O06.3Macrolide Resistance of Mycoplasma Genitalium in France Directly Detected in Clinical Specimens by Real-Time PCR and Melting Curve Analysis

Objectives Mycoplasma genitalium (MG) is a sexually transmitted organism associated with non-gonococcal urethritis in men and several inflammatory reproductive tract syndromes in women. These infections are commonly treated with azithromycin. However macrolide resistance has been reported and is associated with point mutations in domain V of the 23S rRNA gene. In order to evaluate the prevalence of macrolide resistance in MG in French clinical specimens, we first used a recently published High Resolution Melting (HRM) assay. Because wild-type and mutated MG were hardly discriminated in MG-positive clinical specimens, we developed a new molecular assay for the rapid detection of macrolide resistance.MethodsBetween January 2011 and September 2012, 207 urogenital MG-positive clinical specimens were collected from 185 patients. For the detection of macrolide resistance-associated mutations, we designed a real-time PCR based on fluorescence resonance energy transfer (FRET) coupled with melting curve analysis. The assay was first validated on macrolide-resistant MG isolates with characterised A2058G/C and A2059G mutations ( Escherichia coli numbering), then optimised to be applied directly on clinical specimens. Resistant genotypes were confirmed by 23S rRNA gene sequencing.ResultsAmong 207 MG-positive clinical specimens, 136 from 119 patients were amplified with our assay, showing a sensitivity of 65.7% (136/207). A substitution in the 23S rRNA gene was found in 14.2% (17/119) of the patients, with a rate of 14.5% in 2011 and 14% in 2012. Nine and eight clinical specimens harboured the A2059G and A2058G mutations, respectively. In four cases, a mixed population of wild-type and mutated MG was observed.ConclusionMacrolide resistance prevalence of MG is 14.2% in France. Our FRET PCR assay is able to discriminate wild-type from resistant genotype in one reaction directly in clinical specimen. It will allow clinicians to shorten the time to initiate effective treatment and contribute to reduce transmission of resistant strains.