Needle lost in the haystack: multiple reaction monitoring fails to detect Treponema pallidum candidate protein biomarkers in plasma and urine samples from individuals with syphilis.

Background: Current syphilis diagnostic strategies are lacking a sensitive manner of directly detecting Treponema pallidum antigens. A diagnostic test that could directly detect T. pallidum antigens in individuals with syphilis would be of considerable clinical utility, especially for the diagnosis of reinfections and for post-treatment serological follow-up. Methods: In this study, 11 candidate T. pallidum biomarker proteins were chosen according to their physiochemical characteristics, T. pallidum specificity and predicted abundance. Thirty isotopically labelled proteotypic surrogate peptides (hPTPs) were synthesized and incorporated into a scheduled multiple reaction monitoring assay. Protein extracts from undepleted/unenriched plasma (N = 18) and urine (N = 4) samples from 18 individuals with syphilis in various clinical stages were tryptically digested, spiked with the hPTP mixture and analysed with a triple quadruple mass spectrometer. Results: No endogenous PTPs corresponding to the eleven candidate biomarkers were detected in any samples analysed. To estimate the Limit of Detection (LOD) of a comparably sensitive mass spectrometer (LTQ-Orbitrap), two dilution series of rabbit cultured purified T. pallidum were prepared in PBS. Polyclonal anti- T. pallidum antibodies coupled to magnetic Dynabeads were used to enrich one sample series; no LOD improvement was found compared to the unenriched series. The estimated LOD of MS instruments is 300 T. pallidum/ml in PBS. Conclusions: Biomarker protein detection likely failed due to the low (femtomoles/liter) predicted concentration of T. pallidum proteins. Alternative sample preparation strategies may improve the detectability of T. pallidum proteins in biofluids.

Candidate Treponema pallidum biomarkers uncovered in urine from individuals with syphilis using mass spectrometry.

AIM: A diagnostic test that could detect Treponema pallidum antigens in urine would facilitate the prompt diagnosis of syphilis. MATERIALS & METHODS: Urine from 54 individuals with various clinical stages of syphilis and 6 controls were pooled according to disease stage and interrogated with complementary mass spectrometry techniques to uncover potential syphilis biomarkers. RESULTS & CONCLUSION: In total, 26 unique peptides were uncovered corresponding to four unique T. pallidum proteins that have low genetic sequence similarity to other prokaryotes and human proteins. This is the first account of direct T. pallidum protein detection in human clinical samples using mass spectrometry. The implications of these findings for future diagnostic test development is discussed. Data are available via ProteomeXchange with identifier PXD009707.

Assessment of a real-time PCR test to diagnose syphilis from diverse biological samples.

OBJECTIVES: To investigate the contribution of a real-time PCR assay for the detection of Treponema pallidum in various biological specimens with the secondary objective of comparing its value according to HIV status. METHODS: Prospective cohort of incident syphilis cases from three Swiss hospitals (Geneva and Bern University Hospitals, Outpatient Clinic for Dermatology of Triemli, Zurich) diagnosed between January 2006 and September 2008. A case-control study was nested into the cohort. Biological specimens (blood, lesion swab or urine) were taken at diagnosis (as clinical information) and analysed by real-time PCR using the T pallidum 47 kDa gene. RESULTS: 126 specimens were collected from 74 patients with primary (n = 26), secondary (n = 40) and latent (n = 8) syphilis. Among primary syphilis, sensitivity was 80% in lesion swabs, 28% in whole blood, 55% in serum and 29% in urine, whereas among secondary syphilis, it was 20%, 36%, 47% and 44%, respectively. Among secondary syphilis, plasma and cerebrospinal fluid were also tested and provided a sensitivity of 100% and 50%, respectively. The global sensitivity of T pallidum by PCR (irrespective of the compartment tested) was 65% during primary, 53% during secondary and null during latent syphilis. No difference regarding serology or PCR results was observed among HIV-infected patients. Specificity was 100%. CONCLUSIONS: Syphilis PCR provides better sensitivity in lesion swabs from primary syphilis and displays only moderate sensitivity in blood from primary and secondary syphilis. HIV status did not modify the internal validity of PCR for the diagnosis of primary or secondary syphilis.

Effectiveness and cost-benefit of enhancements to a syphilis screening and treatment program at a county jail.

BACKGROUND: High rates of syphilis are found in inmates of county jails. Treatment of this infected transient population necessitated the development of a rapid protocol. GOAL: To evaluate a rapid screening and treatment protocol for syphilis in a county jail. STUDY DESIGN: Over a 2-year period 18,442 inmates were screened for syphilis with a nontreponemal test and record search for treatment history. Confirmatory test results were reviewed following treatment. Cost was defined as deflated marginal outlays. Benefit was calculated as the discounted expected cost of treatment of congenital, late, and neurosyphilis. RESULTS: The sensitivity, specificity, and positive predictive value of the protocol were 99.6%, 80.8%, and 79.3%, respectively. Of 257 confirmed cases, 183 were offered treatment in jail. The percentage of short-term inmates treated increased following implementation. The cost-benefit ratio was 9.14:1. CONCLUSIONS: The protocol was highly effective in patient identification and treatment delivery, and cost-effective as well.

The role of immune complexes in early syphilis and in the Jarisch-Herxheimer reaction.

Circulating immune complexes (CIC) were detected in one of 11 patients with primary syphilis and in 5 of 12 patients with secondary syphilis. The level of CIC was significantly increased in patients with secondary syphilis. Four weeks later a significant decline in CIC was found. No relationship was demonstrated between CIC and affection of the skin, lymph nodes, or kidneys. An increased albumin excretion rate was demonstrated before treatment. No differences were found in the excretion rate of beta-2-microglobulin before or after treatment. Nineteen patients had a Jarisch-Herxheimer reaction during treatment. An increase in CIC was found in 5 patients and a decrease in 7 patients. No correlation could be demonstrated between the level of or changes in CIC and the severity of the Jarisch-Herxheimer reaction.