Background: Bovine tuberculosis is a chronic bacterial disease of cattle that occasionally affects human.A unique mutation at position 169 (C®G) in Mycobacterium bovis differentiates it from Mycobacterium tuberculosis. For the first time, PCR-RFLP method was used in this study to separate the two organisms.Methods: In this study, 98 tuberculosis patients were assessed using microscopic evaluations, culture and antibiogram. Then, nucleotide changes in position 169 were investigated using Allele-specific PCR and PCR-RFLP methods. Finally, the results were compared with spoligotyping results.Findings: Antibiogram revealed 28 subjects (28.57%) to be resistant and 54 (55.1%) to be sensitive to pyrazinamide while and 16 cases (16.23%) showed no growth. Of 28 resistant patients, 3 (3.1%) were diagnosed with M. Bovis using the classical methods. Using molecular methods, 95 (96.93%) out of 98 subjects were diagnosed with M. tuberculosis and 3 (3.06%) with M. bovis. Allele-specific PCR and PCR-RFLP matched with spoligotyping results.Conclusion: Our result showed the incidence of M. bovis infection to be higher in Iranian population (3.1%) than other studied cases (0.5-1%). In addition, we showed that Allele-specific PCR is the method of choice because it is faster and cheaper than PCR-RFLP. However, PCR-RFLP could be a proper alternative to prevent probable mismatches in Allele-specific PCR.

Background and Objectives Pyrazinamide is one of the effective drugs for treating tuberculosis. Inclusion of this drug in the treatment regimen shortens the duration of tuberculosis treatment. The mutation in PNCA gene causes loss of pyrazinamidase activity, the most important resistance mechanism in Mycobacterium tuberculosis isolates. Therefore, the purpose of this study is to investigate the phenotypic and genotypic resistance to pyrazinamide antibiotic in multidrug-resistant (MDR) Mycobacterium tuberculosis isolates in Khuzestan province between 2017 and 2022. Subjects and Methods This study was conducted on 40 isolates of Mycobacterium tuberculosis in Ahvaz TB Regional Laboratory. Drug sensitivity of the isolates was determined by the relative method in the antibiotic pyrazinamide in the isolates. Then, the frequency of PNCA gene mutations related to pyrazinamide resistance in MDR isolates was determined using sequencing. Results According to the results of the drug sensitivity to rifampin and isoniazid antibiotics, there were 25 isolates, of which 16 strains were resistant to two drugs, namely isoniazid and rifampin (MDR-TB), and 9 were single-drug resistant isolates (8 isolates resistant to rifampin and 1 isolate resistant to Isoniazid). Of the 17 samples that had the PNCA gene and were resistant to pyrazinamide, 13 had mutations in the PNCA gene while 4 did not have any mutations. The most common mutation was a non-synonymous mutation in which the amino acid Val had changed to Phe. Conclusion According to the results of this study, there is a high frequency of resistance to pyrazinamide in MDR strains. Also, there is a high percentage of single-resistance mutations in the PNCA gene, while there is lower prevalence of mutations in the panD and rpsA genes, which provide quick and accurate information about the sensitivity to pyrazinamide for MDR-TB and mono-resistant isolates. Therefore, the best method for detecting resistance to pyrazinamide is sequencing and whole PNCA DNA sequence to confirm pyrazinamide resistance instead of the usual methods covering mutated hotspots.

Background: One of the common clinical manifestations of extra-pulmonary tuberculosis is tuberculous meningitis (TBM). New methods, which are simple and effective, are necessary for early diagnosis of TBM. Objective: This study aimed to assess the use of fluorescence microscopy and polymerase chain reaction (PCR) techniques targeting PNCA and IS6110. PCR is a fast and reliable technique for diagnosing tuberculosis (TB), particularly in paucibacillary specimens such as smear-negative sputum for diagnosing patients with Mycobacterium tuberculosis. Materials and Methods: The PNCA and IS6110 multiplex-PCR methods were applied to analyze sputum using a specific pair of primers designed to amplify the insertion sequence, PNCA, and IS6110 in the M. tuberculosis genome. A total of 200 sputum samples were collected from patients with TB. Out of 200 patients, 54. 0% (n = 108) and 46. 0% (n = 92) were males and females at Fatima Jinnah General and Chest Hospital, Quetta, respectively. Results: M. tuberculosis was detected as 15 (16. 3%) and 18 (16. 7%) for PNCA and IS6110 using PCR and fluorescence microscopy, respectively. M. tuberculosis DNA detected by PCR and smear-negative fluorescence microscopy was 16. 50% (n = 33) and 14. 70% (n = 18), respectively. In other words, PCR assay detected the increased prevalence of M. tuberculosis. Risk factors revealed high exposure to receiving TB infection which was associated with urban areas (OR = 1. 07, 95% CI: 0. 34–, 3. 35), TB in family (OR = 1. 22, 95% CI: 0. 38-3. 88), over crowdedness (OR = 2. 93, 95% CI: 0. 37-23. 7), malnutrition (OR: 1. 39,95% CI: 0. 52-3. 74), and rural areas (OR: 1. 54, 95% CI: 0. 72-3. 31). Conclusion: A wide range of molecular assays are now being developed and reviewed for detecting TB. We conclude that the use of PNCA and IS6110 PCR assays is beneficial in the quick diagnosis of TB meningitis.

Background: Pyrazinamide is one of the most important first-line medications for the treatment of tuberculosis and an alternative intake for MDR-TB and XDR-TB patients. Objectives: The purpose of this study was to evaluate resistance to pyrazinamide in the isolates resistant to the Mycobacterium tuberculosis drug in patients in the city of Isfahan. Methods: In this study, the drug susceptibility test was performed with pyrazinamide using the proportion method and PZA assay on 47 isolates resistant to Mycobacterium tuberculosis. Then, the mutations of the PNCA and rpsA genes of the isolates resistant to pyrazinamide were evaluated by the sequencing method. Results: According to the proportion method, 19 cases were resistant to pyrazinamide, 16 of which had mutations in their PNCA and rpsA genes. Besides, five new mutations were recorded, and three isolates lacked mutations in the mentioned genes. Conclusions: Pyrazinamide resistance is high in MDR-TB and INH mono-resistant isolates. Therefore, evaluating the susceptibility to pyrazinamide in patients with MDR-TB before the initiation of treatment with pyrazinamide is considered essential.