Improved detection by next-generation sequencing of pyrazinamide resistance in mycobacterium tuberculosis isolates

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.