Molecular characterization of circulating treponema pallidum clusters in pediatric patient

Abstract

Syphilis is a public health problem with a sustained increase being the incidence of congenital syphilis (Sc) of 1.7 ‰ live births. Currently there are no techniques with sufficient sensitivity and specificity for the diagnosis of Sc Our group recently started with maternities a multicenter study to evaluate the use of molecular biology techniques (MBT) such as PCR, for the diagnosis of Sc. In Argentina there aren’t data about MBT in the diagnosis of syphilis or about the strains of Treponema pallidum pallidum (Tpp) in the pediatric population. Our objective was to evaluate the use of PCR in the diagnostic of syphilis in children and examine the clusters of Tpp by DNA sequencing. Pilot study in 5 pediatric cases of syphilis. Different lesion swabs (total= 8 samples) were processed for DNA extraction (QIAamp DNA Blood Mini Kit) followed by PCR for the genes of Tpp: Tp47kDA (conventional PCR) and dnaA using Taqman® probes (real time PCR - qPCR). Additionally, nested PCR for the TP0136 and TP0548 genes were performed in the samples and the sequences were subsequently purified and sequenced by commercial kit (BigDye™) in a genetic analyzer (3500 analyzer).Then, edition and alignment analysis were performed compared to the reference sequences of cluster SS14 (GenBank CP004011.1) and Nichols (GenBank CP004010.2). The different MBT (PCR, nested PCR and qPCR) concordance in an 88 % while 5 samples (at least one swab per patient) from different regions (soft palate, perianal, palms) were positive, 2 samples (perianal, tongue) were negative. Only 1 sample (perianal) was only positive by qPCR. In the sequencing analysis the predominant Tpp clade was Nichols (n= 4) over SS14 (n= 1). The results of PCR determination in blood of these patients is still in process. This study is the first one conducted in the pediatric population in our country for the detection of syphilis by BMT. As recently studies in Argentina reports, the Nichols clade in our study was greater as 10%, although in our pediatric population the prevalentce was higher (80 %) that in adults (26,8 %). The application of BMT in the detection of Sc is a promising alternative; the development of a large-scale and multicentric study will asses the effectiveness of the BMT and provide valuable information on local transmission networks of Tpp.