Scythes Iusti2006 001

CAN WE RELIABLY DIAGNOSE SYPHILIS?J. Scythes, C. M. Jones

Community Initiative for AIDS ResearchToronto, Canada

22nd IUSTI-EUROPE CONFERENCE ON SEXUALLY TRANSMITTED INFECTIONS

Oral communicationThursday 10/19/2006

14:00 Auditorium RichelieuSession 1: Syphilis and GUD

Abstract N°O.001



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.



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).

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, Canada

Biology 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.

SYPHILIS AND HIV: SEROPREVALENCE AND CLINICAL OBSERVATIONS IN HIV CLINIC, TORONTO

Fralick RA, Scythes JB, Notenboom RH, MacFadden DKThe Toronto Hospital, Western Division, Community Initiative for AIDS Research, Laboratory Services Branch,

Ontario Ministry of Health, Toronto, Canada

10th 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.



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 < 80/ul.



Methods:

(2) Serum aliquots from a further 250 patients from the University of Toronto's AIDS Epi-Study were similarly tested in 1992.

RELIABILITY OF SYPHILIS TESTS IN PATIENTS WITH HIV

Notenboom R; MacFadden DKLaboratory Services Branch, Ministry of Health, Toronto, Canada

VIII 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.



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.



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.

TREPONEMAL BASED SCREENING FOR SYPHILIS—DETECTING LATENT CASES

N. denHollander, R. Berry, M. FearonOntario Public Health Laboratory, Toronto, Canada

2000 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.



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.



Methods:

(4) Recently, 183 patients from two dermatology clinics in Budapest were tested for T. pallidum DNA with a nested PCR.

EXPERIENCE OF MOLECULAR SCREENING OF SYPHILIS BY PCR

Karoly Nagy, John B Scythes, Istvan Horvath, Bela Kemeny, Elyas Talha, Robert Notenboom, Attila Horvath National Inst. Dermato-Venerology, Hungary; Phoenix Bio-Tech Corp., Canada

14th 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.

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 Symposium

Budapest, 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, Hungary

Review of Dermato-Venerology (Hungary) 2003; 79 (2):65-68



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.



Conclusions:

Diagnostically, early acute syphilis is one thing and latent syphilis is quite

another.



RPR(+) findings were extremely rare among these nearly 1500 high-risk

patients.



Screening for syphilis with anti-lipoidal tests, based on a 1906 concept, is

inappropriate.



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.



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.

Sobering quotes

"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

"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

"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

"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

"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


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Further material online at

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