Background: The emergence of drug-resistant strains of Mycobacterium tuberculosis (MTB) is an increasing problem in developed and developing countries. The aims of the present study were to identify various types of mutations in KATG region from 28 MDR strains isolated from sputum of tuberculosis patients.Materials and Methods: Twenty-eight rifampin-resistant strains isolated from sputum of patients with active pulmonary tuberculosis were obtained from various geographic regions of Iran. Drug susceptibility was determined by using the BACTEC system. DNA extraction, standard PCR identification, KATG gene amplification, DNA sequencing and analysis were done.Results: There was no mutation in 2 strains. In 20 strains, mutation was shown to be in codon 315. Three types of mutations were detected consisting of AGC®ACC (Ser®Thr) (80%), AGC®AGG (Ser®Arg) (5%) and AGC®AAC (Ser®Asn) (15%). Furthermore, one type of mutation was seen in codons 311, 299, and 323. Twelve strains showed one mutation in codon 315 (46%), 7 strains 2 mutations (27%), 5 isolate 3 mutations (19%) and in 2 strains 4 mutations (8%) were observed in different codons. Nine silent mutations was demonstrated in codon 311 (GAC®TAC).Conclusion: This research demonstrated that mutations were mostly seen in codons 315 and 299 indicating resistance to isoniazide.

Background: The aim of this study was to investigate the significance of mutation in codon 315 of KATG gene and its correlation with high-level of resistance to isoniazid, nuclotide and amino acid changes in mycobacterium tuberculosis (MTB) isolates randomly collected from sputums of 42 patients with active pulmonary tuberculosis in different regions of Belarus. Materials and Methods: Drug susceptibility testing was determined using the CDC standard conventional proportional method. DNA Extraction, KATG gene amplification, and DNA sequencing analysis were performed.Results: Six isolates (14%) bearing multi-mutations in three codons (309,315 and 316), 26 Isolates (61.9%) demonstrated multi-mutations in all or two of the above codons, and 8 (19%) were found to have a single mutation in 315. Four types of mutations were identified in codons 315: AGC®ACC (n=36)85%, AGC®AGG (n=1) 2.3%, AGC®AAC (n=2) 4.7%, AGC®GGC (n=1) 2.3%, one type of mutation in 316: GGC®AGC (n=18)41.4%, and four types of mutations in 309: GGT®GGT (n=7)16.1%, GGT®GCT (n=4)9.2%, GGT®GTC (n=3)6.9%, GGT®GGG (n=1)2.7%. In 2 (4.7%) isolates mutations were identified in codons 463, 357, and in codons 454, 357 respectively.Conclusion: MTB in patients from Belarus were found to have high-level resistance to isoniazid in the isolates with mutations in codon 315 (> 10 mg/mL).

Background: Mycobacterium tuberculosis is a pathogen that causes Tuberculosis and can invade various organs in infected patients. Its high morbidityandhigh mortality seriously threatenhumanhealth. In recent years, the continuous emergence of drug-resistant Tuberculosis bacteria has brought severe challenges to the prevention and control of Tuberculosis. Objectives: This study aimed to characterize the most frequent mutations of the KATG, inhA, and ahpC genes in isoniazid (INH)-resistant M. tuberculosis clinical isolates in Shanghai Pulmonary Hospital, China, and investigate the relationship between gene mutations and the minimum inhibitory concentrations (MICs) of INH against M. tuberculosis. Methods: We collected 92 INH-resistant and 30 INH-susceptible clinical isolates of M. tuberculosis. The drug resistance profiles of M. tuberculosis clinical isolates against common anti-tuberculosis drugs were determined and sequencing analysis was performed. Results: Of 92 INH-resistant strains, mutations in the KATG and inhA genes were observed in 64 (69. 6%) isolates and five (5. 4%) isolates, respectively, and only had one (1. 1%) strain both KATG and inhA mutations. Among them, 62 (67. 4%) strains carried a single mutation at codon 315 of the KATG gene and a new mutation site was found in the KATG gene of two strains. We detected a single mutation site at codon 271 and three simultaneous mutation sites at codons 315, 431, and 439. Only one (3. 3%) of the 30 isoniazid-sensitive strains had the KATG mutation. The AhpC mutation was detected in no experimental strains. The KATG Ser 315 Thr (AGC315ACC) mutation occurred in 53 (68. 8%) out of 77 strains with high MICs ( 1 g/mL) of isoniazid-resistant M. tuberculosis while five (33. 3%) out of 15 strains with low MICs (less than 1 g/mL) had KATG Ser 315 Thr (AGC315ACC) mutation. Conclusions: Isoniazid-resistant strains were dominated by Ser315! Thr (AGC! ACC) substitution, which seems to be associated with multidrug resistance and high-level resistance to INH. Multisite mutations are related to multidrug-resistant M. tuberculosis and the discovery of new mutation sites provides a new basis for the detection of drug-resistant M. tuberculosis.

Objective: The Rifampicin resistance and susceptibility of Mycobacterium tuberculosis are caused by mutations in the 81-base pair region of the rpoB gene encoding the bsubunit of RNA polymerase.Methods: Isoniazid resistance of M. tuberculosis is related to mutations in inha, oxyR and ahpC genes which 30 to 90 percent of Isoniazid resistance is occurred in 3015 codons of KATG gene. The rpoB and KATG sequences of 30 isolates were analyzed to identify the mutations and compare the mutations with their related susceptibilities.Results: In this research, we investigated the location and type of rpoB and KATG mutations in Mycobacterium tuberculosis which had been achieved from Pasteur Institute of Tehran. PCR Amplification and DNA sequencing methods were performed. In this assay, from 507 to 537 codons and 315 codons of rpoB and KATG genes were sequenced and also mutations were analyzed, respectively.

Background: Tuberculosis is one of the infectious diseases worldwide that has resurgence by the AIDS epidemic and led to the rise of drugresistant tuberculosis patients. Thus, it seems essential to monitor the drug susceptibility in tuberculosis patients. The new High Resolution Melting (HRM) method is simple, rapid and inexpensive for detection of the mutations responsible for drug resistance in Mycobacterium tuberculosis isolates. In this study, we used HRM method to detect mutations in samples collected from tuberculosis patients.Materials and Methods: Three thousand sputum samples were collected from patients with suspected tuberculosis referred to Iran Remedial Center over a period of 2 years, out of which 2000 samples were found positive for M. Tuberculosis on direct smear. After extraction of genomic DNA from sputums, HRM method was used to detection of mutations in KATG and inhA genes.Results: Our findings showed that 120 out of 2000 positive smear samples were resistant to isoniazid due to mutations in KATG and inhA genes, out of which, 25 mutation was found in inhA gene and 95 mutation in KATG gene.Conclusion: The HRM method is quick, easy and affordable without need of culture and any post PCR process for diagnosing of drug resistance in tuberculosis clinical samples.

Background & Objectives: Although isoniazid is the most efficient in killing the tuberculosis bacilli, resistance to this drug also develops most readily. Mutations in KATG, inhA and ahpC are responsible for isoniazid resistance in a large proportion of tuberculosis cases. The frequency of these mutations varies with population samples, however. This study provided the first molecular characterization of Isoniazid-resistance in M. Tuberculosis strains that is widely applicable for rapid drug resistance detection.Materials and Methods: The study was a descriptive and analytical and the presence of mutations in specific regions of the KATG, inhA and ahpC genes was analyzed in 32 M. tuberculosis Isoniazidresistant strains in Isfahan and Tehran. To determine the mutations in codon 315 of KATG, PCR-RFLP technique was performed. In this way, 355 bp PCR products were digested by MspI and MspA1I.Mutations in inhA and ahpC genes were detected by sequencing.Results: The frequency of mutations in the KATG 315(Ser-Thr), inhA and ahpC were detected in 71.9%, 18.9% and 6.2% of the 32 Isoniazid resistant isolates, respectively. Mutation was not found in one of the isolates.Conclusion: The PCR-RFLP with MspI, being able to detect KATG Ser315Thr substitution, can identify more Isoniazid-resistant strains with mutations at codon 315 in the KATG. Elucidation of the molecular basis of Isoniazid resistance in M. tuberculosis has led to the development of different genotypic approaches for the rapid detection of Isoniazid resistance in clinical isolates. The results also suggest that the detection of the Ser315Thr in the KATG gene may be used as a rapid screening method for identifying Isoniazid-resistant clinical M.tuberculosis isolates recovered from Isfahan and Tehran tuberculosis centers.

Objectives: The efforts on control of tuberculosis have been encountered with serious problems by emergence of drug resistant Mycobacterium Tuberculosis (MT), and rapid diagnosis of resistance can play an essential role in control and prevention of the disease. Isoniazid is a first line drug in treatment of TB and development of resistance against this drug are increasingly reported. In this study, rapid diagnosis of Isoniazid resistant MT was investigated by PCR-RFLP method.Methods: A total of 25 Isoniazid - resistant and 25 Isoniazid - susceptible MT isolated at Tuberculosis and lung disease research center were screened for Isoniazid resistance. In the first step MIC of isolates to INH was determined by proportion method. After that, genomic DNA was extracted from all isolates and a fragment of Kat G gene was amplified by PCR using specific primers. Screening for mutation on Ser 315 and Arg 463 codons in the Kat G gene was carried out by digestion of PCR product with restriction enzyme Msp I.Results: Among 25 INH- resistant isolates, 14 isolates (56%) had mutation in Ser315 locus and 5 isolates (20%) showed mutation in Arg 463 locus, whereas 6 strains (24%) didn’t show any mutation in these codons.Mutation in both 315 and 463 codons were not found in any isolates. In the susceptible isolates, no any mutation was detected in the studied codons.Conclusion: The results of this study indicated that PCR-RFLP method is able to detect resistance to INH in 76% cases and so, it can be used for rapid diagnosis of Isoniazid resistant MT isolates.