title: Evaluation of pyrosequencing for detecting extensively drug-resistant Mycobacterium tuberculosis among clinical isolates from four high-burden countries.
abstract: Reliable molecular diagnostics, which detect specific mutations associated with drug resistance, are promising technologies for the rapid identification and monitoring of drug resistance in Mycobacterium tuberculosis isolates. Pyrosequencing (PSQ) has the ability to detect mutations associated with first- and second-line anti-tuberculosis (TB) drugs, with the additional advantage of being rapidly adaptable for the identification of new mutations. The aim of this project was to evaluate the performance of PSQ in predicting phenotypic drug resistance in multidrug- and extensively drug-resistant tuberculosis (M/XDR-TB) clinical isolates from India, South Africa, Moldova, and the Philippines. A total of 187 archived isolates were run through a PSQ assay in order to identify M. tuberculosis (via the IS6110 marker), and to detect mutations associated with M/XDR-TB within small stretches of nucleotides in selected loci. The molecular targets included katG, the inhA promoter and the ahpC-oxyR intergenic region for isoniazid (INH) resistance; the rpoB core region for rifampin (RIF) resistance; gyrA for fluoroquinolone (FQ) resistance; and rrs for amikacin (AMK), capreomycin (CAP), and kanamycin (KAN) resistance. PSQ data were compared to phenotypic mycobacterial growth indicator tube (MGIT) 960 drug susceptibility testing results for performance analysis. The PSQ assay illustrated good sensitivity for the detection of resistance to INH (94%), RIF (96%), FQ (93%), AMK (84%), CAP (88%), and KAN (68%). The specificities of the assay were 96% for INH, 100% for RIF, FQ, AMK, and KAN, and 97% for CAP. PSQ is a highly efficient diagnostic tool that reveals specific nucleotide changes associated with resistance to the first- and second-line anti-TB drug medications. This methodology has the potential to be linked to mutation-specific clinical interpretation algorithms for rapid treatment decisions.
authors.affiliation: P. D. Hinduja National Hospital & Medical Research Centre, Department of Microbiology, Veer Sarvarkar Marg, Mahim, Mumbai, India k.ajbani03@gmail.com.
authors.forename: Kanchan
authors.initials: K
authors.lastname: Ajbani
authors.affiliation: California Department of Public Health, Microbial Diseases Laboratory, Richmond, California, USA.
authors.forename: Shou-Yean Grace
authors.initials: SY
authors.lastname: Lin
authors.affiliation: P. D. Hinduja National Hospital & Medical Research Centre, Department of Microbiology, Veer Sarvarkar Marg, Mahim, Mumbai, India.
authors.forename: Camilla
authors.initials: C
authors.lastname: Rodrigues
authors.affiliation: California Department of Public Health, Microbial Diseases Laboratory, Richmond, California, USA.
authors.forename: Duylinh
authors.initials: D
authors.lastname: Nguyen
authors.affiliation: California Department of Public Health, Microbial Diseases Laboratory, Richmond, California, USA.
authors.forename: Francine
authors.initials: F
authors.lastname: Arroyo
authors.affiliation: Department of Medicine, University of California, San Diego, La Jolla, California, USA.
authors.forename: Janice
authors.initials: J
authors.lastname: Kaping
authors.affiliation: Department of Medicine, University of California, San Diego, La Jolla, California, USA.
authors.forename: Lynn
authors.initials: L
authors.lastname: Jackson
authors.affiliation: Department of Medicine, University of California, San Diego, La Jolla, California, USA.
authors.forename: Richard S
authors.initials: RS
authors.lastname: Garfein
authors.affiliation: Department of Medicine, University of California, San Diego, La Jolla, California, USA.
authors.forename: Donald
authors.initials: D
authors.lastname: Catanzaro
authors.affiliation: Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Eisenach
authors.affiliation: Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa.
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Victor
authors.affiliation: Microbiology and Morphology Laboratory, Institute of Phthisiopneumology, Chisinau, Moldova.
authors.forename: Valeru
authors.initials: V
authors.lastname: Crudu
authors.affiliation: Tropical Disease Foundation/Makati Medical Center, Makati City, Philippines.
authors.forename: Maria Tarcela
authors.initials: MT
authors.lastname: Gler
authors.affiliation: National Tuberculosis Reference Laboratory, Johannesburg, South Africa.
authors.forename: Nazir
authors.initials: N
authors.lastname: Ismail
authors.affiliation: California Department of Public Health, Microbial Diseases Laboratory, Richmond, California, USA.
authors.forename: Edward
authors.initials: E
authors.lastname: Desmond
authors.affiliation: Department of Medicine, University of California, San Diego, La Jolla, California, USA.
authors.forename: Antonino
authors.initials: A
authors.lastname: Catanzaro
authors.affiliation: Department of Medicine, University of California, San Diego, La Jolla, California, USA.
authors.forename: Timothy C
authors.initials: TC
authors.lastname: Rodwell
mesh_terms: D000995:Antitubercular Agents / Q000627:therapeutic use*; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D002374:Catalase / Q000235:genetics; D027081:DNA Gyrase / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D012321:DNA-Directed RNA Polymerases; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D054908:Extensively Drug-Resistant Tuberculosis / Q000188:drug therapy* / Q000382:microbiology; D006801:Humans; D007538:Isoniazid / Q000627:therapeutic use; D008826:Microbial Sensitivity Tests / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D010088:Oxidoreductases / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; C505805:rpoB protein, Mycobacterium tuberculosis; D010088:Oxidoreductases; D002374:Catalase; C480353:katG protein, Mycobacterium tuberculosis; C085257:InhA protein, Mycobacterium; D012321:DNA-Directed RNA Polymerases; D027081:DNA Gyrase; D007538:Isoniazid