Objectives To develop a simple DNA sequencing test for simultaneous identification and antimicrobial resistance (AMR) detection of multiple sexually transmitted infections (STIs). Methods Real-time PCR (qPCR) was initially performed to identify Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT), Mycoplasma genitalium (MG) and Trichomonas vaginalis (TV) infections among a total of 200 vulvo-vaginal swab samples from female sex workers in Ecuador. qPCR positive samples plus qPCR negative controls for these STIs were subjected to single gene targeted PCR MinION-nanopore sequencing using the smartphone operated MinIT. Results Among 200 vulvo-vaginal swab samples 43 were qPCR positive for at least one of the STIs. Single gene targeted nanopore sequencing generally yielded higher pathogen specific read counts in qPCR positive samples than qPCR negative controls. Of the 26 CT, NG or MG infections identified by qPCR, 25 were clearly distinguishable from qPCR negative controls by read count. Discrimination of TV qPCR positives from qPCR negative controls was poorer as many had low pathogen loads (qPCR cycle threshold >35) which produced few specific reads. Real-time AMR profiling revealed that 3/3 NG samples identified had gyrA mutations associated with fluoroquinolone resistance, 2/10 of TV had mutations related to metronidazole resistance, while none of the MG samples possessed 23S rRNA gene mutations contributing to macrolide resistance. Conclusions Single gene targeted nanopore sequencing for diagnosing and simultaneously identifying key antimicrobial resistance markers for four common genital 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.

No abstract

Objectives: To develop a simple DNA sequencing test for simultaneous identification and antimicrobial resistance (AMR) detection of multiple sexually transmitted infections (STIs). Methods: A total of 200 vulvo-vaginal swab samples from 200 female sex workers in Ecuador were initially tested by real-time PCR for Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT), Mycoplasma genitalium (MG) and Trichomonas vaginalis (TV). Samples positive for these STIs and controls were subjected to single gene targeted PCR MinION-nanopore sequencing by using the smartphone operated MinIT. Results: Among 200 vulvo-vaginal swab samples 43 were positive for at least one of the STIs by real-time PCR. Single gene targeted nanopore sequencing generally yielded higher pathogen specific reads in positive samples than negative controls. Of the 26 CT, NG or MG infections identified by real-time PCR, 25 were clearly distinguishable from the negative controls using sequence read count. Discrimination of TV positives from controls was poorer as many had low pathogen loads (cycle threshold > 35) with associated low sequence read counts. AMR analysis workflow indicated that 11% of the classified reads aligned with antibiotic resistance genes, all of which identified fluoroquinolone resistance in NG. Conclusions: Single gene targeted nanopore sequencing for diagnosing and simultaneously identifying key antimicrobial resistance markers for four common STIs, associated with genital tract discharge in women 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.