Maningi, Nontuthuko ExcellentDaum, Luke T.Rodriguez, John D.Mphahlele, MatsiePeters, Remco P.H.Fischer, Gerald W.Chambers, James P.Fourie, Petrus Bernardus2016-02-182015-12Maningi, NE, Daum, LT, Rodriguez, JD, Mphahlele, M, Peters, RPH, Fischer, GW, Chambers, JP & Fourie , PB 2015, 'Improved detection by next-generation sequencing of pyrazinamide resistance in mycobacterium tuberculosis isolates', Journal of Clinical Microbiology, vol. 53, no. 12, pp. 3779-3783.0095-1137 (print)1098-660X (online)10.1128/JCM.01179-15http://hdl.handle.net/2263/51455Technical 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.en© 2015 American Society for Microbiology. All Rights Reserved.Pyrazinamide resistanceDrug resistancePyrazinamide (PZA)Mycobacterium tuberculosis (MTB)Multi-drug resistant tuberculosis (MDR-TB)Next generation sequencing (NGS)Postprint Article