Improved detection by next-generation sequencing of pyrazinamide resistance in mycobacterium tuberculosis isolates
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Date
Authors
Maningi, Nontuthuko Excellent
Daum, Luke T.
Rodriguez, John D.
Mphahlele, Matsie
Peters, Remco P.H.
Fischer, Gerald W.
Chambers, James P.
Journal Title
Journal ISSN
Volume Title
Publisher
American Society for Microbiology
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.
Description
Keywords
Pyrazinamide resistance, Drug resistance, Pyrazinamide (PZA), Mycobacterium tuberculosis (MTB), Multi-drug resistant tuberculosis (MDR-TB), Next generation sequencing (NGS)
Sustainable Development Goals
Citation
Maningi, 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.