title: Novel mutation detection IN rpoB OF rifampicin-resistant Mycobacterium tuberculosis using pyrosequencing.
abstract: Tuberculosis (TB) remains a major global public health problem particularly severe in parts of Asia and Africa, where often it is present in HIV-AIDS patients. Although rifampicin-resistant (RIFr) TB is slow to emerge due to the low rate of mutation of its target leading to RIFE being a marker of TB that is already resistant to other anti-TB drugs, and such cases are prone to treatment failure. More than 95% of rifampicin resistance is associated with mutations in Mycobacterium tuberculosis (MTB) rpoB, with 97% of mutations occurring within the 81 bp rifampicin-resistant determining region (RRDR) of this gene. In this study, we employed pyrosequencing technique to identify mutations in RRDR and 5 codons beyond of 39 MTB strains, comprising of 14 multi-drug resistance TB (MDRTB) and 3 RIF susceptible (RIFs) MTB from the Center of Disease Control (CDC), Ratchaburi Province, and 19 mono RIFr MTB, 1 MDRTB and 2 poly-drug resistant MTB from the Chest Institute, Ministry of Public Health, Thailand. Mu- tations in 8/22 samples from the Chest Institute and 13/14 from CDC were able to be identified. Six point mutations were detected, with Ser531Leu mutation accounting for 13, the silent mutation at Gly536 for 4, deletion of Gly523 for 2, combination of His526Cys and novel Leu533Arg for 1, and a novel Leu538Arg for 1. Mutation analysis of the 81 bp fragment and 5 codons beyond in MTB rpoB using pyrosequencing provides a useful approach in predicting RIFr phenotype allowing early diagnosis and appropriate drug therapy.
authors.forename: Pyar Kyi
authors.initials: PK
authors.lastname: Htike Min
authors.forename: Pannamthip
authors.initials: P
authors.lastname: Pitaksajjakul
authors.forename: Natthakan
authors.initials: N
authors.lastname: Tipkrua
authors.forename: Waranya
authors.initials: W
authors.lastname: Wongwit
authors.forename: Pornrutsami
authors.initials: P
authors.lastname: Jintaridh
authors.forename: Pongrama
authors.initials: P
authors.lastname: Ramasoota
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins; D012321:DNA-Directed RNA Polymerases; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D006801:Humans; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012293:Rifampin / Q000494:pharmacology*; D017422:Sequence Analysis, DNA; D018088:Tuberculosis, Multidrug-Resistant / Q000235:genetics
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; C505805:rpoB protein, Mycobacterium tuberculosis; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin