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

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.

Design of peptides interfering with iron-dependent regulator (IdeR) and evaluation of Mycobacterium tuberculosis growth inhibition.

OBJECTIVES: Tuberculosis (TB), a disease caused by Mycobacterium tuberculosis (Mtb), stayed a global health thread with high mortality rate. Since TB has a long-term treatment, it leads high risk of drug resistant development, and there is an urgent to find new drugs. The aim of this study was designing new inhibitors for a new drug target, iron dependent regulator, IdeR. MATERIALS AND METHODS: Based on the interaction most populated amino acids of IdeR to the related gene operators, 50 short peptides were modeled. Bonding affinity of short peptides toward DNA were studied by docking. Top 10 best predicted bonding affinity were selected. DNA binding assay, microplate alamar blue assay, colony counting, quantitative real time- PCR (qRT-PCR) of IdeR corresponding genes, cell wall-associated mycobactin and whole-cell iron estimation were done to prove the predicted mechanism of in silico potent constructs. RESULTS: Amongst the 10 synthesized short peptide candidates, glycine-valine-proline-glycine (GVPG) and arginine-proline-arginine (RPR) inhibited Mtbin vitro at 200 µM concentration. qRT-PCR showed mbtB 58-fold over expression that resulted in Mtb growth inhibition. Cell wall-associated mycobactin and whole-cell iron estimation confirmed the results of qRT-PCR. CONCLUSION: We introduced two new lead compounds to inhibit Mtb that are promising for the development of more potent anti-tubercular therapies.