Treatment of TB has become very difficult due to the prevalence of antibiotic resistance to first-line anti-TB medications. M. Tuberculosis can use the various mechanisms for resistance against anti-tuberculosis agents. One of the most important drug resistance mechanisms in this bacterium is mutations in genes encoding anti-tuberculosis drugs target proteins [14]. Infection with drug-resistant strains, especially in malnourished people or patients infected with HIV, is fatal, so the prevalence of MDR-TB is a serious risk for TB control worldwide [15]. The overall resistance rate in the present study was 15.38%, and the prevalence of MDR strains was estimated to be 3.84%, but the important point about MDR isolate was the collection of this isolate from Ghaemshahr in Mazandaran province, while Golestan province has the highest number of tuberculosis and considered as one of the most important provinces of TB infection in Iran.
In a study conducted by Nasiri et al. in 2014 on 6426 tuberculosis patients in 5 provinces of Iran, the overall resistance rate was 16.3%, while 6.3% of their isolates reported as MDR-TB [8]. In another study conducted in northern Iran, 5.5% of M. tuberculosis isolates were resistant to quinolones, rifampin, kanamycin, and amikacin, while 4.7% and 7.3% of the isolates were resistant against streptomycin and isoniazid, respectively [16]. Also, in a systematic study from 1998–2014 in Iran, the average prevalence of resistance rates against isoniazid, streptomycin, rifampin, and ethambutol were 26%, 23%, 22.5%, and 16%, respectively, while the highest resistance was related to isoniazid [17]. However, in our study, no resistance to ethambutol and streptomycin was observed, which could be due to the lower use of these drugs in the initial treatment of patients in this area.
In the present study, 65.38% of the patients were male indicating that the incidence of this disease in this region is almost more common in males, while another study by Lienhardt et al. found that TB was highly correlated with gender, while it was more common in males [18]. We also showed that the level of literacy of individuals can be an important factor in infecting patients, because approximately 70% of the patients studied were illiterate. On the other hand, although we did not find a significant relationship between the number of family members and the risk of M. tuberculosis infection, however the highest number of our patients was observed in families with 4 and 5 members. A study conducted by Chia et al. in 2015 about the genetic mutations and their role in the development of antibiotic resistance in clinical isolates of M. tuberculosis showed that the prevalence of mutation in the KatG, mabA-InhA, and rpoB genes was 19.4%, 70.1%, and 98.5%, respectively [19]. Sequencing results in our study indicated the presence of a mutation in the codon 315 of the KatG gene in the MDR isolate of M. tuberculosis, which converted the amino acid serine to threonine, while there is no evidence of the existence of two or more copies of this gene in the H37RV strain genome [20]. However, Duong et al. showed that the mutation in codon 315 of the katG gene can lead to the high isoniazid resistance in M. tuberculosis clinical isolates [20]. This mutation has been one of the most common causes of isoniazid resistance in clinical isolates of Mycobacterium tuberculosis in other parts of the world [21–23]. On the other hand, in a Russian study by Lipin et al. on drug-resistant M. tuberculosis strains, a mutation in the ropB gene was observed in 92% of MDR strains, while the highest mutation rates (89% of the isolates) were observed in codons 516, 526, and 531 [24]. Also, mutation in katG gene was detected in 95.4% of their MDR isolates, while the highest mutations were observed in codon 315 [24]. However, in the present study, mutations in codon 450 and nucleotide 1349 of the rpoB gene were observed in two isolates resistant to rifampin, while one of these isolates was MDR, and as a result of this mutation, the amino acid serine was converted to tryptophan. It should be noted that this gene is also a single copy in the genome of H37RV strain [20]. The results of these studies and our study show that mutation in codon 315 of katG gene is the most common mutation in all parts of the world, while the locus of mutation in ropB gene was different in our study and other researches.
The risk of infection following exposure to M. tuberculosis is mainly influenced by external factors, social characteristics, and individual behaviors. The most important internal risk factors for tuberculosis are immunosuppressive diseases, diabetes, malnutrition, smoking, alcohol consumption, and inhalation of polluted air [25]. One of the important aspects of this study was the assessment of risk factors in tuberculosis patients. However, age conditions play an important role in the development of M. tuberculosis infection, while children and people aged 15 to 25 years are highly susceptible to tuberculosis [25, 26]. The age distribution in tuberculosis patients in the present study ranged from 20 years to 86 years. Undoubtedly, in order to study the relationship between this factor and tuberculosis, there is a need to study in different age groups in a large number of patients.
Besides, one of the risk factors associated with tuberculosis was diabetes, while out of 26 patients, 5 (19.2%) had diabetes mellitus. This result has been confirmed in other studies. For example, Stevenson and colleagues in 2007 showed the link between tuberculosis and diabetes mellitus in India [27]. However, other studies in other parts of the world have reported diabetes as the most common risk factor for TB [25, 28]. Due to the increasing number of people with diabetes in different parts of the world and also in Iran, the study of M. tuberculosis infection in these people is very necessary and essential measures should be considered to prevent TB infection and treatment of these high risk persons [25, 27].
Other important factors reported in relation to tuberculosis included smoking [29]. In this study, smoking and hookah consumption among the patients were 42.3% and 46.1%, respectively. The results of two other studies have shown that smoking in a dose-dependent manner is directly related to pulmonary tuberculosis [29, 30]. Smoking can impair the function of the humoral immune system and respiratory cilia of alveolar cells and can lead to a decrease in the number of CD4 + lymphocytes resulting to the cellular immune system suppression [25]. Also, due to the effect of the nicotine on T cells' anergy and cellular immune function, it predisposes individuals to infection with intracellular microbes such as M. tuberculosis [25].