P360 Single gene targeted nanopore sequencing for simultaneous identification and antimicrobial resistance detection of sexually transmitted infections

Background World Health Organization guidelines to treat sexually transmitted infections (STIs) with the right antibiotic, at the right dose and the right time are undermined by spread of antimicrobial resistance (AMR). Accurate and rapid diagnostics simultaneously predicting antibiotic susceptibility and diagnosis may help address this challenge. We report early data from a novel approach using single gene targeted nanopore sequencing for simultaneous STI identification and AMR detection among female sex workers (FSWs) in Ecuador. Methods Real-time PCR (RT-PCR) was performed on vulvo-vaginal swab samples from 200 FSWs to identify Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT), Mycoplasma genitalium (MG) and Trichomonas vaginalis (TV) infections. Samples positive and negative (controls) for these STIs were amplified and barcoded by PCR, targeting genes: gyrA (NG), ntr6 (TV) and 23S rRNA (MG), which confer resistance to fluoroquinolones, metronidazole and macrolides respectively, and omp1 (CT). A DNA library, constructed from a pool of these barcoded samples, was sequenced on Oxford Nanopore Technologies (ONT) MinION sequencer controlled by the smartphone operated ‘MinIT’ hand-held processor. Data were analysed using ONT AMR workflow, with manual BLAST confirmation. Results Among PCR positives analysed, 25/26 of CT, NG or MG infections were clearly distinguishable from controls using sequence read counts, but discrimination of TV positives from controls was challenging as many had low pathogen loads (RT-PCR: cycle threshold>35) with associated low sequence read counts. Manual BLAST confirmed 3/3 NG, 2/10 TV, and 0/11 MG had fluoroquinolone, metronidazole and macrolide resistance-associated mutations respectively. Overall library preparation and sequencing time took approximately about five hours. Conclusions Single gene targeted nanopore sequencing for diagnosing and simultaneously identifying key AMR markers for four common STIs shows promise. Further work to optimise accuracy, reduce costs and improve speed may allow sustainable approaches for managing STIs and emerging AMR in resource poor and laboratory limited settings.