O4.3 - MgSeq: Enabling M. genitalium identification and antimicrobial resistance detection in genital and extra-genital samples from female sex workers in Ecuador using multiplex PCR and nanopore sequencing

Background

Mycoplasma genitalium (MG) is an important sexually transmitted infection (STI) associated with reproductive health sequelae in women, with high rates of antimicrobial resistance (AMR) to macrolides and fluoroquinolones. The small 570-590 kb genome allows for development of rapid molecular-epidemiology technologies more commonly used for viruses, such as for covid-19. We developed a multiplex multi-gene tiled-PCR scheme enabling identification and detection of resistance associated mutations (RAMs) and clade-typing.

Method

A multiplex PCR panel to generate 2kb amplicons tiling across twenty-six MG genes was developed using a web-based primer design tool, optimised using co-culture positive reference and clinical MG strains. The panel was used to amplify MG-positive DNA from clinical samples previously collected from 250 female sex workers (FSWs) and 250 non sex workers (NSWs) from Ecuador, collected two years previously from vaginal, anorectal and pharyngeal sites, frozen and later transported to the U.K. Amplicons were barcoded and sequenced using Oxford Nanopore MinION. Kraken taxonomic identifier was used to confirm MG DNA presence. Phylogenetic trees were built using IQTREE and visualised using Figtree.

Results

Approximately 50kb of MG genome was amplified using 29 primer pairs in three primer pools. Proportion of MG amongst all FSWs was 9.6% (24/250). DNA extracts from 13 vaginal, 12 anorectal and 1 pharyngeal sample, representing 17 FSWs, 6 of whom had 2 samples included, and 2 NSWs were sequenced. An unrooted phylogenetic tree identified three distinct clinical clades, with positive control and reference sequences confirmed as adjacent. Any two MG sequences derived from separate anatomical sites in the same individual were always of the same clade. However, there was no clear clustering by geolocation and no known clinically significant RAMs in 23S rRNA, gyrA, or parC were detected. However, 38.5% (10/26) samples harboured parC P62S, implicated in evolution of fluoroquinolone resistance.

Conclusion

MgSeq is a new, potentially rapid method for long-read MG DNA sequencing, from highly fragmented, low quality clinical samples. Close clustering of sequences derived from multiple sites in a FSWs suggest anatomical contamination or mechanical sharing through sexual practices. Adaptation of this method could be utilised for other important STIs.