title: Next-generation ion torrent sequencing of drug resistance mutations in Mycobacterium tuberculosis strains.
abstract: A novel protocol for full-length Mycobacterium tuberculosis gene analysis of first- and second-line drug resistance was developed using the Ion Torrent Personal Genome Machine (PGM). Five genes-rpoB (rifampin), katG (isoniazid), pncA (pyrazinamide), gyrA (ofloxacin/fluoroquinolone), and rrs (aminoglycosides)-were amplified and sequenced, and results were compared to those obtained by genotypic Hain line probe assay (LPA) and phenotypic Bactec MGIT 960 analysis using 26 geographically diverse South African clinical isolates collected between July and November 2011. Ion Torrent sequencing exhibited 100% (26/26) concordance to phenotypic resistance obtained by MGIT 960 culture and genotypic rpoB and katG results by LPA. In several rifampin-resistant isolates, Ion Torrent sequencing revealed uncommon substitutions (H526R and D516G) that did not have a defined mutation by LPA. Importantly, previously uncharacterized mutations in rpoB (V194I), rrs (G878A), and pncA (Q122Stop) genes were observed. Ion Torrent sequencing may facilitate tracking and monitoring geographically diverse multidrug-resistant and extensively drug-resistant strains and could potentially be integrated into selected regional and reference settings throughout Africa, India, and China.
authors.affiliation: Longhorn Vaccines & Diagnostics, San Antonio, Texas, USA. LTD@lhnvd.com
authors.forename: Luke T
authors.initials: LT
authors.lastname: Daum
authors.forename: John D
authors.initials: JD
authors.lastname: Rodriguez
authors.forename: Sue A
authors.initials: SA
authors.lastname: Worthy
authors.forename: Nazir A
authors.initials: NA
authors.lastname: Ismail
authors.forename: Shaheed V
authors.initials: SV
authors.lastname: Omar
authors.forename: Andries W
authors.initials: AW
authors.lastname: Dreyer
authors.forename: P Bernard
authors.initials: PB
authors.lastname: Fourie
authors.forename: Anwar A
authors.initials: AA
authors.lastname: Hoosen
authors.forename: James P
authors.initials: JP
authors.lastname: Chambers
authors.forename: Gerald W
authors.initials: GW
authors.lastname: Fischer
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics; D001431:Bacteriological Techniques / Q000379:methods*; D024881:Drug Resistance, Bacterial; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000235:genetics; D009154:Mutation; D020125:Mutation, Missense; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D013019:South Africa; D014376:Tuberculosis / Q000382:microbiology
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D050505:Mutant Proteins