Mycoplasma genitalium is a cause of non-gonoccocal urethritis (NGU) in men and cervicitis and pelvic inflammatory disease in women. Recent international data also indicated that the first line treatment, 1 gram stat azithromycin therapy, for M. genitalium is becoming less effective, with the corresponding emergence of macrolide resistant strains. Increasing failure rates of azithromycin for M. genitalium has significant implications for the presumptive treatment of NGU and international clinical treatment guidelines. Assays able to predict macrolide resistance along with detection of M. genitalium will be useful to enable appropriate selection of antimicrobials to which the organism is susceptible and facilitate high levels of rapid cure. One such assay recently developed is the MG 23S assay, which employs novel PlexZyme™ and PlexPrime™ technology. It is a multiplex assay for detection of M. genitalium and 5 mutations associated with macrolide resistance. The assay was evaluated in 400 samples from 254 (186 males and 68 females) consecutively infected participants, undergoing tests of cure. Using the MG 23S assay, 83% (331/440) of samples were positive, with 56% of positives carrying a macrolide resistance mutation. Comparison of the MG 23S assay to a reference qPCR method for M. genitalium detection and high resolution melt analysis (HRMA) and sequencing for detection of macrolide resistance mutations, resulted in a sensitivity and specificity for M. genitalium detection and for macrolide resistance of 99.1/98.5% and 97.4/100%, respectively. The MG 23S assay provides a considerable advantage in clinical settings through combined diagnosis and detection of macrolide resistance.

Mycoplasma genitalium is a significant pathogen for which first-line treatment is becoming less effective due to increased resistance to macrolides. As conventional culture and antimicrobial susceptibility testing is not feasible for routine detection of this pathogen, molecular markers such as detection of mutations in the 23S rRNA gene have been described to predict resistance. Recently, a novel multiplex quantitative PCR (qPCR) assay, ResistancePlus MG, has been described for the simultaneous detection of Mycoplasma genitalium and macrolide resistance. In the current study, the clinical performance of the assay was evaluated on 1,089 consecutive urine and anogenital swab samples in symptomatic and asymptomatic male and female patients. Overall, 6.0% were positive for M. genitalium, with 63.1% having macrolide resistance-associated mutations. Compared to the laboratory-validated qPCR method targeting the 16S rRNA gene and Sanger sequencing to determine 23S rRNA mutations, the sensitivity and specificity of M. genitalium detection were 98.5% and 100% and for detection of macrolide resistance mutations were 100.0% and 96.2%, respectively. This assay offers a considerable advantage in clinical settings for M. genitalium testing by making the results of macrolide resistance and mutation analyses simultaneously available, which is increasingly important with escalating macrolide resistance.

BackgroundWhilst qPCR provides an extremely powerful tool for genetic analysis, some applications such as multiplexing variant alleles (eg SNPs, point mutations or deletions), remain challenging using current primer/probe systems. The novel design features of PlexPrimers allow sensitive, multiplexed analysis of variant alleles even when these are tightly clustered.MethodPlexPrimers were combined with PlexZymes in qPCR assays for the detection of SNPs in human absorption, distribution, metabolism, and excretion (ADME) genes; clustered mutations in the 23S rRNA gene which confer antibiotic resistance to Mycoplasma genitalium; and deletions within the human epidermal growth factor receptor (EGFR) gene.ResultsThe combination of PlexPrimers and PlexZymes allowed robust multiplexing of targets which resulted in 100% concordance with results obtained using hydrolysis probe kits for 14 SNPs in the ADME genes. A 7-plex qPCR assay targeting M. genitalium, 5 clustered mutations associated with macrolide resistance and an internal control, allowed efficient amplification of all targets, with all 5 mutations detected in a single channel. Finally, the strategy was employed to analyse common EGFR mutants with high sensitivity, detecting deletions present at only 0.01%.ConclusionPlexPrime is a novel technology for the detection of genetic variants. Unlike previous strategies, the combination of PlexPrimers with PlexZymes enables both allele-specific detection and allele-specific amplification in qPCR. The study demonstrated highly sensitive and specific detection of mutations and SNPs, and superior multiplexing capacity. The ability to multiplex clustered genetic variants reduces the time to result providing more actionable information.

M ycoplasma genitalium is a sexually transmitted bacterium that causes urethritis in men and is associated with potentially serious sequelae in women (1). The levels of antibiotic resistance to the first-line (azithromycin) and second-line (moxifloxacin) treatments are high and increasing (2, 3). Two commercial nucleic acid amplification tests for the diagnosis of M. genitalium were recently released, the Hologic (Bedford, MA, USA) Aptima Mycoplasma genitalium (4) and SpeeDx (Sydney, Australia) ResistancePlus MG (5) tests. In a single multiplexed reaction, the ResistancePlus MG assay reports both the detection of M. genitalium and the presence/absence of five macrolide resistance mutations in the 23S rRNA gene, facilitating individualization of antimicrobial therapy. The ResistancePlus MG test has been validated by the Roche LightCycler 480 II real-time PCR system (LC480) (5, 6). The aim of this study was to evaluate the SpeeDx ResistancePlus MG (550) diagnostic kit (Sydney, Australia) on the Applied Biosystems 7500 (ABI 7500) Fast platform, a commonly used diagnostic quantitative PCR (qPCR) platform. A retrospective analysis was performed on samples collected from 1 May 2016 to 21 June 2016 (initially tested by 16S rRNA gene qPCR, including quantitation [7]). All positive samples (n ϭ 111) and a random selection of negative samples (n ϭ 100) were included in this study. Sample types included urine and swabs (anorectal, vaginal, cervical/endocervical, urethral) and originated from The Royal Women's Hospital (Melbourne, Australia), the Melbourne Sexual Health Centre, or external referrals. Samples were previously extracted on the MagNA Pure 96 (Roche) for diagnostic purposes and extracts stored at Ϫ30°C. For the test assay, reaction mixtures (20-l final volume) were assembled with 5 l of sample and the Plex Mastermix from the ResistancePlus MG (550) diagnostic kit, before analysis on the ABI 7500 Fast platform. Cycling consisted of 95°C for 2 min, followed by 10 cycles of 95°C for 5 s and 61°C for 30 s (Ϫ0.5°C per cycle) and 40 cycles of 95°C for 5 s and 52°C for 40 s. Detection occurred in three channels: (i) the FAM channel (FAM [6-carboxyfluorescein] dye, 495 to 520 nm) for detection of M. genitalium through the MgPa gene, (ii) the JOE channel (JOE [6-carboxy-4=,5=-dichloro-2=,7=-dimethoxyfluorescein] dye, 529 to 555 nm) for detection

most clinically important genetic markers conferring resistance to antibiotics used for the treatment of gonorrhoea. Methods We designed a fluorescent dye real-time PCR assay combined with high resolution melting (HRM) analysis. Several triplex and duplex reactions included target sequences for: two NG-specific genes (porA and opa); targets specific for the penA mosaic XXXIV (A501, G545S), associated with reduced cephalosporin susceptibility; single nucleotide polymorphisms conferring resistance to ciprofloxacin (GyrA S91F), azithromycin (23S rRNA A2059G and C2611T) and spectinomycin (16S rRNA C1192T and 5S rRNA T24P). We tested >50 characterised NG isolates, including: clinical isolates resistant to ciprofloxacin, azithromycin, spectinomycin, ceftriaxone (strain F89), strains with reduced cephalosporin susceptibility, the wild-type reference strain ATCC49226, and commensal Neisseria spp. Results HRM analysis correctly identified: all NG strains and all mutations, except for the A501P mutation in strain F89. However, cross-reactions with commensal Neisseria spp. occurred for: penA mosaic XXXIV (n = 3 species) and 23S rRNA (n = 3 species). Conclusion Our multiplex PCR assay accurately identified NG and detected the most frequent mutations associated with antimicrobial resistance in cultured isolates. The assay provides results significantly quicker than current culture-based methods. The analytical sensitivity and specificity of the assay for use with urethral, rectal, pharyngeal and vaginal specimens should be evaluated in the near future.