CAN WE RELIABLY DIAGNOSE SYPHILIS? J. Scythes, C. M. Jones Community Initiative for AIDS Research Toronto, Canada

1
CAN WE RELIABLY DIAGNOSE SYPHILIS?J. Scythes, C.
M. JonesCommunity Initiative for AIDS
ResearchToronto, Canada
22nd IUSTI-EUROPE CONFERENCE ON SEXUALLY
TRANSMITTED INFECTIONS Oral communication Thursd
ay 10/19/2006 1400 Auditorium Richelieu Session
1 Syphilis and GUD Abstract NO.001
2
CAN WE RELIABLY DIAGNOSE SYPHILIS?J. Scythes, C.
M. JonesCommunity Initiative for AIDS
ResearchToronto, Canada
Objectives To ascertain if syphilis screening
reliably detects latent disease, including in the
HIV context, by comparing standard syphilis
testing with newly-developed techniques, using
either recombinant antigen based treponemal
serology or direct detection by DNA amplification.
3
CAN WE RELIABLY DIAGNOSE SYPHILIS?J. Scythes, C.
M. JonesCommunity Initiative for AIDS
ResearchToronto, Canada
Methods (1) 500 patients at a downtown
Toronto HIV clinic were screened from 1988-1992
with both RPR and quantified treponemal tests,
including MHA-Tp (TPHA).
4
SYPHILIS - A ROLE IN SEXUALLY ACQUIRED
AIDS?D.K. MacFadden, R.H. Notenboom, J.
ScythesDept. of Medicine, University of Toronto,
Laboratory Services Branch, Ministry of Health,
Community Initiative for AIDS Research, Toronto,
Ontario, CanadaBiology and Pathogenicity of
Treponemes ConferenceUniversity of Birmingham,
England, 11-13 April 1989
Numerous inconsistencies have been noted in HIV
epidemiology between the various risk groups.
Clinical signs of HIV infection seem to appear
much later in previously healthy heterosexuals.
Marked differences in both expression and
progression of HIV disease between the sexually
and non-sexually acquired forms have been
reported. These differences might well be
explained by a hypothetical interrelationship
between STDs and AIDS thereby explaining the
predisposition to HIV infection both in North
American homosexuals and the heterosexual
population of sub-Saharan Africa. Extensive
historical data supports the role of an STD as a
co-factor allowing significant viral expression.
Certain aspects of HIV epidemiology may therefore
be explained by concomitant infection by
treponemes and HIV. Although the natural history
of HIV infection in North American homosexuals is
well documented, it appears that infection by
non-sexual means results in less serious disease
or prolongation of a clinically latent period.
This is supported by evidence that infectivity
rates of immunologically intact individuals, e.g.
needle-stick exposures, hemophiliacs and
transfusion recipients are less than that of
immunologically impaired transfusion recipients,
e.g. hypogammaglobulinemia, leukemia, etc.
Seroconversions in the immunologically intact
groups may carry a less serious prognosis re
progression to AIDS. The reported differences
between sexually and non-sexually acquired AIDS
may result from failure to diagnose treponemal
disease either due to the inadequacies of
treponemal testing or due to secondary effects of
HIV infection. a) Of 43 patients in a gay
primary practice, 13 reported a history of
treatment for syphilis 6 of these were negative
in all syphilis tests (VDRL, MHA-TP, FTA and
TPI). b) Furthermore, 83 HIV antibody positive
and not end-stage homosexual patients, in order
of presentation, at an HIV clinic were all VDRL
negative 12 were negative in treponemal tests
despite a history of treated syphilis. 24
reported a history, i.e. 50 lost antibody. c)
To investigate the concommitance of HIV antibody
and treponemal antibody status, we tested 100
homosexuals blinded. Only one out of the 10
patients with AIDS was treponemal antibody
positive.
5
SYPHILIS AND HIV SEROPREVALENCE AND CLINICAL
OBSERVATIONS IN HIV CLINIC, TORONTOFralick RA,
Scythes JB, Notenboom RH, MacFadden DKThe
Toronto Hospital, Western Division, Community
Initiative for AIDS Research, Laboratory Services
Branch, Ontario Ministry of Health, Toronto,
Canada10th International Meeting of the
International Society for STD ResearchHelsinki,
Finland, August 27 - September 1, 1993
Objective To determine the reliability of
syphilis testing in HIV positive patients
attending the Toronto Hospital's Immunology
Clinic. Methods Chart review and analyses of
history of syphilis and current CD4
counts. Results Out of 500 active patients, 398
with no history of syphilis and CD4 counts over
200 excluded. Among the remaining 102 patients no
primary or secondary syphilis was diagnosed 53
had reactive treponemal tests (CD4 count range
6-1015, mean 272). No patient had documented
clinical or serological relapse despite 11
patients (20.4 of reactive treponemal tests) who
had reactive treponemal tests without a history
of syphilis. Follow up of these patients showed
no clinical signs or serological evidence of
active syphilis. Six patients with a history of
syphilis had nonreactive treponemal tests (CD4
count range 27-170, mean 78). Reactive
non-treponemal tests and the late sequelae of
syphilis infection in HIV positive population are
much lower than expected. Reinfection and
super-infection are difficult to detect, and
potentially atypical in the immunosuppressed or
in those with previous syphilis. Significantly 11
patients had reactive treponemal tests yet no
history of syphilis. Conceivably there are many
HIV patients like this. The immune response to
syphilis is cell-mediated. The 6 patients with a
history of syphilis, nonreactive treponemal tests
and lower CD4 T-lymphocyte counts represent an
important sub group who have lost their
treponemal antibodies. Conclusion Further
clinical study is needed to determine their
susceptibility to active tertiary syphilis.
6
CAN WE RELIABLY DIAGNOSE SYPHILIS?J. Scythes, C.
M. JonesCommunity Initiative for AIDS
ResearchToronto, Canada
Results (1) None of 500 patients were RPR(),
while 60/500 were TPHA (), 11 of whom had no
syphilis history. Six with a syphilis history
were TPHA (-) with CD4 lt 80/ul.
7
CAN WE RELIABLY DIAGNOSE SYPHILIS?J. Scythes, C.
M. JonesCommunity Initiative for AIDS
ResearchToronto, Canada
Methods (2) Serum aliquots from a further 250
patients from the University of Toronto's AIDS
Epi-Study were similarly tested in 1992.
8
RELIABILITY OF SYPHILIS TESTS IN PATIENTS WITH
HIVNotenboom R MacFadden DKLaboratory
Services Branch, Ministry of Health, Toronto,
CanadaVIII International Conference on AIDS/III
STD World Congress, Amsterdam, the Netherlands,
19-24 July 1992
OBJECTIVE To assess the extent of treponemal
antibody decrease in persons at risk for HIV and
to determine whether such loss is a specific
one. METHODS Paired sera collected at a 3-4
year interval from patients attending primary
care facilities and/or an HIV clinic were tested
for syphilis by Microhemagglutination for
Treponema pallidum (MHA-TP). Paired sera of which
at least the first specimen was reactive in the
MHA-TP were also tested blinded for HIV,
Toxoplasma gondii (Tg) and mumps. Additional
tests for syphilis were carried out as well
Fluorescent Treponemal Antibody-Absorbed Test
(FTA-Abs), Captia IgG for T. pallidum and
quantitative MHA-TP. RESULTS A) HIV ab
patients showed a significantly larger proportion
with decreasing MHA-TP titers than HIV ab-
patients (24/40 vs. 1/15). In the HIV ab group,
we found 6 seroreversions to non-reactive by
MHA-TP, none in HIV ab- group. B) Among HIV ab-
patients no changes in anti Tg, anti mumps or
anti Tp were found, while among HIV ab patients
we found 2 instances in which all four
serological markers were decreased. In 6 cases,
(HIV ab) changes in anti mumps titers were not
parallel by changes in Tp tests. CONCLUSION
Serological tests may underestimate the incidence
of (past) syphilis in HIV ab patients. The
decrease in treponemal antibodies in these
patients is not due to a generalized loss of
antibody.
9
CAN WE RELIABLY DIAGNOSE SYPHILIS?J. Scythes, C.
M. JonesCommunity Initiative for AIDS
ResearchToronto, Canada
Results (2) Of 125/250 Epi-Study patients
with HIV, 24 had dropping treponemal titres
during B-cell polyclonal activation, while only
one HIV(-) case lost treponemal antibody
selectively in this way.
10
CAN WE RELIABLY DIAGNOSE SYPHILIS?J. Scythes, C.
M. JonesCommunity Initiative for AIDS
ResearchToronto, Canada
Methods (3) 557 patients from another
downtown HIV/STD clinic were screened in 2000 as
above, and also monitored with the Trep-Chek, a
more sensitive, FDA-approved, recombinant
ag-based EIA test.
11
TREPONEMAL BASED SCREENING FOR SYPHILISDETECTING
LATENT CASESN. denHollander, R. Berry, M.
FearonOntario Public Health Laboratory, Toronto,
Canada2000 General Meeting of the American
Society for Microbiology Los Angeles,
California, May 21-25, 2000
Background With the advent of national plans to
eliminate syphilis from the United States and
Canada it becomes vital that all cases (new and
old) be identified and where necessary treated.
Most laboratories in North America use
non-treponemal based screening tests for
syphilis. We asked how many cases of "unknown"
syphilis infection were being missed using this,
the oldest of all syphilis assays. Methods We
selected 557 "high risk" (from STD Clinics)
specimens from our routine syphilis submissions
which were non-reactive in our standard
non-treponemal test Rapid Plasma Reagin (RPR)
and re-tested them using a new automatable EIA.
The EIA (Trep-Chek Phoenix Bio-Tech Corp)
utilizes recombinant antigens from Treponema
pallidum. All specimens were also tested using a
microhemagglutination assay for T. pallidum
(MHA-Tp) and EIA positives were confirmed using a
T. pallidum specific Western blot (WB).
Sensitivity of the new assay was checked against
15 patients with proven early primary syphilis
(direct fluorescence confirmed, dark-field
positive smears from primary lesions) and a panel
of 16 treated, confirmed syphilis patients. A
panel of 119 "biological false positive"
specimens (RPR reactive but MHA-Tp non-reactive)
was also tested. Results The panel of 16
treated, confirmed patients were all positive by
EIA. Of the 15 early primary syphilis cases 14
were positive by EIA. Of the 119 BFP specimens
108 were negative by EIA but 10 of the remaining
11 were either positive or equivocal in the WB
(thus not true BFP's). Thus the initial
sensitivity and specificity for the new EIA was
97 and 99 respectively. In the "high risk"
population there were 16 positive and 11
indeterminate EIA cases (4.85 of those tested).
Of these only 9 were MHA-Tp reactive. However
fully 24 of these 27 EIA positives were either
positive or equivocal in the WB (our Gold
Standard assay). Most importantly 20 of these 24
"confirmed" positive syphilis patients were not
previously identified in our 20 yr database thus
potentially new cases. Conclusions Standard
non-treponemal based assays are clearly under
sensitive (as apparently are some treponemal
assays like the MHA-Tp) potentially allowing some
syphilis cases to go undetected and untreated.
12
CAN WE RELIABLY DIAGNOSE SYPHILIS?J. Scythes, C.
M. JonesCommunity Initiative for AIDS
ResearchToronto, Canada
Results (3) Of 557 clinic patients, none were
RPR(), while 27 had evidence of syphilis by
Trep-Chek, 24 of whom were WB () or equivocal.
Nine were TPHA(). Only 4/27 had ever been
followed up.
13
CAN WE RELIABLY DIAGNOSE SYPHILIS?J. Scythes, C.
M. JonesCommunity Initiative for AIDS
ResearchToronto, Canada
Methods (4) Recently, 183 patients from two
dermatology clinics in Budapest were tested for
T. pallidum DNA with a nested PCR.
14
EXPERIENCE OF MOLECULAR SCREENING OF SYPHILIS BY
PCRKaroly Nagy, John B Scythes, Istvan Horvath,
Bela Kemeny, Elyas Talha, Robert Notenboom,
Attila Horvath National Inst. Dermato-Venerology,
Hungary Phoenix Bio-Tech Corp., Canada14th
biennial meeting of the International Society for
Sexually Transmitted Diseases ResearchOttawa,
Canada, July 27-30 2003
Objectives To evaluate nested PCR on DNA
purified from whole blood, comparing PCR results
with syphilis (sy) serology and patient
history. Methods Part of the T. pallidum (Tp)
genome encoding the 47kD membrane protein was
amplified by PCR (Zoechling et. al). Positive
controls were obtained from a LuesII condyloma
inoculated into rabbit scrota, and from a
quantified suspension of Nichols Tp.
Seventy-eight persons were then tested 70 gay
males, 2 LuesII cases, and 6 HIV()
outpatients. Results The scrotum test was
PCR(), as was the Nichols preparation. Among the
70 men, all RPR(-) and TPHA(-), PCR detected 4 sy
cases. Repeat sy serology in these 4 men was (-),
except one weak TPHA. Both LuesII cases were
serology() and PCR(). One HIV() case found
among the 70 men was sy PCR(), but our 6 HIV()
outpatients were all sy PCR(-). No sy PCR() case
had a history except the LuesII cases. PCR
detected sy in 4/78, for a prevalence of
5.6. Conclusions Syphilis remains a difficult
target, and probably facilitates HIV acquisition
and progression. Serology missed 4 latent cases
in our small series. Other techniques for direct
detection of Tp require costly expertise. Our
whole blood PCR did not cross-react with Lyme. It
can be used in amniotic fluid and CSF. Can
therapy for sy cure these inconclusive cases? Are
they more vulnerable to HIV? Are they infectious?
Perhaps sy PCR should be evaluated for
screening.
15
A NEW GOLD STANDARD FOR SYPHILIS?J. B. Scythes,
C. M. Jones, R. H. NotenboomCommunity Initiative
for AIDS Research, Phoenix BioTech Corporation
European Academy of Dermatology and Venereology
II. Spring SymposiumBudapest, Hungary, 29 April
1 May 2004
Novel PCR screening on whole blood has found T.
pallidum DNA in erythrocytes of young gay men in
Budapest who are repeatedly negative in all other
standard syphilis tests, including treponemal
antibody screening (TPHA). This follows on
historical evidence suggesting syphilis has been
under-diagnosed ever since screening with
non-specific anti-lipoidal antigens began in
1906.... 105 gay males attending a dermatology
practice in Budapest have all been screened with
four tests RPR, TPHA, HIV Elisa, and the whole
blood syphilis nested PCR. Seven cases of
syphilis (average age 26) were detected using
syphilis PCR, but only two with a treponemal
assay, the accepted standard for detecting latent
syphilis. The results of all tests were as
follows 4 cases were PCR () TPHA (-) RPR (-)
HIV (-) 1 case was PCR () TPHA () RPR () HIV
(-) 1 case was PCR () TPHA () RPR (-) HIV (-) 1
case was PCR () TPHA (-) RPR (-) HIV
() Interestingly, the only HIV () case out of
these 105 men was also syphilis PCR (), as were
the only two TPHA () treated cases. One of these
TPHA () cases was the only reactor in the RPR
test in the entire cohort, none of whom had signs
or symptoms of early syphilis. The probability
that these PCR results are a chance observation
is very remote. Others have proven persistent
syphilis using this technique. (15) The clinical
implications of all these findings remain
unknown. Reference SYPHILIS PCR MOLECULAR
DETECTION OF T. PALLIDUM IN SERONEGATIVE
CASESTalha E, Kemeny B, Horvath I, et al.,
National Inst. Dermato-Venerology, HungaryReview
of Dermato-Venerology (Hungary) 2003 79 (2)65-68
16
CAN WE RELIABLY DIAGNOSE SYPHILIS?J. Scythes, C.
M. JonesCommunity Initiative for AIDS
ResearchToronto, Canada
Results (4) 13/183 of the Budapest group were
syphilis PCR(), while only four had ever been
treated. These four were the only TPHA ()
persons in the group.
17
CAN WE RELIABLY DIAGNOSE SYPHILIS?J. Scythes, C.
M. JonesCommunity Initiative for AIDS
ResearchToronto, Canada
Conclusions Diagnostically, early acute
syphilis is one thing and latent syphilis is
quite another.
18
CAN WE RELIABLY DIAGNOSE SYPHILIS?J. Scythes, C.
M. JonesCommunity Initiative for AIDS
ResearchToronto, Canada
RPR() findings were extremely rare among these
nearly 1500 high-risk patients.
19
CAN WE RELIABLY DIAGNOSE SYPHILIS?J. Scythes, C.
M. JonesCommunity Initiative for AIDS
ResearchToronto, Canada
Screening for syphilis with anti-lipoidal tests,
based on a 1906 concept, is inappropriate.
20
CAN WE RELIABLY DIAGNOSE SYPHILIS?J. Scythes, C.
M. JonesCommunity Initiative for AIDS
ResearchToronto, Canada
The persistence of treponemal antibody may
indicate ongoing syphilis infection despite
therapy, and such patients may need re-treatment
and follow-up using gene amplification.
21
CAN WE RELIABLY DIAGNOSE SYPHILIS?J. Scythes, C.
M. JonesCommunity Initiative for AIDS
ResearchToronto, Canada
Some patients with latent syphilis seem to have
little or no treponemal antibody. It is therefore
unclear to what extent undetected latent syphilis
overlaps with HIV, and HIV/AIDS.
22
Sobering quotes
23
"In spite of 400 years of study, we still do not
know the actual importance of syphilis as a cause
of death. To what extent does death directly from
syphilis masquerade under other diagnoses or to
what extent is syphilis an indirect cause of
death from other conditions? Is it justifiable to
assume, as did Osler, that syphilis actually
ranks first, instead of its apparent tenth, among
killing infections?" Moore JE. Unresolved
Clinical Problems of Syphilology. Am J Syph
Gonor and Ven Dis 1939 23(1)701-11
24
"Within 2 years after infection, untreated
syphilis produces immune changes in the host
which, with rare exceptions, are permanent and
make it impossible for tissues to react to
subsequent infection with development of early
syphilitic lesions." Thomas EW. Syphilis Its
Course and Management. New York Macmillan,
1949. p. 10
25
"Far from eradicating syphilis, antibiotics are
driving the disease underground and increasing
the difficulty of detection. Although the
incidence of disease has more than tripled since
1955, the chancre and secondary rash no longer
are commonly seen. Undoubtedly, some of these
lesions are being suppressed and the disease
masked by the indiscriminate use of antibiotics.
The ominous prospect of a widespread resurgence
of the disease in its tertiary forms looms
ahead." Pereyra AJ, Voller RL. A graphic
guide for clinical management of latent syphilis.
Calif Med 1970 112(5)13-8
26
"A substantial proportion of HIV-infected men
may have unrecognized, latent, inadequately
treated syphilis. These findings support more
aggressive treatment of T. pallidum infection in
this patient population." Musher DM, Hamill
RJ, Baughn RE. Effect of human immunodeficiency
virus (HIV) infection on the course of syphilis
and on the response to treatment. Ann Intern Med
1990 113(11)872-81
27
"The clinical manifestations of syphilis, which
have taken various forms over the centuries, have
now been transformed to mimic the appearance of
the opportunistic infections and cancers that may
accompany HIV infection, as well as the clinical
symptoms of AIDS itself." Larsen SA. Syphilis
Diagnosis Dynamics. 9th International Meeting of
the ISSTDR 1991 Banff, Alberta, abstract no.
C-12 273
28
CAN WE RELIABLY DIAGNOSE SYPHILIS?J. Scythes, C.
M. Jones
Community Initiative for AIDS Research32 Beaty
Avenue Toronto, Canada M6K 3B4 E-mail
ltjscythes_at_infinity.netgt Further material online
athttp//cbc.ca/ideas/Aidshttp//colman.net/ea
dv