A 5' -nuclease genotyping assay for identification of macrolide-resistant Mycoplasma genitalium in clinical specimens

Rapid and sensitive detection of macrolide resistance inMycoplasma genitaliumis required for guidance of adequate antimicrobial treatment. Previous studies have confirmed that single base mutations at position 2058 or 2059 in domain V of the 23S rRNA gene ofM. genitaliumresults in high-level macrolide resistance. Sequencing of PCR products remains the gold standard for identification of mutations conferring resistance to macrolides, but is laborious and time-consuming. The aim of the present study was to develop a 5' -nuclease genotyping assay to detect single nucleotide polymorphisms in the 23S rRNA gene ofMycoplasma genitaliumassociated with macrolide resistance by combining PCR with hydrolysis probes and subsequent endpoint genotyping analysis. The 5' -nuclease genotyping assay was used as a referral test to be used onM. genitaliumpositive samples and validated on 259 positive samples, of which 253 (97.7%) could be successfully sequenced. With the newly developed assay, 237/259 (91.5%) of the investigatedM. genitaliumpositive samples could be genotyped. Both the positive and the negative predictive values were 100% when evaluated on successfully genotyped samples. The newly developed assay discriminated macrolide-resistantM. genitaliumin clinical specimens possessing A2058G, A2058C, A2058T and A2059G with a sensitivity of 94.4% (95% CI: 90.7-98.2) and a specificity of 92.7% (95% CI: 87.8-97.6) when evaluated on successfully sequenced samples. The assay can correctly guide antimicrobial treatment ofM. genitaliuminfections.