P196 Development of Taqman based multiplex qPCR assay for simultaneous detection of N.gonorrhoeae, C.trachomatis and T.vaginalis in the endocervical/vaginal samples

Abstract

<h3>Background</h3> Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT) and Trichomonas vaginalis(TV) are among the major causative agents of sexually transmitted infections (STIs). Huge fraction of patients with NG, CT and TV infections are asymptomatic making syndromic case management(SCM) becomes unreliable. Taqman based triplex qPCR assay for simultaneous detection of NG, CT and TV, besides being dependable diagnostic tool, can effectively differentiate between mono-infections and co-infections of these pathogens. <h3>Methods</h3> Endo-cervical dry swabs were procured from non-pregnant females visiting Department of Obstetrics &amp; Gynaecology, VMMC and Safdarjung Hospital, Delhi, India as per ICMR ethical guidelines. DNA was isolated from the samples and presence of the causative agent, if any, was determined using in-house developed Multiplex qPCR assay by employing specific primer-probe sets targeting each pathogen. Analytical sensitivities and specificities were determined for each uniplex assay. In-house developed assay was clinically validated in comparison to published NAAT based assays. <h3>Results</h3> A 108 bp region of orf-1 gene of NG, 133 bp region of pld gene of CT and 147 bp region of pfo-B gene of TV genomic DNA were utilized for identification. Analytical sensitivity of NG uniplex assay was determined to be 10ag of recombinant DNA (3–4 DNA equivalents) and for both CT and TV, it was determined to be 100ag of DNA (30 DNA equivalents). 85 clinical samples have been examined so far and the assay detects NG and CT DNA with 83% and 60% sensitivities, 99% and 100% specificities, 83% and 100% PPV and, 99% and 98% NPV respectively. Two clinical samples among them were co-infected with NG and CT. <h3>Conclusion</h3> In-house developed multiplex qPCR assay provides an opportunity for diagnosis of three highly prevalent and curable STIs in only one assay. The results following validation are motivating enough to project it as a potentially reliable diagnostic tool for their detection.