Detection of Isoniazid and Rifampin Resistance in Mycobacterium tuberculosis Clinical Isolates from Sputum Samples by High-Resolution Melting Analysis

• Published in: Current microbiology Vol. 79; no. 9; p. 257
• Main Authors: Parsa, Shadi, Yaghoubi, Atieh, Izadi, Nafiseh, Sabet, Faezeh, Nik, Leila babaei, Derakhshan, Mohammad, Rezaee, Seyed Abdolrahim, Meshkat, Zahra, Hoseini, Seyed Javad, Jmehdar, Saeid Amel, Kiani, Fatemeh, Samiei, Amin, Soleimanpour, Saman
• Format: Journal Article
• Language: English
• Published: New York Springer US 01-09-2022; Springer Nature B.V
• Subjects: Amino acids; Biomedical and Life Sciences; Biotechnology; Clinical isolates; Gene sequencing; Genetic analysis; High resolution; Isoniazid; KatG gene; Life Sciences; Melting; Microbiology; Multidrug resistance; Multidrug resistant organisms; Mutation; Mycobacterium tuberculosis; Rifampin; RpoB protein; Sputum; Tuberculosis
• ISSN: 0343-8651; 1432-0991
• DOI: 10.1007/s00284-022-02960-z

The effective management of multidrug-resistant tuberculosis (MDR-TB) and the need for rapid and accurate screening of rifampin (RIF) and isoniazid (INH)-resistant
Mycobacterium tuberculosis
(Mtb) isolates are the most fundamental and difficult challenges facing the global TB control. The present study aimed to compare the diagnostic accuracy of high-resolution melting-curve analysis (HRMA) in comparison to multiplex allele-specific PCR (MAS-PCR) and xpert MTB/RIF as well as the conventional drug-susceptibility test (DST) and gene sequencing for the detection of INH and RIF resistance in the Mtb isolates. In the present study, a total of 431
Mtb
isolates including 11 MDR (%2.55), 7 INH resistance (%1.62), two RIF resistance (%0.46), and 411 sensitive isolates were phenotypically confirmed. HRMA assay identified
katG
gene mutations and the
mabA-inhA
promoter region in 15 of 18 INH-resistant samples and
rpoB
gene mutations were successfully evaluated in 11 out of 13 RIF-resistant samples. The sensitivity and specificity of the HRMA method were 83.3% and 98.8% for INH and 84.6% and 99% for RIF, respectively. The most common mutation in RIF-resistance-determining region (RRDR) occurred at codon 531 (TCG → TTG)(84.6%) and then at codon 513 (CAA → GTA)(7.6%) and 526 (CAC → TAC) (7.6%), which resulted in the amino-acid changes. Also, 88.8% of INH-resistant samples had mutations in the
katG
gene and the
mabA-inhA
promoter region, of which the highest mutation occurred at codon 315 (AGC → ACC) of the
katG
gene. In conclusion, all these results indicated that the sensitivity and specificity of the HRM method were increased when the
katG
gene and the
mabA-inhA
promoter region were used as a target.