Study setting and population
Taylor Square Private Clinic was established in 1981 as Australia’s first private sexual health clinic and has one of the highest case-loads of HIV-infected MSM patients in Australia [11]. All high-risk patients are routinely screened for T pallidum, Chlamydia trachomatis, Neisseria gonorrhoeae and HIV according to guidelines [12]. All patients in this study were consecutive high-risk MSM with lesions suggestive of early syphilis between 2004 and 2010, though in the early years of the study the test was in limited supply so it was only used for the highest-risk men. Heterosexual patients were excluded because syphilis is rare in heterosexuals in Australian cities. There was no standardized specimen collection protocol and several different doctors collected specimens.
T pallidumPCR
Specimen collection
A dry swab specimen was obtained from genital, anal or oral ulcers; or from the surface of rashes, papular lesions, or mucosal erosions suggestive of secondary syphilis; then placed in transport medium and sent at room temperature to the Institute of Clinical Pathology and Medical Research (ICPMR) for syphilis PCR testing. Blood for syphilis serology was collected at the same time.
DNA extraction
DNA was extracted from clinical specimens using the NucliSENS easyMAG (BioMeurieux) total nucleic acid extraction system with off board lysis. In brief, swabs were placed in individual lysis tubes, vortexed vigorously and incubated at room temperature for 10 minutes. Lysis solution was then combined with magnetic silica in an extraction cartridge and DNA extracted in the easyMAG using Boom technology. DNA extracts were stored at −80°C prior to PCR testing.
Assay design
The T pallidum PCR targets a 146 base pair region of the gene encoding the 47kDa lipoprotein (Genbank accession number: M88769). The assay uses a 6 carboxyfluorescein (FAM) labelled hydrolysis probe and end point fluorescence detection to identify amplification of products.
The primers [Syph1: 1130AGG GGA AGG TGC TGA CCA TAG1150; & Syph2: 1275GGG AGT GAA ATC CGC AGA GAG1255] and hydrolysis probe [1166(6-FAM) AGC CTA AGC TTG TCA GCG ATC AAG C (BHQ1)1191] were designed using Primer3 and manufactured by Sigma Aldrich.
The PCR was performed in a 50uL reaction containing sample DNA, 3.5mM MgCl2, 200nM each primer, 100nM probe & HotStar Taq Master Mix (Qiagen) [containing 1.5mM MgCl2; 200uM each dNTP & HotStar taq DNA polymerase]. Baseline fluorescence was determined using the FlourTracker (Stratagene) before amplification in a Eppendorf Mastercycler under the following conditions: 95°C × 15 min; 50 cycles of [96°C × 10 sec, 60°C x 1min, 72C × 1min]; 72°C × 5min. Post amplification fluorescence was again measured in the FluorTracker and the ratio of post- to pre-amplification fluorescence calculated.
Samples with fluorescence ratios greater than 3.4 were considered positive and confirmed by gel electrophoresis on a 2% agarose gel run for 40 minutes at 200V. Fluorescence ratios less than 1.5 indicated a negative sample, and samples with ratios between 1.5 and 3.4 were deemed equivocal and the assay repeated.
Controls
Individual samples were tested in parallel with inhibition and contamination controls and as both neat and diluted DNA to ensure detection of both weak and strong specimens. Inhibition controls consisted of sample DNA spiked with positive material, while contamination controls utilised no template reagent samples. Each run also contained a serial dilution of positive control to identify the limit of detection of the assay. The positive control was produced by extraction of DNA from a T pallidum (Nichols) suspension which had been previously used in the T pallidum immobilization assay. Initial experiments indicated the assay had a limit of detection of 10–100 organisms and was specific for T pallidum species.
Syphilis serology
Sera were initially screened with fully automated one-step Immulite Treponemal EIA (Siemens Healthcare Diagnostics) which uses one major T pallidum recombinant antigen (p17). Specimens positive by EIA were then subjected to confirmatory testing with a RPR test (Reditest, Biokit) and a T pallidum particle agglutination assay (TPPA) (Serodia TPPA, Fujirebio) and fluorescent treponemal antibody absorption (FTA-ABS) test (MarDx®). Men that had previously been treated for syphilis were screened with the RPR test.
Narrow definition of a syphilis case
A MSM patient was classified as having primary or secondary syphilis if:
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1.
His serology was positive by EIA and TPPA at the time of the PCR swab, after being negative in the previous year, or
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2.
His RPR titre had risen 4-fold (two titres) or more if he had previously been treated for syphilis.
However, as this definition excludes cases that are in the serological window period, for this study we used a broader case definition:
Broader definition of a syphilis case
A MSM patient was classified as having primary or secondary syphilis if he had consistent symptoms and signs and:
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1.
His serology was positive by both the EIA and TPPA at the time of the PCR swab or within 6 weeks of treatment, after being negative in the previous year, or
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2.
His RPR titre rose 4-fold (two titres) or more if he had previously been treated for syphilis.
This expanded (longitudinal) definition of early syphilis was developed because many cases were treated presumptively (before serological confirmation) on clinical suspicion or as soon as a positive PCR test result became available. Direct detection tests such as dark-field microscopy [13] and PCR [14] for syphilis have been documented to sometimes precede the serological response, as with all infections.
We conservatively settled on a 6-week cut-off as the minimum period for someone to be (a) treated for syphilis, (b) to have a long-acting injected penicillin or a 2-week course of oral doxycycline ‘wash out’, (c) to become newly-infected with syphilis, and (d) to thereafter mount a serological response. Any seroconversion to syphilis within 6 weeks was, therefore, deemed to result from a T pallidum infection at the time of the initial swab collection and treatment.
Uninfected case definition
A MSM patient was deemed to not have syphilis if the T pallidum PCR test was negative and:
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1.
His T pallidum EIA test remained negative, or
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2.
His RPR titre did not rise 4-fold (two titres) or more if he had previously been treated for syphilis.
Analysis
Clinical and laboratory data that were extracted from the patient management system included HIV status, syphilis stage, treatment, previous syphilis, syphilis serology and PCR results.
The performance (sensitivity and specificity) of the Treponema pallidum PCR in lesions suggestive of primary syphilis and secondary syphilis was then assessed against the definitions of a syphilis case (above), and by HIV status. For the purpose of comparability with other studies, we also assessed the performance of the PCR test against the narrower definition of syphilis that was limited to base-line clinical signs and serology. We calculated 95% confidence intervals (CIs) using the binomial approximation method.
Using a Chi-square test we compared the performance of the PCR test in HIV-positive and HIV-negative men.
All analyses were conducted using Stata statistical software version 9 [15].