AUTHOR=He Wenyin , Yuan Ying , Liang Junyu , Fan Xuejiao , Li Lei , Pan Xingfei 

TITLE=Detection of macrolide and fluoroquinolone resistance-associated 23S rRNA and parC mutations in Mycoplasma genitalium by nested real-time PCR

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=13

YEAR=2023

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2023.1271392

DOI=10.3389/fcimb.2023.1271392

ISSN=2235-2988

ABSTRACT=<sec><title>Background</title><p>Traditional drug susceptibility testing cannot be performed in clinical laboratories due to the slow-growing characteristics of <italic>Mycoplasma genitalium</italic> when cultured <italic>in vitro</italic>. 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 <italic>Mycoplasma genitalium</italic> infected patients. Furthermore, resistance to fluoroquinolone has become another emerging clinical challenge.</p></sec><sec><title>Objective</title><p>Sequencing 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 <italic>23S rRNA</italic> and <italic>parC</italic> genotypes in relation to the macrolide and fluoroquinolone resistance.</p></sec><sec><title>Results</title><p>105 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 <italic>Ureaplasma urealyticum</italic>, <italic>Mycoplasma hominis</italic>, <italic>Chlamydia trachomatis</italic>, <italic>Neisseria gonorrhoeae</italic>, <italic>Human papillomavirus</italic>, <italic>Herpes simplex virus</italic>, <italic>Candida albicans</italic> and <italic>Ureaplasma parvum</italic>, but the <italic>23S rRNA</italic> assay cross-reacted with <italic>Mycoplasma pneumoniae</italic>. Compared with sequencing results, the sensitivity of <italic>23S rRNA</italic> 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 <italic>kappa</italic> value was 0.96 (<italic>P</italic> &lt; 0.001); the sensitivity of <italic>parC</italic> 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 <italic>kappa</italic> value of 0.92 (<italic>P</italic> &lt; 0.001).</p></sec><sec><title>Conclusions</title><p>The results of this sensitive and rapid alternative for identifying resistant genotypes of <italic>Mycoplasma genitalium</italic> are intuitive and easy to interpret, especially for mixed MG populations. Although the relevant <italic>23S rRNA</italic> primers need further adjustment, this reliable method would provide an effective diagnostic tool for the selection of antibiotics in clinical practice.</p></sec>