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Introduction Tuberculosis (TB) is still one of important health problems in the developing countries, including Iran. Every year, around 8 million people catch TB and 2.5 million person loss their lives because of this disease [1]. TB is one of the most ancient human known diseases and one of the most common causes of death, especially in developing countries [2]. Although today the cause of this disease, as well as its vaccine and some very effective medicines to cure it have been discovered, TB still remains as a serious health scourge in the world [3]. Due to its geographical position and vicinity to countries with a high incidence of TB and because it accepts immigrants, Golestan province at the northern part of country is one of the infested provinces of the country. Today, drug resistance is considered as a serious problem in TB control programs in most countries. The risk of first-line treatment multi-drug-resistant tuberculosis (MDR-TB) and second-line treatment extensive-drug-resistant tuberculosis (EDR-TB) is considered a big problem in TB control. According World Health Organization (WHO) record, out of 9.2 million new cases of TB infection, 500000 cases were drug-resistant TB in 2006. Resistance to anti-TB drugs is considered one of the big challenges of public health which threatens the successful implementation of DOTS (Directly Observed Treatment, Short-course) plan. In average, the cost of diagnosing and free treatment of each TB patient is 2000,000 Iranian rial (less than US$70), but the cost of treatment of drug-resistant TB alone is 250,000,000 Iranian rial (more than US$8000) and takes 6 to 24 months. The admission fee and costs of frequent tests and cultivations required for the treatment of cure-resistant TB are excluded from this cost. Also, ultimately only 40 to 60 per cent of the treatment will be successful and rest of the patients are doomed to die, while maintaining the potency of transmitting the disease to others [4]. In terms of microbiology, the drug resistance to tubercle bacilli takes place when a genetic mutation occurs in bacilli, but the more important issue is that partial or incorrect treatment by those in charge of the disease causes drug sensitive bacillus to be destroyed due to consumption of antiTB drugs with inappropriate compound and insufficient amount, instead the resistant strains to survive and multiply in presence of inappropriate treatments and change into prevalent strains in patients’ bodies [5]. Multi-drug-resistant tuberculosis (MDR-TB) is a kind TB in which the bacillus shows resistance, at least, to two anti-TB drugs; that is, Izoniazid and Rifamoin. These drugs are used as the First-line drugs in treatment of TB in TB patients. XDR-TB is a rare type of MDR-TB. This type of TB is determined by resistance against Izoniazid and Rifamoin, as well as Fluoroquinolones and at least one of the injectable Second-line drugs such as Amikacin, Kanamaycin and Capreomycin. Because in this type of TB there is resistance against first- and second-line drugs, treatment options effective for this problem are restricted [6]. Currently, determination of level of MDR-TB and EDRTB and evaluation of their spread and control are considered as one of the main criteria of quality and success of TB control programs in countries [7]. Considering the high outbreak of TB in Iran, particularly in Golestan province, the emerge of treatment-resistant TB, especially MDR, and the possibility of spread of these strains to other parts of Iran and considering the increase of probability of death and failure of treatment amongst resistant TB patients , increase of time and costs of treatment and more important, the serious threat of TB control due to resistant strains, despite numerous studies in other countries, few studies have been conducted in Iran. Abbasi and GolAlipour [8], in a study conducted in GorganIran, from 260 newly infected patients (new cases) 31 patients were still smear-positive after the end of fourth month of treatment, which after implementation of second phase of treatment (Cat II) only 2 patients were sputum smear-positive, and after antibiogram test, it was determined that only one patient had been resistant to Izoniazid and Rifamoin. And the prevalence and rate of drug-resistance of mycobacterium strains under the study showed that the resistance is not yet serious in Gorgan; that is why it calls for careful attention to prevent development and spread of resistant strains [8]. Liwani et al. [9] found that the outbreak of MDRTB was 3.4% in Golestan province, which is within national statistics. By MGIT (Mycobacteria Growth Indicator Tube), isolation and antibiogram of MDR strains toke 26 days in average which can shorten the time of diagnosing and determining the drug-resistance of TB bacillus [9]. Considering geographical situation of Golestan province as well as its close vicinity to countries with high TB outbreak and because it receives immigrants the need for conducting this study is felt strongly; as a result, the present study aiming to initial drug-resistance in new smear-positive pulmonary TB patients (new cases) and factors affecting it was conducted in Golestan province in 2011-2012 so that with determining prevalence of resistance the best treatment method could be chosen and the spread of it could be prevented. Considering TB outbreak, particularly in Golestan province and the arrival of new combination drugs to Iran, the present study was conducted in 2011 and 2012 to compare the result of treatment in TB patients cured with old separate drugs and new combination drugs in Golestan province. Methods This study is of cross-sectional and analytic type which has been conducted in Golestan province during 2011-2012 to determine initial drug resistance in smear-positive pulmonary TB patients (new cases), the factors affecting it, and their resistance pattern. The population under study included all new smear-positive pulmonary TB patients who came to health center of Gorgan County. Patients whose direct sputum smear microscopy test were positive (at least two positive sputum smear) and had not already received anti-TB treatment or less than one week had passed from commence of their TB treatment entered the study. Extra pulmonary TB cases were excluded from the study. The method of sampling was simple random. Demographic information including age, sex, place of residence, level of education, level of monthly income, patient’s inmate records and the initial sputum positivity rating were recorded on a questioner. To culture samples on Lowenstein-Jensen medium, all samples were initially decontaminated using Petrov’s method. Also, acid-fast staining was performed using Zeihl Neelsen’s method. Since the aim of this study is to determine initial drug susceptibility in patients infected with pulmonary TB induced by Mycobacterium tuberculosis strains (MTB), as a result the species of strain isolated from sample was determined by using biochemical tests. Different tests, such as Niacin test, Nitrate reduction test, and thermostable catalase (68°C), were used to determine species. After isolating mycobacterium tuberculosis strain, the effect of first-line anti-TB drugs including Izoniazid, Rifamoin, Ethambutol, and Streptomycin on strains under the study was examined. The method we used to determine the drug susceptibility of isolated strains is the proportional method. This method is standard and reference and is confirmed by WHO (World Health Organization) and CLSI (Clinical and Laboratory Standards Institute). This method is performed on strains for first- and second-line drugs. The method needs at least 3-4 weeks incubation [1012]. In proportional method, all of these strains, after suspension is made, are cultured on Lowenstein-Jensen medium with and without antibiotic (control medium) and results are read after 28 and 42 days. Standard Mycobacterium tuberculosis H37Rv is used as quality control. If the ratio of number of bacillus resistant to both drugs to the number of alive bacillus in inoculated material is less than the level called critical ratio for resistance, the bacillus is considered susceptible to that drug and if this ratio is equivalent or higher than that, the strain is considered resistant. Finally, drug-resistances were examined by statistical tests as well as resistance frequency distribution based on demographic specifications. Obtained information was entered into version 16 of SPSS app (SPSS Inc., Chicago, IL, USA) and was analyzed based on descriptive and analytical statistics. In this study the confidence interval was 95% and P<0.05 was considered significant. Results In this study, 266 patients have the initial condition to enter the study, that antibiogram test was performed on 214 patients. Out of this number, the antibiogram test of samples from 189 patients was successful in terms of performance, such that from 214 test cases, 10 cases were NonTuberculosis Mycobacterium (NTB) and 15 cases led to failure of bacteria growth in antibiogram control medium (failed). Out of 189 successful antibiogram tests, 12 patients did not come back after samples were sent and their demographic information was not recorded and finally, these cases were announced missing (6.3% missing data). So, only demographic information of 177 patients has been recorded in this study. Figure 1 shows the schematic view of study plan and the number of people omitted from the study and the reason for their omission. The average age of patients was 48.4 ± 21.3 years, with the range of 9-96 years. Most of participants were men (56.5%) and were married (50.8%). Only 2.8% of patients were nonIranian. 54.8% of patients lived in villages and 45.2 lived in cities. 38 people of patients (21.5%) had mentioned imprisonment records. Other demographic information of patients has been given in table 1. From 189 patients infected with TB (new case) on whom the antibiogram test was performed in this study, 21 patients (11.1%) showed all types of resistance, which may be against one, two or more drugs at the sane, and others (88.9) were sensitive against antibiotics under the study. It is to mention that 4 patients (2.1%) were resistant to Rifampin and Izoniazid (MDR) at the same time. Table 2 shows the initial drugresistance pattern of the patients of this study. Discussion TB is one of the prevalent infectious diseases in current era and is the second most common cause of death after HIV in human beings [13]. Currently, TB has turned into a health scourge in human communities, with the presence of resistance against anti-TB drugs and drug-resistance prevalence in TB is a major health concern in the world [14]. The present study showed that out of 189 patients, 21 patients (11.1%) had all type of resistance, which this resistance can be against one, two, or few drugs simultaneously, and the rest of patients (88.9%) were susceptible to antibiotics under the study. It is to mention that 4 patients (2.1%) were resistant to Rifampin and Izoniazid (MDR) at the same time. In the study conducted by Abbasy and et al. [8] in 1997-1999 in Golestan province, they showed that from 260 patients infected with TB, 31, 9, and 2 patients were still sputum smear-positive, respectively, after the end of second month of treatment, at the beginning of fifth month of treatment, and after the implementation of second phase of treatment, which after Antibiogram it was determined that only one patient was resistant to Rifampin and Izoniazid and another patient was resistant to all drugs, which translates into very low prevalence of drug-resistance (less than 0.5%) [8]. It could be said that prevalence of initial anti-TB drug-resistance is in significant growth and this can have undesirable effects on the treatment of TB patients. In the study conducted by Peimani and et al. [15], which was conducted on TB patients in Tabriz-Iran in 2011, it was shown that only one incidence of multi-drug-resistance had been determined and level of resistance was low in Tabriz province, and therefore, the rate of negative-sputum increase in patients is within the expected level. The scourge of drug resistance in the world and possibility of diagnosis in our country (Iran) show the necessity of screening and proper treatment to improve the status-quo [15]. Also, findings of a very large research conducted in 36 countries of the world to examine anti-TB drug-resistance showed that American countries such as the Dominican Republic had high level of resistance; the cause of this prevalence can be attributed to poorness of TB control system, although immigration issue can be mentioned as a reason too [7]. Also, in African countries with very high level of HIV infection and poorness of TB control system, despite what expected, the level of resistance was very low; the reason why the resistance prevalence is low perhaps could be attributed to the treatment implemented under supervision of health system. Western European countries, prevalence of multi-drug-resistance is very low and has been reported around 1 per cent, but in some countries, such as Span, 28% of TB patients have HIV infection simultaneously [16]. In Asian countries, in which TB is an endemic disease, resistance prevalence is so different; in some countries like Korea, it is very low and in some countries like India, multi-drug-resistance has been reported 13.3% [6]. The study conducted to determine percentage of drug-resistance in pulmonary TB patients in Istanbul and compare it with researches conducted in Iran and some other countries showed that 91 tuberculosis mycobacterium strains were isolated from sputum of patients suspected to pulmonary TB came to Pasha Faculty of Medicine &Surgery in Istanbul by standard method. Susceptibility of isolated strains to four main antiTB drugs, that is Izoniazid, Rifampin, Ethambutol, and Streptomycin were examined by indirect proportional method using Lowenstein Jensen culture medium. The general resistance level was 44 per cent. The highest level of initial resistance was 21.1% to Streptomycin and after that was 15.8% to Izoniazid, 5.3% to Rifampin, and 2.6% to Ethambutol respectively. The level of initial MDR (Multi-Drug-Resistance) was 2.6%. Maximum secondary resistance was 33.3% to Izoniazid and after that was 28.6% to Streptomycin, 23.8% to Rifampin, and 14.3% to Ethambutol respectively. The level of secondary MDR was 11.9% [17]. In another research, the goal was to demonstrate tuberculosis mycobacterium resistance pattern to four anti-TB drugs in children infected to pulmonary TB. Amongst 350 children (0-15 years of age) with confirmed TB, 7 persons had resistance to at least one of the anti-TB drugs. Among these 7 children, 6 patients were Afghan refugee and one person was Iranian. In this group, 85.7% showed resistance to RMP, 4.71% to INH, 1.57% to SM, and 28.6% to EMB. Also, 28.5% showed resistance to all four anti-TB drugs –RMP, EMB, SM, and INH, 2.14% to three drugs – INH, SM, and RMP, 5.28% to two drugs – RMP and INH, and 2.14% to RMP and 14.2% to SM [18]. In this study, it was demonstrated that 14 per cent of patients with Fars ethnicity, 9 per cent of patients with Sistanian ethnicity, and 8.4 per cent of patients with Turkoman ethnicity were resistant against TB. And also, 21% of individuals with inmate records had one of resistance types. Conclusion The drug-resistance outbreak and level of studied Mycobacterium tuberculosis strains show that failure to pay urgent and careful attention to existing drug-resistances in strains isolated from TB patients will lead to irrecoverable increase of prevalence and spread level of multi-drugresistant strains. Obtained results were compared with some studies conducted in Iran and other countries. Due to high prevalence of drugresistance, especially in developing countries, wide researches for effective control of TB are necessary in these countries. Acknowledgments Executives and collaborators of the present study feel urgently to gratitude from anonymous patients whose laboratory data and files were used in this study. Also, we appreciate the material and spiritual supports deputy of research and technology, deputy of education, and infectious diseases research center of Golestan University of Medical Sciences bestowed upon us during conducting this study. Conflict of Interests The authors declare that they have no competing interests. References: .1 Moniri R‚ Rasa SH MS. [A survey on type of mycobacterium and drug resistance rates of mycobacterium tuberculosis strains in Kashan (Persian)]. . Journal of Shahid Sadoughi University of Medical Sciences and Health Services. 2001;9(1):67-70. .2 Cohn DL ‚Bustreo F RM. Drug resistant tuberculosis: review of the worldwide situation and the WHO/IVATLD global surveillance project. International Union Against Tuberculosis and Lung Disease. 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