Evaluating the prevalence and risk factors for macrolide resistance in Mycoplasma genitalium using a newly developed qPCR assay.

Joyce F Braam,Maarten F Schim Van Der Loeff,David J Hetem,Clarissa E Vergunst,Martijn S Van Rooijen,Sylvia M Bruisten,Sophie Kuizenga Wessel,Alje P Van Dam,Roel H T Nijhuis,Remco Ph Peters

doi:10.1371/journal.pone.0240836

Abstract

Mycoplasma genitalium (MG) is a sexually transmitted bacterium in which macrolide resistance is rapidly increasing, limiting treatment options. We validated a new assay to detect the presence of macrolide resistance-associated mutations in MG (MG-MRAM). In 2018, symptomatic and asymptomatic clients visiting sexually transmitted infections (STI) clinics in Amsterdam or The Hague were tested for MG using transcription mediated amplification (TMA) assays. The sensitivity to detect MG of the newly developed MG-MRAM qPCR was compared to the MgPa qPCR, both in relation to the TMA assay. For the sensitivity and specificity to detect relevant mutations the MG-MRAM qPCR was compared to 23SrRNA sequencing analysis. The qPCR was subsequently used to determine the presence of MG-MRAM at different anatomical locations and to identify risk factors for MG-MRAM. MG-positive clients (402) providing 493 MG-positive samples were included. In total 309/493 (62.7%) samples from 291 (72.4%) clients were successfully typed with the MG-MRAM qPCR. The MG-MRAM qPCR had a sensitivity of 98.6% (95%CI 91.1%-99.9%) and specificity of 94.1% (95%CI 78.9%-99.0%) to detect MG-MRAM compared to sequencing analysis. Infection with MG-MRAM was detected in 193/291 (66.3%) clients: in 129/178 (72.5%) men and 64/113 (56.6%) women (p = 0.005). Prevalence of MG-MRAM was significantly higher in men, clients with a higher education, HIV-positive clients and clients with >10 sexual partners in the previous six months, but in multivariable analysis no factor was significantly associated with MG-MRAM presence. Since MG-MRAM prevalence was very high, testing for MG-MRAM is essential if treatment for MG is considered, and can be performed with this sensitive and specific qPCR test in routine diagnostics.

Highlights

Mycoplasma genitalium (MG) is a sexually transmitted organism which infects 3.1–4.5% of Dutch clients undergoing screening for sexually transmitted infections (STI) [1, 2]
Prevalence and risk factors for macrolide resistance in Mycoplasma genitalium enrolled during the study period is described by Hetem et al 2020
We describe a new qPCR assay with locked nucleic acids (LNA) probes to detect MG-wild type (WT) and MG-macrolide resistance-associated mutations (MRAM) for the most commonly occurring mutations in the 23SrRNA gene of MG

Summary

Introduction

Mycoplasma genitalium (MG) is a sexually transmitted organism which infects 3.1–4.5% of Dutch clients undergoing screening for sexually transmitted infections (STI) [1, 2]. The most common clinical manifestation of MG infection is non-gonococcal urethritis (NGU). A meta-analysis from 2011 showed that MG was strongly associated with NGU (pooled OR 5.5 [95% CI: 4.4–7.0]) [3]. In women MG has been associated with an increased risk of cervicitis, pelvic inflammatory disease, preterm birth, and spontaneous abortion [4]. According to European guidelines, uncomplicated MG infections should be treated with azithromycin 500 mg PO on day one followed by 250 mg on days 2–5 [5]. Single dose treatment (1000 mg) with azithromycin is often the preferred treatment of NGU in many countries, including the Netherlands [6]. Chlamydia trachomatis (CT) infections are treated with 1000 mg azithromycin, but often without excluding co-infection with MG [6]

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