Development of a Real-Time PCR Assay To Detect Treponema pallidum in Clinical Specimens and Assessment of the Assay's Performance by Comparison with Serological Testing

Abstract

The incidence of infectious syphilis in men who have sex with men and human immunodeficiency virus-infected patients has increased steadily in Victoria, Australia, since 2002. A TaqMan real-time PCR assay targeting the polA gene of Treponema pallidum (TpPCR) was developed. The analytical sensitivity of the assay was estimated to be 1.75 target copies per reaction. Initially, the assay was used to test a variety of specimens (excluding blood) from 598 patients. Of the 660 tests performed, positive PCR results were obtained for 55 patients. TpPCR results were compared with serology results for 301 patients being investigated for early syphilis. Of these patients, 41 were positive by both TpPCR and serology, 246 were negative by both TpPCR and serology, 4 were TpPCR positive but negative by serology, and 10 were TpPCR negative but showed evidence of recent or active infection by serology. Directly compared with serology, TpPCR showed 95% agreement, with a sensitivity of 80.39% and a specificity of 98.40%. Potential factors leading to the discrepant results are discussed. Concurrent serology on 21 patients with TpPCR-positive primary syphilitic lesions demonstrated that a panel of current syphilis serological tests has high sensitivity for the detection of early syphilis. We found that TpPCR is a useful addition to serology for the diagnosis of infectious syphilis. Direct comparison with other T. pallidum PCR assays will be required to fully assess the limitations of the assay.Since 2002, there has been a marked increase in the number of cases of locally acquired syphilis infection in Victoria, Australia (2). Most cases were detected in men who have sex with men, with a disproportionate number of cases in human immunodeficiency virus (HIV)-infected patients (3).Clinical suspicion, and recognition of the symptoms and signs of syphilis, supported by serology is the mainstay of syphilis diagnosis, as direct detection methods such as dark-field microscopy and direct immunofluorescence are relatively insensitive, require fresh high-quality specimens, and are unsuitable for use on specimens from mucosal sites or if superinfection is present (4). Serology may be problematic in the early stages of primary syphilis, as rapid plasma reagin assay (RPR) responses may take some time to develop (4), particularly in HIV-infected patients, and few laboratories in Australia routinely use immunoglobulin M (IgM) assays.The Victorian Infectious Diseases Reference Laboratory (VIDRL) acts as the state reference laboratory for syphilis serology, confirming positive results from other laboratories and offering less commonly used tests, such as IgM assays. The laboratory also provides primary diagnostic services to a number of Melbourne sexually transmitted infection (STI) clinics and receives large numbers of specimens for culture or PCR for other agents of STI, allowing easy access to specimens for the development and assessment of novel STI detection assays. Our aim was to develop a robust, sensitive, and specific realtime PCR assay to d...

Clinical Prediction and Diagnosis of Neurosyphilis in HIV-Infected Patients with Early Syphilis

“…Our results suggest that in early syphilis, the RPR titer magnitude may not be as predictive of NS as it is during the later stages of disease, and it probably should not be used as the sole criterion for deciding whether to perform an LP. Several studies have evaluated PCR for the diagnosis of NS in adults (10,19,(22)(23)(24)(25)(26)(27)(28)(29). However, the results obtained in these studies are difficult to compare to our results for several reasons.…”

Diagnóstico de la infección por Treponema pallidum en pacientes con sífilis temprana y neurosífilis mediante reacción de la polimerasa en cadena

“…La DFA-TP es un método de gran sensibilidad y especifi cidad pero el reactivo que requiere no está disponible para los laboratorios de diagnóstico, sino que sólo para los laboratorios de referencia 19 . Debido a las limitaciones de los métodos disponibles en la actualidad, el diagnóstico de sífi lis es poco especí-fi co. En este contexto, se ha planteado el uso de NAATs basados en la reacción de la polimerasa en cadena (RPC) para la detección de T. pallidum [20][21][22] . Estos tests se basan principalmente en la detección de dos genes: el gen tpN47 que codifi ca para la proteína de membrana de 47 kDa [22][23][24][25][26] y el gen polA que codifi ca para la ADN polimerasa I 21,27 .…”

“…Debido a las limitaciones de los métodos disponibles en la actualidad, el diagnóstico de sífi lis es poco especí-fi co. En este contexto, se ha planteado el uso de NAATs basados en la reacción de la polimerasa en cadena (RPC) para la detección de T. pallidum [20][21][22] . Estos tests se basan principalmente en la detección de dos genes: el gen tpN47 que codifi ca para la proteína de membrana de 47 kDa [22][23][24][25][26] y el gen polA que codifi ca para la ADN polimerasa I 21,27 . La RPC dirigida al gen de la proteína de 47 kDa se ha utilizado ampliamente para detectar T. pallidum en distintos tejidos, mostrando sensibilidades similares al RIT en muestras de úlceras genitales y en sangre total 15,23 .…”

Laboratorial Diagnosis of Syphilis

“…Real-time PCR was performed in genital lesion specimens, but not in serum. When compared with serology results, polA gene real-time PCR presented the sensitivity of 80.39% and the specificity of 98.40% (Leslie et al, 2007). Another study found sensitivity of 72.8% and specificity of 95.5% in patients with clinical diagnosis of primary syphilis, the detection of secondary syphilis was low, and the sensitivity was 43.0% (Heymans et al, 2010).…”

“…Another study found positivity ratio of 39.1% for ttp47 PCR (260 bp) and 31.1% for polA PCR (378 bp), in blood samples from patients with latent syphilis (Castro et al, 2007). The real-time PCR had been developed for detection of T.pallidum DNA in clinical samples, they targeted polA gene (Heymans et al, 2010;Koek et al, 2006;Leslie et al, 2007) or tpp47 gene (Gayet-Ageron et al, 2009). The first test was validated using an analytical panel (n = 140) and a clinical panel of genital samples (n = 112) from patients attending a sexually transmitted infections clinic.…”