How to Interpret Syphilis Test Results Thursday, August 30, 2007 In response to the syphilis outbreak in Alberta, as defined

by the Provincial He alth Officer, the Provincial Laboratory is collaborating with regional diagnostic laboratories, to implement new serological test methods for the diagnosis of syphilis, effective September 4th, 2007. The Old Method for Syphilis Serology In the old syphilis serology process, the initial test was the Rapid Plasma Reag in (RPR) test. The RPR test is not treponeme-specific, but measures antibody against the glycolipid cardiolipin. Th is antibody is an indicator of tissue damage and gives a useful measure of the activity of the infection. The R PR assay may also be positive in other conditions however, such as pregnancy, infectious mononucleosis, hepatitis , malaria, connective tissue diseases, esp. lupus, and IV drug abuse. A positive RPR must therefore be confir med by a Treponeme-specific test, such as TPPA or FTA-ABS. A major limitation of the RPR screening test is t hat it is slow to rise in primary syphilis, and may fade out in late or tertiary syphilis, thereby missing cases ( 4). The New Lab Process The new test algorithm is summarized briefly in the adjacent figure. The enzyme immunoassay (EIA) is the cornerstone of the new algorithm, and this is the main difference between the old and new processes. The Syphilis EIA is a treponeme-STD Clinics, specific test, similar to the old TPPA and FTA-ABS tests, but and Regions other than Capital and Calgary is more objective, more accurate and can be automated. The EIA uses recombinant syphilis antigens, and detects IgM or IgG antibodies against Treponema pallidum. These antibodies arise during the primary stage, and persist in most cases for the life of the patient. ProvLab has evaluated a ProvLab number of syphilis EIA tests, and in collaboration with the regional labs, will be switching to EIA as the first screening negative test. EIA results will usually be available within 1-2 days of Previous case? Report receipt of the serum at the lab. negative Since the RPR titre tends to parallel disease activity, it is a yes no useful indicator of response to therapy by observing a fall in titres over time (6). It is also useful, in seropositive persons, Perform RPR ti tre Syphilis EIA

to detect re-infection or treatment failure by observation of rising titres. For this reason, any EIA-positive patient will negative positive also be tested by RPR at the Provincial Lab. Report Perform RPR titre The INNO-LIA assay is a new supplementary test which negative measures antibodies to three recombinant Treponema pallidum antigens and one synthetic peptide antigen, and has Perform been reported to be extremely accurate (1,3). As it measures INNO-LIA treponemal antibody, it is expected to remain positive for life. This assay will be run 1-2 times per week to confirm new positive cases, and ens ure optimal performance of the EIA. Previously confirmed cases will not be retested by INNO-LIA (but will get R PR to monitor response to treatment). positive CLS or DKM L Community patients, Capital and Calgary Syphilis EIA Control antigens The INNO-LIA assay, from Hagedorn et al. (3) Treponemal antigens

2 Stage of Disease The RPR may be useful in helping to stage syphilis infection, but must be consid ered together with epidemiologic history, clinical presentation, timing of exposure, history of ade quate treatment and subsequent serologic response. Expert consultation should be sought to assist with this. Other subspecies T. pallidum subsp. pertenue, yaws T. pallidum subsp. carateum pinta T. pallidum subsp. endemecium bejel These non-venereal treponemal infections were largely controlled in the 1960 s, bu t are reported to be on the increase related to deteriorating social conditions. There are no laboratory tes ts readily available that can differentiate these infections from syphilis or each other. Consider patient ori gin and travel, patient age (these are often pediatric), distribution of lesions. Generally speaking because of the difficulty in distinguishing between reactive serologic tests for syphilis and non-venereal trepanematoses, m ost clinicians in Canada would manage the patient as a case of syphilis (often late latent syphilis). For a goo d review see Farnsworth and Rosen, Clinics in Dermatology, 24: 181-90, 2006. (2). Source: Farnsworth, Clinics in Dermatology 2006 Neurosyphilis This is often a difficult diagnosis, which should be made in consultation with a medical expert in this area. A reactive VDRL test on CSF is a specific indicator of neurosyphilis. Sensitivit y of VDRL for neurosyphilis is <50% however, so a negative result is not useful. Congenital syphilis This can also be a difficult diagnosis; consult an expert in STDs or pediatric i nfectious diseases for advice on management and follow up. The RPR titre typically declines by 3 months of age an d be reverts to non-reactive by 6 months if the infant is not infected. If the titres are stable or increase after 6 12 months of age, the child should be evaluated (including CSF examination) and treated as for congenital sy philis. Passively transferred treponemal antibodies can be present in an infant up to 15 months; a reactive tr eponemal test after 18 months is diagnostic of congenital syphilis; few data are available at the present time on seroreversion of the syphilis EIA test.

Interpretation of Syphilis Test Results: Syphilis Test Result: Comments: Darkfield microscopy positive Presence of spirochaetes with characteristic motil ity in exudate from genital ulcer or condyloma lata confirms the diagnosis. Expertise and equipment exists only at the STD Clinics for this test. Low sensitivity; a negative test does not rule out the diagnosis. Direct fluorescent antibody microscopy positive Fluorescent antibody detection of spirochaetes in exudate from genital ulcer or condyloma lata confirms the diagnosis. Performed on air dried slide of exudate sent to ProvLab. Low sensitivity; a nega tive test does not rule out the diagnosis. PCR positive (swab of exudate in universal transport medium) To be implemented in late 2007/early 2008. A positive Polymerase Chain Reaction (PCR) confirms presence of T. pallidum in lesion. Medium sensitivity; a negative test does not rule out the di agnosis. EIA negative For secondary or early latent syphilis, 100% of patients are expect ed to have treponemal antibody, therefore this result rules out these conditions. In primary infection, a small percentage may not yet have developed an antibody response. If this is a clinical likelihood, repeat serology in 2-4 weeks. For a few patients who are effectively treated ear ly in infection, treponemal antibody may revert to negative. EIA negative RPR positive Biological false positive, not syphilis. RPR tests may be non-specifically react ive in other conditions such as pregnancy, collagen vascular diseases (SLE), infectious mono, hepatitis, and IV drug abuse. EIA positive RPR negative INNO-LIA positive This patient has treponemal antibody but not reaginic antibody. Possible early p rimary infection, as RPR is slower to convert. Another likely scenario is late infection, as RPR may revert to negativ e over time, while treponemal antibodies usually persist for life. Absence of reaginic antibody does not necessarily indicate inactive infection. Also consider endemic treponematoses, see below. Detailed clinical review includ ing travel and treatment history is required. Has this patient had HIV or other STD tests? EIA positive RPR negative INNO-LIA negative or indeterminate This discrepant result is uncommon and difficult to interpret. Consider early se roconversion, and repeat serology in two weeks. Reactivity in EIA may be non-specific but this is rare. Reactivity may be fading due to very remote infection. EIA positive RPR Reactive INNO-LIA positive Treponemal and reaginic antibody consistent with any stage of infection. Managem ent is based on historical and clinical features, see treatment guidelines, links below.

If RPR titre is >32 dils, consider the possibility of neurological infection (5) . Has this patient had HIV or other STD tests? RPR titre fails to drop by fourfold. Rate of drop of RPR titre depends on durati on of infection and original titre. See Romanowski et al, (6) and Cdn Guidelines on STI for details. Some patients remain low RPR-positive indefinitel y. Also consider re-infection or neurosyphilis, particularly if titre rises 4 fold. Consultation with a specialist recommended. CSF VDRL positive In conjunction with positive blood serology, a positive VDRL t est is a specific indicator for neurosyphilis. The sensitivity is very low (<50%), therefore a negative VDRL result is of no va lue. August 30, 2007 P Tilley MD p.tilley@provlab.ab.ca

For details on management of syphilis and other STD s please see the Canadian Guid elines on Sexually Transmitted Infections, 2006 (http://www.phac-aspc.gc.ca/std-mts/sti_2006/qr-ess entiel_e.html ) and the Alberta STD Treatment Guidelines ( http://www.health.gov.ab.ca/professionals/STD Treatment.pdf ). Contacts For advice on management of a patient with an STI, contact your local infectious diseases physician, or the following STD specialists: Dr. Ameeta Singh, (780) 415-2825 Dr. Ron Read, (403) 944-7575 Dr. Barbara Romanowski (780) 436-4900 For information on lab testing, please contact your local microbiologist-on-call , or the Provincial Lab virologist-on-call at (403) 944-1200 or (780) 407-7121. Alternatively, you may r each me at the following numbers: Peter Tilley MD FRCPC Program Leader, STD s, Provincial Lab (403) 944-1203, p.tilley@provlab.ab.ca Reference List 1. Ebel, A., L. Vanneste, M. Cardinaels, E. Sablon, I. Samson, B. K. De, F. Hulstaert, and M. Zrein. 2000. Validation of the INNO-LIA syphilis kit as a confirmatory assay for Treponema pallidum antibodies. Journal of Clinical Microbiology 38:215-219. 2. Farnsworth, N. and T. Rosen. 2006. Endemic treponematosis: review and update. [Review] [97 refs]. Clinics in Dermatology 24:181-190. 3. Hagedorn, H. J., A. Kraminer-Hagedorn, B. K. De, F. Hulstaert, H. Pottel, and M. Zrein. 2002. Evaluation of INNO-LIA syphilis assay as a confirmatory test for syphilis. Journal of Clinical Microbiology 40:973-978. 4. Larsen, S. A., B. M. Steiner, and A. H. Rudolph. 1995. Laboratory diagnosis and interpretation of tests for syphilis. [Review] [219 refs]. Clinical Microbiology Reviews 8:1-21. 5. Marra, C. M., C. L. Maxwell, S. L. Smith, S. A. Lukehart, A. M. Rompalo, M. Eaton, B. P. Stoner, M. Augenbraun, D. E. Barker, J. J. Corbett, M. Zajackowski, C. Raines, J. Nerad, R. Kee, and S. H. Barnett. 2004. Cerebrospinal fluid abnormalities in patients with syphilis: association with cl inical and laboratory features. Journal of Infectious Diseases 189:369-376. 6. Romanowski, B., R. Sutherland, G. H. Fick, D. Mooney, and E. J. Love. 1991. Serologic response to treatment of infectious syphilis.[see comment]. Annals of Internal Medicine 114:1005-1009.

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