Ravibalan Thirumurugan1, Maruthai Kathirvel2, Kommoju Vallayyachari2, Kesavan Surendar2, Antony V Samrot3, Muthuraj Muthaiah4. 1. Department of Microbiology, Intermediate Reference Laboratory, State TB Training and Demonstration Centre, Government Hospital for Chest Diseases, Gorimedu, Puducherry 605006, India; Department of Biotechnology, Sathyabama University, Jeppiaar Nagar, Rajiv Gandhi Salai, Sholinganallur, Chennai 600119, Tamil Nadu, India. 2. Department of Biotechnology, Sathyabama University, Jeppiaar Nagar, Rajiv Gandhi Salai, Sholinganallur, Chennai 600119, Tamil Nadu, India. 3. Department of Microbiology, Intermediate Reference Laboratory, State TB Training and Demonstration Centre, Government Hospital for Chest Diseases, Gorimedu, Puducherry 605006, India. 4. Department of Biotechnology, Sathyabama University, Jeppiaar Nagar, Rajiv Gandhi Salai, Sholinganallur, Chennai 600119, Tamil Nadu, India. Electronic address: stdcirlpdy@gmail.com.
Abstract
BACKGROUND: rpoB gene mutations in Mycobacterium tuberculosis (MTB) make the bacteria resistant to rifampicin. Thus, these mutations are surrogate markers for multi-drug resistance (MDR). The objective of this study was to evaluate an allele-specific multiplex-polymerase chain reaction (MAS-PCR) assay to detect mutations at codons 516, 526 and 531 of the rpoB gene. METHODS: In total, 127 M. tuberculosis clinical isolates were subjected to standard drug susceptibility tests. A MAS-PCR assay was then performed to detect mutations in the rpoB gene. Three different allele-specific PCR assays were performed (single-step MAS-PCR) and the amplified products were sequenced. RESULTS: Of the 127 isolates, 69 (54.3%) were multidrug resistant M. tuberculosis (MDR-TB), 21 (16.5%) were rifampicin mono-resistant and 37 (29.1%) were drug susceptible. The frequency of mutations at codons 531, 526 and 516 was 54.4%, 18.9% and 5.6%, respectively. A triple mutation was found in 4 (4.4%) isolates. Mutations in regions other than the 81-bp region were observed at codons 413 (11.1%), 511 (12.2%) and 521 (15.6%) of the rpoB gene. CONCLUSIONS: The simplicity and specificity of the MAS-PCR assay allows for easy implementation in clinical laboratories to detect rifampicin drug resistance in MDR-TB strains.
BACKGROUND: rpoB gene mutations in Mycobacterium tuberculosis (MTB) make the bacteria resistant to rifampicin. Thus, these mutations are surrogate markers for multi-drug resistance (MDR). The objective of this study was to evaluate an allele-specific multiplex-polymerase chain reaction (MAS-PCR) assay to detect mutations at codons 516, 526 and 531 of the rpoB gene. METHODS: In total, 127 M. tuberculosis clinical isolates were subjected to standard drug susceptibility tests. A MAS-PCR assay was then performed to detect mutations in the rpoB gene. Three different allele-specific PCR assays were performed (single-step MAS-PCR) and the amplified products were sequenced. RESULTS: Of the 127 isolates, 69 (54.3%) were multidrug resistant M. tuberculosis (MDR-TB), 21 (16.5%) were rifampicin mono-resistant and 37 (29.1%) were drug susceptible. The frequency of mutations at codons 531, 526 and 516 was 54.4%, 18.9% and 5.6%, respectively. A triple mutation was found in 4 (4.4%) isolates. Mutations in regions other than the 81-bp region were observed at codons 413 (11.1%), 511 (12.2%) and 521 (15.6%) of the rpoB gene. CONCLUSIONS: The simplicity and specificity of the MAS-PCR assay allows for easy implementation in clinical laboratories to detect rifampicin drug resistance in MDR-TB strains.
Authors: M Gidado; N Nwokoye; P Nwadike; P Ajiboye; R Eneogu; S Useni; J Onazi; A Lawanson; E Elom; A Tubi; J Kuye Journal: Public Health Action Date: 2018-03-21
Authors: James Peek; Mirjana Lilic; Daniel Montiel; Aleksandr Milshteyn; Ian Woodworth; John B Biggins; Melinda A Ternei; Paula Y Calle; Michael Danziger; Thulasi Warrier; Kohta Saito; Nathaniel Braffman; Allison Fay; Michael S Glickman; Seth A Darst; Elizabeth A Campbell; Sean F Brady Journal: Nat Commun Date: 2018-10-08 Impact factor: 14.919