Specific detection of Treponema pallidum in clinical samples: validation of a qPCR assay combining two genomic targets

Abstract

ObjectivesWe evaluated a real-time quantitative PCR (qPCR) for detection of the Treponema pallidum (TP) genome in clinical samples through simultaneous detection of two genomic targets.MethodsWe performed qPCR with TaqMan technology using two TP genes, polA and tpp47, as targets, with an internal positive control. The qPCR assay was compared with syphilis diagnosis based on a combination of clinical examination, serological results and inhouse nested PCR (nPCR). Samples were analysed at the National Reference Center for STIs at Cochin Hospital in Paris.ResultsIn total, from October 2010 to December 2016, 320 documented clinical samples (mucosal and cutaneous swabs) were collected from patients with or without syphilis attending STI centres in France. The qPCR had an overall sensitivity of 89% (95% CI 85.1% to 92.1%), a specificity of 100%, a positive predictive value of 100% and a negative predictive value of 88% (95% CI 84.3% to 91.5%). The agreement between qPCR and nPCR results was 94% (κ=0.88, 95% CI 0.83 to 0.93). Calibration of the qPCR assay, by cloning both the polA and tpp47 genes, defined the detection threshold as 1 copy/µL of DNA elution.ConclusionsWe validated a new qPCR for detecting the TP genome in clinical samples with excellent sensitivity and specificity. The cloning of polA and tpp47 genes for calibration would be interesting in the evaluation of bacterial loads in samples.

Comparison of molecular and serological assays on cerebrospinal fluid for the diagnosis of neurosyphilis

“…DNA was extracted from each sample with the InnuPREP Blood DNA‐IPC16 Mini kit on an InnuPure® C16 instrument (Analytikjena, Jena, Germany), in accordance with the manufacturer's instructions. DNA extracts were tested by nPCR targeting the tpp47 gene of TP, and real‐time quantitative PCR (qPCR) targeting both the tpp47 and polA genes of TP, as previously described 10,11 …”

“…DNA extracts were tested by nPCR targeting the tpp47 gene of TP, and real-time quantitative PCR (qPCR) targeting both the tpp47 and polA genes of TP, as previously described. 10,11…”

Treponema pallidum resistance to azithromycin in France: A nationwide retrospective study from 2010 to 2022

“…Samples were tested by nested PCR (nPCR) or real-time PCR, targeting the TP genes tpp47 and polA, as previously described. 4,5 All TP-positive samples were analysed for the presence of the A2058G mutation by amplification with nPCR followed by sequencing, as previously described. 6 Of the 413 routinely positive samples between 2010 and 2022, the 23S rRNA gene was amplified for 299 samples (72.4%).…”

Evaluation of the usefulness of routine molecular biology for the diagnosis of primary syphilis by assessing the serological status of patients with PCR‐confirmed syphilitic ulcers

“…previously described. 2 The diagnosis of primary syphilis was based on the presence of suggestive ulceration (without skin lesions compatible with secondary syphilis) associated with a positive qPCR. The 100% specificity of this PCR assay enabled us to rule out false positives results.…”

“…The 100% specificity of this PCR assay enabled us to rule out false positives results. 2 In total, 174 patients had a qPCR-positive sample with a diagnosis of primary syphilis (Figure 1). The median age was 37 years (IQR: 28-44.3), 97.7% were men (170) and 85.6% of patients (149) identified themselves as men who have sex with men.…”