Development of a Real-Time PCR Assay for Detection of Macrolide Resistance Mutations in Mycoplasma genitalium and Its Application for Epidemiological Surveillance in Russia.

Abstract

A real-time PCR assay based on probe fluorescence quenching by a primer was developed and validated for detection of macrolide resistance (MR)-associated mutations in the 23S rRNA gene of Mycoplasma genitalium. The assay involves identification of any nucleotide substitutions at positions 2058, 2059, and 2611 of 23S rRNA (Escherichia coli numbering) by the probe-based melting curve analysis immediately after amplification and was capable of detecting target mutations in clinical specimens and spiked samples with 92% sensitivity and 100% specificity. We applied this new assay to assess the prevalence of MR-associated mutations in 949 nonduplicate urogenital samples positive for M. genitalium by routine diagnostic PCR, which were collected from symptomatic patients in five cities in the European part of Russia during the period 2009-2019. Forty-three (4.92%) samples revealed the presence of MR mutations, and no trend toward an increase in resistance prevalence was observed over the 10-year period of the study. The most commonly detected mutations were A2058G (26/43; 60.47%) and A2059G (13/43; 30.23%), while other mutations were rare: A2058T (3/43; 6.98%) and C2611T (1/43; 2.33%). The data obtained underline the need for regular epidemiological monitoring to ensure effective patient management, rational use of antibiotics, and prevention of further spread of resistance.