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

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.

SUBMITTER: Braam JF

PROVIDER: S-EPMC7575077 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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

Braam Joyce F JF Hetem David J DJ Vergunst Clarissa E CE Kuizenga Wessel Sophie S van Rooijen Martijn S MS Nijhuis Roel H T RHT Schim van der Loeff Maarten F MF van Dam Alje P AP Bruisten Sylvia M SM

PloS one 20201020 10

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 d ...[more]

PMID: 33079948

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Project description:BackgroundTraditional drug susceptibility testing cannot be performed in clinical laboratories due to the slow-growing characteristics of Mycoplasma genitalium when cultured in vitro. Sanger sequencing is the standard method for detecting drug resistance-associated mutations. It has been used in some laboratories to guide the choice of macrolide antibiotics for Mycoplasma genitalium infected patients. Furthermore, resistance to fluoroquinolone has become another emerging clinical challenge.ObjectiveSequencing analysis can detect unknown mutations, but it is time-consuming, requires professional analytical skills and the appropriate testing equipment. The main objective of this study was to establish a nested real-time PCR method for the simultaneous detection of 23S rRNA and parC genotypes in relation to the macrolide and fluoroquinolone resistance.Results105 MG-positive samples and 27 samples containing other pathogens were used for validation. The limit of the nested real-time PCR detection was 500 copies/reaction and there was no cross-reaction with Ureaplasma urealyticum, Mycoplasma hominis, Chlamydia trachomatis, Neisseria gonorrhoeae, Human papillomavirus, Herpes simplex virus, Candida albicans and Ureaplasma parvum, but the 23S rRNA assay cross-reacted with Mycoplasma pneumoniae. Compared with sequencing results, the sensitivity of 23S rRNA was 100% (95% CI; 93.3 -100), the specificity was 94.3% (95% CI; 79.4 - 99.0), the overall consistency was 98% (95% CI; 92.5 - 99.7) and kappa value was 0.96 (P < 0.001); the sensitivity of parC was 100% (95% CI; 93.4 - 100), the specificity was 89.7% (95% CI; 71.5 - 97.3) and the overall consistency was 96.9% (95% CI; 90.7 - 99.2) with a kappa value of 0.92 (P < 0.001).ConclusionsThe results of this sensitive and rapid alternative for identifying resistant genotypes of Mycoplasma genitalium are intuitive and easy to interpret, especially for mixed MG populations. Although the relevant 23S rRNA primers need further adjustment, this reliable method would provide an effective diagnostic tool for the selection of antibiotics in clinical practice.

Dataset Information

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

Publications

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

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