Abstract:
Technical limitations of common tests used for detecting pyrazinamide (PZA) resistance in
Mycobacterium tuberculosis (MTB) isolates pose challenges for comprehensive and accurate
descriptions of drug resistance in patients with multi-drug resistant tuberculosis (MDR-TB) . In
this study, a 606 base pair fragment (comprising the pncA coding region plus promoter) was
sequenced using Ion Torrent next generation sequencing (NGS) for detecting associated PZA
resistance mutations in 90 re-cultured, MDR-TB isolates from an archived series collected in
2001. These 90 isolates were previously Sanger sequenced, with 55 (62%) designated as carrying
wild type pncA gene and 33 (38%) showing mutations. Also earlier, PZA susceptibility of the isolates was determined using the Bactec 460 TB system and the Wayne test. In this study,
isolates were re-cultured and susceptibility testing performed in Bactec 960 MGIT. Concordance
between NGS and MGIT results was 87% (n = 90), and with the Bactec 460, Wayne test, and
pncA gene Sanger sequencing, 82% (n = 88), 83% (n = 88), and 89% (n = 88), respectively.
NGS confirmed the majority of pncA mutations detected by Sanger sequencing, but revealed
several new and mixed-strain mutations that resolved discordancy in other phenotypic results.
Importantly, in 53% (18/34) of these isolates, pncA mutations were located in the 151-360
region, and warrants further exploration. In these isolates, with known resistance to rifampicin,
NGS of pncA improved PZA resistance detection sensitivity to 97% and specificity to 94% using
NGS as the gold standard, and helped to resolve discordant results from conventional
methodologies.
