Point-of-Care Amenable Detection of Mycoplasma genitalium and Its Antibiotic Resistance Mutations

Mycoplasma genitalium (MG) is an emerging sexually transmitted bacterium. Due to its fastidious and slow-growing nature, MG is difficult to detect through culture-based diagnostics. Like Neisseria gonorrheae, another bacterial pathogen linked to sexually transmitted infections (STIs), MG has developed resistance to macrolide and fluoroquinolone antibiotics used to treat STIs. The ability to detect MG and identify genomic mutations associated with antibiotic resistance simultaneously can enable antibiotic stewardship and mitigate the spread of antibiotic-resistant MG. Toward this end, we first developed a multiplexed probe-based PCR-melt assay that detects MG and the presence of macrolide resistance mutations in the 23S rRNA gene and fluoroquinolone resistance mutations in the parC gene. Each target was identified via its unique combination of fluorescence label and melting temperature. This approach allowed differentiation between the different types of mutations at the genes of interest. Following initial assay optimization, the assay was integrated into a droplet magnetofluidic cartridge used in a portable platform to integrate automated sample extraction, PCR amplification, and detection. Lastly, we demonstrated that the integrated assay and droplet magnetofluidic platform could detect MG and antibiotic resistance-associated mutations in clinical isolates spiked into urine samples in 40 min.