Development and Evaluation of a Line Probe Assay for Rapid Identification of pncA Mutations in Pyrazinamide-Resistant Mycobacterium tuberculosis Strains

Abstract

Resistance of Mycobacterium tuberculosis to pyrazinamide (PZA) derives mainly from mutations in the pncA gene. We developed a reverse hybridization-based line probe assay with oligonucleotide probes designed to detect mutations in pncA. The detection of PZA resistance was evaluated in 258 clinical isolates of M. tuberculosis. The sensitivity and specificity of PZA resistance obtained by this new assay were both 100%, consistent with the results of conventional PZA susceptibility testing. This assay can be used with sputa from tuberculosis patients. It appears to be reliable and widely applicable and, given its simplicity and rapid performance, will be a valuable tool for diagnostic use.

Prevention of False Resistance Results Obtained in Testing the Susceptibility of Mycobacterium tuberculosis to Pyrazinamide with the Bactec MGIT 960 System Using a Reduced Inoculum

“…In addition, we routinely used disposable Pasteur pipettes to seed the inoculum (14), which was dispensed regardless of any timewasting procedure, such as leaving the seed MGIT to settle for 15 min and then taking the inoculum from the top of the seed tube instead of deeper down, close to the sediment. In case of severe resistance, our results were also in agreement with pncA mutation analysis, thus suggesting that molecular detection of PZA resistance is going to be the way forward for clinical laboratories as soon as rapid line probe assays are commercially available (26,27).…”

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

“…The katG probes covered 90 mutations related to INH resistance. On the PZA strip, 47 S probes covered regions of M. tuberculosis pncA (pncA-1 to -47), with 2 probes (pncA-16 and -17) containing a silent mutation in pncA (42). Probes inhA-S6 and -S7 and katG-S8 to -S11 on the NTM/MDR-TB strip were the same as inhA-1 and -2 and katG-20, -22, -23, and -24 on the INH strip, respectively.…”

“…LiPA was performed as described previously (3,42), using 121 oligonucleotide probes (see Table S2 in the supplemental material) immobilized onto four strips, called the NTM/MDR-TB, INH, PZA, and FQ strips (Nipro Co., Osaka, Japan). All clinical isolates and all sputum specimens were tested by LiPAs using all four strips, regardless of the results of Table S2.…”

“…Drug resistance in M. tuberculosis is due to mutations, including rpoB mutations, associated with RIF resistance; mutations in katG, the promoter region of the fabG1-inhA operon, fabG1, furA, and inhA, associated with INH resistance; pncA mutations, associated with pyrazinamide (PZA) resistance; and gyrA mutations, associated with resistance to fluoroquinolones (FQ) (47). Hybridization-based line probe assays (LiPAs) detect mutations associated with resistance to RIF (12,21,33,38), INH (3), PZA (42), and FQ (15).…”

Role of pncA and rpsA Gene Sequencing in Detection of Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates from Southern China

“…Such novel mutants accounted for 25% (13 of 52) of all the PZA-resistant isolates. The PCR-single-strand conformational polymorphism (PCR-SSCP) and line probe assay (LiPA) developed previously (30,31) could detect DNA mutations quickly, but they could not tell if the mutation was synonymous or nonsynonymous. No silent mutations were found in the pncA , and 8, reverse transcription-PCR products with primer pairs A, B, C, and D, respectively, using the cDNA as the templates; lanes 3, 5, 7, and 9, PCR products with primer pairs A, B, C, and D, respectively, using the reverse transcription products as the control templates without adding the reverse transcriptase.…”