title: Improved Detection by Next-Generation Sequencing of Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates.
abstract: The technical limitations of common tests used for detecting pyrazinamide (PZA) resistance in Mycobacterium tuberculosis isolates pose challenges for comprehensive and accurate descriptions of drug resistance in patients with multidrug-resistant tuberculosis (MDR-TB). In this study, a 606-bp fragment (comprising the pncA coding region plus the promoter) was sequenced using Ion Torrent next-generation sequencing (NGS) to detect associated PZA resistance mutations in 88 recultured MDR-TB isolates from an archived series collected in 2001. These 88 isolates were previously Sanger sequenced, with 55 (61%) designated as carrying the wild-type pncA gene and 33 (37%) showing mutations. PZA susceptibility of the isolates was also determined using the Bactec 460 TB system and the Wayne test. In this study, isolates were recultured and susceptibility testing was performed in Bactec 960 MGIT. Concordance between NGS and MGIT results was 93% (n = 88), and concordance values between the Bactec 460, the Wayne test, or pncA gene Sanger sequencing and NGS results were 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 to 360 region and warrant further exploration. In these isolates, with their known resistance to rifampin, 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.
authors.affiliation: University of Pretoria, Pretoria, South Africa South African Medical Research Council, Pretoria, South Africa.
authors.forename: Nontuthuko E
authors.initials: NE
authors.lastname: Maningi
authors.affiliation: Longhorn Vaccines & Diagnostics, San Antonio, Texas, USA.
authors.forename: Luke T
authors.initials: LT
authors.lastname: Daum
authors.affiliation: Longhorn Vaccines & Diagnostics, San Antonio, Texas, USA.
authors.forename: John D
authors.initials: JD
authors.lastname: Rodriguez
authors.affiliation: South African Medical Research Council, Pretoria, South Africa.
authors.forename: Matsie
authors.initials: M
authors.lastname: Mphahlele
authors.affiliation: University of Pretoria, Pretoria, South Africa Anova Health Institute, Johannesburg, South Africa.
authors.forename: Remco P H
authors.initials: RP
authors.lastname: Peters
authors.affiliation: Longhorn Vaccines & Diagnostics, San Antonio, Texas, USA.
authors.forename: Gerald W
authors.initials: GW
authors.lastname: Fischer
authors.affiliation: University of Texas at San Antonio, San Antonio, Texas, USA.
authors.forename: James P
authors.initials: JP
authors.lastname: Chambers
authors.affiliation: University of Pretoria, Pretoria, South Africa bernard.fourie@up.ac.za.
authors.forename: P Bernard
authors.initials: PB
authors.lastname: Fourie
mesh_terms: D000581:Amidohydrolases / Q000235:genetics*; D000995:Antitubercular Agents / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial; D060005:Genotyping Techniques / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics; D011718:Pyrazinamide / Q000494:pharmacology*; D012680:Sensitivity and Specificity
chemical_list: D000995:Antitubercular Agents; D011718:Pyrazinamide; D000581:Amidohydrolases; C487496:PncA protein, Mycobacterium tuberculosis