Multiplex Assay for Simultaneous Detection of Mycoplasma genitalium and Macrolide Resistance Using PlexZyme and PlexPrime Technology.

Sepehr N Tabrizi,Elisa Mokany,Suzanne M Garland,Samantha Walker,Alison V Todd,Christopher K Fairley,Lit Y Tan,Jimmy Twin,Marin Poljak,Melanie Bissessor,Catriona S Bradshaw,Mitchell F Balish

doi:10.1371/journal.pone.0156740

Abstract

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.

Highlights

Mycoplasma genitalium was first isolated in 1980 from men with non-gonococcal urethritis (NGU) [1]
This study evaluates the MG 23S assay, which employs novel PlexZymeTM and PlexPrimeTM technology (SpeeDx), is a single well, multiplex qPCR assay, which simultaneously detects M. genitalium and five 23S rRNA mutations associated with macrolide resistance
The assay was evaluated using clinical samples previously screened for both M. genitalium detection by 16S rRNA qPCR and 23S rRNA mutation status by high resolution melt analysis (HRMA) and Sanger sequencing

Summary

Introduction

Mycoplasma genitalium was first isolated in 1980 from men with non-gonococcal urethritis (NGU) [1]. It has since been established as a common sexually transmitted infection responsible for 10–35% of non-chlamydial NGU in men [2] and associated with cervicitis, endometritis, pelvic inflammatory disease (PID) and tubal factor infertility in women [3,4,5,6,7]. Current recommended treatment for uncomplicated M. genitalium infection is a single 1 g oral dose of the macrolide antibiotic, azithromycin [10, 11]. Cure rates following this treatment have declined to about 70% for genital M. genitalium infections, as macrolide resistance has emerged [12,13,14] [15]

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Results

Conclusion