Detection of parC gene mutations associated with quinolone resistance in Mycoplasma genitalium: evaluation of a multiplex real-time PCR assay

Abstract

Introduction. Increasing levels of antibiotic resistance are complicating treatment for the sexually transmitted pathogen Mycoplasma genitalium . Resistance to fluoroquinolones is associated with mutations in the parC gene. Although the precise mutations conferring resistance are not fully understood, the single nucleotide polymorphism (SNP) G248T/S83I is most implicated. Aim. To evaluate the performance of the MG+parC(beta2) assay (SpeeDx, Australia), which detects single nucleotide polymorphisms (SNPs) in the parC gene at amino acid position S83 (A247C/S83R, G248T/S83I, G248A/S83N) and D87 (G259A/D87N, G259T/D87Y, G259C/D87H). Methods. Clinical samples were analysed by MG+parC(beta2) assay and results compared to Sanger sequencing. Sensitivity, specificity, and predictive value for treatment failure were calculated. Results. From analysis of 205 samples, the MG+parC(beta2) assay performed with a high sensitivity 98.2% (95% CI:90.3–100) and specificity 99.3% (95% CI:96.3–100) for parC SNP detection with a kappa of 0.97 (95% CI:0.94–1.00). The predictive value of G248T/S83I detection (the most common SNP, prevalence of 13% in the study population) was analysed with respect to treatment failure (patients received sequential doxycycline-moxifloxacin). The positive-predictive-value for moxifloxacin failure after detection of S83I was only 44% (95% CI:24.4–65.1), while negative-predictive-value was high at 96.9% (95% CI:92.7–99.0), suggesting that other SNPs are contributing to resistance. Conclusion. MG+parC(beta2) performed with high concordance compared to Sanger sequencing. Such qPCR assays can assist in understanding causes of treatment failure, inform the development of diagnostic assays, and can be applied to surveillance of mutations in populations. Due to an incomplete understanding of the basis for fluoroquinolone resistance, such tests do not appear to be ready for clinical application.

Genetic Determinants of Macrolide and Fluoroquinolone Resistance in Mycoplasma genitalium and Their Prevalence in Moscow, Russia

“…The fact that our data demonstrate that these two variants are the most common is in good agreement with observations reported in international studies [ 14 ]. S80N substitution, which is frequently detected in resistant M. genitalium strains, was experimentally proven to contribute to resistance development and therapy failure [ 27 ]. Other mutations are represented by single cases from our sample set and are rather uncommon in general; however, their connection to therapy inefficacy has been confirmed in a number of studies [ 28 , 29 ].…”

Prevalence of macrolide and fluoroquinolone resistance-associated mutations in Mycoplasma genitalium in metropolitan and overseas France

“…In addition, from a technical point of view, amplification and sequencing of fluoroquinolone targets are time-consuming and hardly usable in a routine strategy. Very few commercial kits have been developed to detect mutations in the parC gene 28 29. Kits usually detect groups of mutations at positions Ser83 and Asp87, with no distinction between the ParC alterations likely to be associated with treatment failure, which reduces the clinical interest of their results.…”

Prevalence of Mycoplasma genitalium fluoroquinolone-resistance markers, and dual-class-resistance markers, in asymptomatic men who have sex with men

“…Diagnostic assays that report markers of fluoroquinolone resistance may assist in antibiotic prescribing and stewardship [29,45]. The value of such assays in predicting treatment failure is still unclear, as not all patients with M. genitalium harbouring clinically relevant parC SNPs fail treatment [46]. This is largely because the molecular mechanism of fluoroquinolone resistance in M. genitalium is not fully understood, and other factors, such as organism load or pre-treatment with doxycycline, may influence treatment outcomes [25,41].…”

“…The value of such assays in predicting treatment failure is still unclear, as not all patients with M. genitalium harbouring clinically relevant parC SNPs fail treatment [46]. This is largely because the molecular mechanism of fluoroquinolone resistance in M.…”