Identification of macrolide-resistant Mycoplasma genitalium using real-time PCR.

Abstract

Mycoplasma genitalium is a common cause of non-gonococcal urethritis (NGU) in Western Europe, but is not routinely tested for in all clinics. A high prevalence of macrolide-resistant M.genitalium has been reported. An easy to use test that can predict likely macrolide treatment failure is potentially very valuable. We report the development of a rapid and reliable real-time PCR-assay which detects all relevant resistance loci in the M.genitalium 23S rRNA gene. Mycoplasma genitalium-positive clinical samples were collected between December 2012 and May 2013, from samples sent routinely to the laboratory for diagnostic testing for M.genitalium. The real-time PCR assay was designed using forward amplification primers complementary to all relevant commonly identified 23s rRNA gene mutations, a common reverse amplification primer and a common TaqMan Probe. We report a Taqman assay for detection of common 23S rRNA genotypes at position 2058 and 2059 (Escherichia coli numbering) associated with macrolide resistance, directly from clinical samples. We validated the assay by comparison with DNA sequence determination. Our TaqMan assay detects common genotypes associated with macrolide-resistant M.genitalium, namely, A2058G, A2059G and A2058C. We show association between the presence of resistant M.genitalium and treatment failure, thereby confirming the validity of testing for these mutants to prevent further spread of antimicrobial resistance and to allow informed choice of antibiotics for treatment.