis testingary
• Diagnosis of active infection
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• Screen for infectious syphilis (early
stage)
c
e (earlyr & late)
• Screen for infection at any stage
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• Confirmatory tests li
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• Provide a guideO
to treatment
status and monitor the
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efficacy of treatment
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• DetectCneurological involvement (CSF)
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E congenital infection
• Detect
Lab tests for Syphilis diagnosis
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• Direct detection of Treponema pallidum
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• NB Cannot be cultured in vitro
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• Dark ground microscopy
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•Fluorescent antibody staining c
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•PCR
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• Antibody detection li
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• Detects antibodies
against
pathogenic
treponemes
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•always
reported
as
‘treponemal’ serology
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• Incubation
period
9 - 90 days
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•C
Natural history - many decades
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E • Suspect neurosyphilis - test serum before CSF
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Methods of detecting T. pallidum
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primary infection L
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Dark ground Sensitivity
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microscopy 79-97%
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DFA-Tp
Sensitivity
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O73-?100%
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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)
E
75-95%
Exudate; specialised
equipment; objective; high
specificity (T. pallidum
subsp.); 2-4 hours; no kit
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EIA or TPPA
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Serological tests I
• Non-treponemal tests
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• VDRL slide test (read
microscopically)
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• Rapid plasma reagin
or carbon
antigen test
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(RPR or VDRL/RPR)
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• Wasserman
(CFT)
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• Cardiolipin antigen “Reagin” “Lipoidal”
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VDRL slide test
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Negative
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Positive
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As seen through a microscope
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VDRL Carbon antigen test/RPR
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Serological Tests IIib
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• Treponemal tests
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• Antigen from Nichols strain of
T.
pallidum
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• TPHA (erythrocytese
as carrier) o
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t
n
• TPPA (gelatin particles
as u
carrier)
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a
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• TPLA (automated)
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b
• EIA/CLIA
(native and recombinant antigen)
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Immunoblot (Western Blot or recombinant)
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• FTA-Abs
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E • Rapid immunochromatic strip tests
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EIA (T. pallidum enzyme immunoassay)
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TPPA (or TPHA) test
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What we want in an ideal screening
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test ?
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• Specific (100%)
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• Simple to perform
(automation)
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a
n ofy reagents
• ConsistentO
quality
b
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• Objective
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• Reproducible
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•E Cheap
• Sensitive (100%)
You don’t always get what you want!
Screening with RPR/VDRL ry
a
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• Specificity > 99%
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• Problem of Biological False Positives (pregnancy,
malaria,
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infectious mononucleosis, hepatitis, u
connective
tissue
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disease, IV drug abuse)
c
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• Sensitivity varies by stage
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• 70-85% in primary
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• ~100% in secondary
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stage infection
• 60-80% in late O
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• Prozone
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• Usually negative
after treatment
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• Cheap
and simple
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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)
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• SpecificityID
> 99.5%©
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• Sensitivity
– 90-95% in primary syphilis
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• Easier
to perform and read than TPHA
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E
TPLA
• Automated version (Sekisui Mediace)
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• Variety of EIAs
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• Native vs. recombinant T. pallidum antigens
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• Screening tests detect total IgG and
IgM
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• Specificity > 99.5%
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a
• Sensitivity
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y in all other stages
Oand 100%
• 80-85% in primary
b
D after©treatment (may become neg in HIV)
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• Antibody persists
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C
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• 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)
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Window period
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• Maximum detection of primary syphilis depends
on high index of
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clinical suspicion
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eall serological
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• Window of 1-2 weeks when
screening
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o
tests may be negative e
h
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n
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• Perform a directltest
if thereu
is a lesion
a
n
• Request EIA
IgM
y
O for specific
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and/orID
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• Repeat
test 6 weeks later
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E
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What to use as a confirmatoryratest?
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• Depends on
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• Resources and test volume
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• Screening test used
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n
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a
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y be
• Confirmatory O
test should
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• A treponemal test
of a different type (i.e. using
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different
antigens)
C
S
E • Equivalent sensitivity and specificity
Predictive value as a measure ofy
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the utility of a diagnostic testra
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• Sensitivity; specificity; prevalence
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• Positive predictive value
(PPV)
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a
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• The probability
that a positive
result is a true
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b
result forD
the infection
being tested for
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• 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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Recommendation for confirmatoryrtesting
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TPPA (TPHA) when EIA is used touscreen
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EIA when TPPA (TPHA) is used
to screen
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L
o
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h
t
n
i
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stage
disease;
monitor
Optimal profile to help
a
n
y
treatment; and O
detect re-infection
should include
b
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• Quantitative
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M
C
• (Quantitative)
TPPA (TPHA)
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E• EIA for anti-treponemal IgM
Can we use the immunoblot as ary
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confirmatory test ? ib
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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
C
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E
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Serological tests: active infectionraand
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staging disease L
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• A VDRL/RPR titre of ≥16 and/ortu
a positive IgM test
c need for treatment
indicate active disease ande
the
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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
©
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need
for treatment
C
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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;
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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)
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Reinfection
b
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L
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• 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
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a
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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
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Report
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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
C
S
E
Neg
Treponemal antibody not detected.
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Congenital syphilis ib
L
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• 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
<fourfold higher that those of the
mother
e
r
L
• Congenital infection supported
by:o
e
h
t
– Positive IgM (EIA, 19S
FTA-Abs,
IgM
Immunoblot)
n
i
u
l
n
– Fourfold or greater
increasea
in VDRL or TPPA titre above
y on a second specimen)
O (andbconfirmed
that of the mother
• DefinitiveID
diagnosis
provided by:
©
M
– Demonstration
of T. pallidum in tissues or secretions
C
(umbilical cord, placenta, nasal discharge, skin lesion)
S
E – PCR of CSF, serum, amniotic fluid is also helpful
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Congenital syphilis 3 (follow-up)
b
i
L
e
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u
• If congenital syphilis is indicated
by physical
t
c
e perform
signs or laboratory tests,
CSF
r
L
o
serology (and PCR)
e
h
t
n
i
u
l
• IgM may be negative
at
birth and should be
a
n
y
repeated upOto 12 b
weeks
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• IgG (including
VDRL
and TPPA) tests should
M
C
be
repeated until negative
S
E
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Recommended Reading
b
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L
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• T Herremans et al. 2010 “A review of tdiagnostic
tests in
u
c
congenital syphilis.” Eur. J. Clin. Microbiol.
Infect. Dis. 29 (5)
e r
495-501.
L
o
• ECDC Congenital Syphilise
case definitions:
h
t
n
i
u
l
http://www.ecdc.europa.eu/en/activities/surveillance/sti/Pages/Cas
a
n
e%20definition.aspx
y
O
b
• European guideline
on the management of syphilis (2008, under
D
©
review) I
M
http://www.iusti.org/regions/europe/euroguidelines.htm
C
S
E