Comparison of three real-time PCR methods for detection of macrolide-resistant Mycoplasma genitalium in Sweden

Gullsby, Karolina; Lindh, Linda; Adriaenssens, Ruben; Herrmann, Björn

doi:10.1016/j.diagmicrobio.2021.115349

Cited by 1 publication

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“…However, its value in clinical applications is limited by the complexity of the processes and the lack of professional instrumentation in some laboratories. Although real-time fluorescent PCR-based commercial kits or self-constructed methods are available and evaluated, they usually have limitations, such as detecting only one single target, either 23S rRNA or parC, lack of accurate genotype identification and unpublished primer and probe sets (Wold et al, 2015;Thellin et al, 2019;Le Roy et al, 2020;Gullsby et al, 2021;Shedko et al, 2021). To provide a flexible and rapid guide to clinical antibiotic therapy, this study aims to establish a nested real-time PCR assay for key sites associated with macrolide and fluoroquinolone resistance, including mutations at 23S rRNA A2058, A2059 and parC S83, D87 positions.…”

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confidence: 99%

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

He,

Yuan,

Liang

et al. 2023

Front. Cell. Infect. Microbiol.

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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.

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