Background Current epidemiological studies demonstrate a high prevalence of Mycoplasma genitalium (MG) infections in high-risk groups, especially MSM. Owing to the widespread macrolide resistance the European Guideline on MG infections recommend complementing the molecular detection of MG with an assay capable of detecting macrolide resistance-associated mutations. Macrolide resistance is caused by a single nucleotide polymorphism (SNP) in region V of the 23S rRNA gene. Two nucleotide positions in the 23S rRNA gene have been associated with azithromycin resistance: A2058 and A2059.

Methods The Aptima M. genitaliumAssay® was used for the initial detection on the Panther® system. A hydrolysis probe real-time PCR on theOpen Channelof the PantherFusion® system for automated reflex testing after MG positive results was established and validated. The Open Channelof the instrument allows the use of custom primers and probes on the PantherFusion® system. Minor groove binder (MGB) hydrolysis probes were used for accurate and reliable SNP discrimination at position 2058/2059. The workflow enables an automated analysis process including DNA extraction, PCR setup, and results interpretation. Using the open channel 300 samples could be genotyped within an 8 h working day.

Results 60 MG positive clinical samples were tested. The laboratory developed test (LDT) was able to detect the wild type variant in 20 samples and the A2058G/A2059G mutations in 22 samples. All results were confirmed by amplicon sequencing and commercially available test system. 18 (30%) samples did not show any typing result either using different LDT or commercial test system.

Conclusion MG positive samples can be typed for macrolide resistance using our LDT on the same platform during one run. The combination of a MG high-throughput test followed by macrolide resistance testing improves the efficiency of large-scale epidemiological resistance surveillance. However, highly sensitive TMA assay might result in a significant number of non-typable samples. Further studies are needed to improve the sensitivity and explanatorypower of MG resistance testing.

Disclosure No significant relationships.