Evaluation of a line probe assay for the rapid detection of gyrA mutations associated with fluoroquinolone resistance in multidrug-resistant Mycobacterium tuberculosis

Abstract

The aim of this study was to establish the importance of detecting fluoroquinolone (FQ) resistance in multidrug resistant (MDR) Mycobacterium tuberculosis, and to show the usefulness of a hybridization-based line probe assay (LiPA) for detecting gyrA mutations. Thirty-three MDR M. tuberculosis isolates were collected from a total of sixty MDR isolates identified in Japan over 6 months during a national surveillance study in 2002. Seventeen MDR isolates were collected by the National Center for Global Health and Medicine in Japan over 6 years from 2003 to 2008. These 50 isolates were examined for FQ susceptibility, and analysed by LiPA and gyrA sequencing. Among them, 22 (44 %) showed FQ resistance. All FQ-resistant isolates had at least one mutation in gyrA. The results of the LiPA were fully consistent with the DNA sequencing results. Given that on the basis of our results almost half of the MDR M. tuberculosis isolates in Japan might have resistance to FQ, it is important to monitor FQ resistance in patients with MDR tuberculosis (TB), as well as with drug-susceptible TB, prior to commencing treatment. For the detection of FQ resistance, LiPA is useful and can rapidly and efficiently assess FQ resistance.

Comprehensive Multicenter Evaluation of a New Line Probe Assay Kit for Identification of Mycobacterium Species and Detection of Drug-Resistant Mycobacterium tuberculosis

“…It is also important to detect resistance to PZA and LVX in MDR tuberculosis, as the majority of MDR M. tuberculosis isolates have been reported to be resistant to either PZA or LVX in Japan (4,5).…”

Importance of the Genetic Diversity within the Mycobacterium tuberculosis Complex for the Development of Novel Antibiotics and Diagnostic Tests of Drug Resistance

“…To overcome the limitations imposed by the low growth rate of MTBC, genotypic antibiotic susceptibility tests have been introduced and either have been endorsed by the World Health Organization or are currently under evaluation (11,72,73,98,119,144). Given that these hybridization-based assays interrogate the DNA rather than the amino acid sequence, they detect both synonymous and nonsynonymous mutations, unless the genetic diversity in the target regions was taken into consideration for the design of the respective probes (4,12,24,83,155). For example, additional probes had to be included for the assay manufactured by Nipro Corp. to cover several neutral or silent mutations in pncA (13,108,144).…”

Role of fluoroquinolones in the treatment of tuberculosis

“…Attempts have also been made to correlate the nature and kind of gyrA mutations with the level of resistance to FQs in M. tuberculosis strains with conflicting results. Some studies have reported that M. tuberculosis strains containing D94G mutation in the gyrA gene exhibit high-level resistance to FQs while those with D94A mutation have lower MIC for LFX [93,96,97]. On the contrary, other reports have not found an association of different mutation patterns in gyrA or gyrB genes with the level of FQ resistance in M. tuberculosis isolates [89,94].…”

“…The presence of missense mutations in the so-called quinolone resistance-determining region (QRDR) of gyrA or gyrB genes conferring resistance to FQs was subsequently confirmed by using clinical M. tuberculosis isolates in multiple studies [83][84][85][87][88][89][90][91][92][93][94][95][96][97]. The presence of S95 or T95 at gyrA codon 95 is a naturally occurring polymorphism that is not associated with resistance of M. tuberculosis isolates to FQs.…”

“…Although some of these assays target both gyrA and gyrB genes, the majority of genotypic tests target only QRDR of gyrA gene for simplicity and acceptable levels of sensitivity. The genotypic tests that have been developed include PCR-DNA sequencing [83][84][85], PCR-single strand conformation polymorphism [101], temperature-mediated heteroduplex analysis by using denaturing high-pressure liquid chromatography (HPLC) [102], pyrosequencing [103], microchip array [104], locked nucleic acid probe real-time PCR [105], multiplex PCR amplimer conformation analysis [89] and both, in-house developed as well as commercially available line probe assays [92,96,97]. The FQs are an important component of the therapy regimens for patients with MDR-TB or XDR-TB since their inclusion greatly improves treatment outcome.…”