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

• Published in: Frontiers in cellular and infection microbiology Vol. 13; p. 1271392
• Main Authors: He, Wenyin, Yuan, Ying, Liang, Junyu, Fan, Xuejiao, Li, Lei, Pan, Xingfei
• Format: Journal Article
• Language: English
• Published: Frontiers Media S.A 20-10-2023
• Subjects: 23S rRNA; Cellular and Infection Microbiology; fluroquinolone; macrolide; Mycoplasma genitalium; nested real-time PCR; parC
• ISSN: 2235-2988; 2235-2988
• DOI: 10.3389/fcimb.2023.1271392

Background
Traditional 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.
Objective
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
23S rRNA
and
parC
genotypes in relation to the macrolide and fluoroquinolone resistance.
Results
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
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).
Conclusions
The 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.