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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.
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