[Prevalence of non-tuberculosis mycobacteria in patients referring to Mycobacteriology Research Center of Iran]

Considering the increasing significance of diseases due to NTM all over the world, we investigated the burden of such diseases in our region. The aim of this study was to assess NTM prevalence from different clinical samples during a period of 8 years in Massih Daneshvari Hospital, in Iran. This descriptive study was performed on 8322 samples obtained from pulmonary TB patients in Mycobacteriology Research Center from 2004 -2012. Using Tb1 and Tb2 primers, a 190 bp fragment of IS6110 gene was amplified in order to identify Mycobacterium species. Specimens with negative IS6110 PCR results were analyzed with PCR-RFLP using hsp65 gene, for NTM investigation. Out of 8322 samples, we identified 124 [1.5%] strains of NTM. The mean age of the patients was 57 +/- 18/9 years [age range: 7 - 88 years]. 55/6% of the patients were male. The most common species detected in our study were Mycobacterium simiae [44.3%], Mycobacterium chelonae [16.9%] and Mycobacterium kansasii [12.9%]. We found a high prevalence rate of Mycobacterium simiae among our patients. Treatment protocols for NTM are different from the protocols for Mycobacterium tuberculosis complex, so early diagnosis of these species will be of great importance

[Investigation of the relationship between polymorphism of IFN-gammaR1 [-611],IFN-gamma[2109] and IL-12B[-1188] genes and pulmonary TB]

A large number of factors are involved in the development of TB, but the most important one belongs to the host genetic factors. One of the genetic factors is cytokine gene polymorphisms. The results of recent studies indicate that IL-12 and IFN-gamma play a central role in regulating the type and level of immune response in mycobacterial infections. Mutations in these genes may be associated with susceptibility to pulmonary TB. The aim of this study was to investigate the frequencies of IFN- gamma [2109], IFN-gamma R1 [-611] and IL-12B [-1188] genes polymorphisms and their relationships with susceptibility to pulmonary TB in Iranian population. This was a case-control study. Thirty TB patients with positive smears hospitalized in TB departments of Masih Daneshvari Hospital and 30 healthy controls with no history of TB were selected for this study. Genotypes of IFN- gamma [2109], IFN-gamma R1 [-611] and IL-12B [-1188] genes were determined by using PCR-RFLP method. The PCR-products were analyzed by use of restriction enzymes. The data were analyzed by means of SPSS and Hardy - Weinberg equilibrium. Considering IFN-gamma R1 [-611] and IL-12B [-1188] genes there was a significant difference between the control and study groups [P < 0.05], but in regard to IFN- gamma [2109], this difference was not detected between the two groups. Mutation in the regions of -611 of IFN-gamma R1 and -1188 of IL-12B genes may increase the host susceptibility to mycobacterium tuberculosis and genotyping of these regions can be used for screening of the high risk individuals

[Evaluation of genetic pattern of non-tuberculosis mycobacterium using VNTR method]

Epidemiological studies of Non-tuberculosis Mycobacterium is important because of the drug resistance pattern and worldwide dissemination of these organisms. One of genetic fingerprinting methods for epidemiological studies is VNTR [Variable Number Tandem Repeat]. In this study genetic pattern of atypical Mycobacterium was evaluated by VNTR method for epidemiologic studies. 48 pulmonary and non pulmonary specimens separated from patients with the symptoms of pulmonary tuberculosis [PTB] and identified as Non-tuberculosis Mycobacteriumby phenotypic and PCR-RFLP methods were selected for this study. Clinical samples and their standard strains were evaluated according to VNTR pattern using the 7 genetic loci including ETR-B. ETR-F. ETR-C. MPTR-A. ETR-A. ETR-E. ETR-D. The results of VNTR method showed that none of the 7 loci had any polymorphism in the standard strains of atypical mycobacterium. Some of these variable number tandem repeat in 42 clinical samples of non-tuberculosis Mycobacterium were polymorphic while the PCR product [for any loci] was not found in the remaining 6 specimens. Although the used genetic loci of this study were suitable for epidemiological studies of Mycobacterium tuberculosis, these loci were not able to determine the diversity of genetics of non-tuberculosis Mycobacterium Therefore, it seems necessary that other loci be studied using VNTR method

Association between TNF-alpha [-857] gene polymorphism and susceptibility to tuberculosis

TNF-alpha as a pro-inflammatory cytokine play a key role in host defense against tuberculosis [TB]. Presence of mutation in TNF-alpha gene can influence the effectiveness, performance and capability of immune responses against this infection. The Aim of this study was to investigate the frequency of TNF-alpha alleles and its relationship with susceptibility to TB and TNF-alpha gene variations. A case-control study was conducted and 103 healthy controls and 93 TB patients were enrolled. Genotype of TNF-238, TNF -244, TNF-308, TNF -857 and TNF-863 were distinguished using PCR-RFLP method. TNF-857 and TNF-863 were in high frequency mutation regions in a population level, and a significant difference at TNF-857 was noticed between the two groups of case and control. Presence of mutation in TNF-857 region probably increases the host susceptibility to mycobacterial infection. Genotyping of these regions in combination with other factors can be used for screening of high risk persons. According to high distribution of mutations in TNF-857 and TNF-863 regions, further studies on association of these regions is suggested

[Identifying Mycobacterium tuberculosis resistance to rifampin by multiplex specific PCR method]

Mycobacterium tuberculosis is still considered the most common cause of death cases related to pathogenic infectious factors in the world. Rifampin is among the most important first-line drugs to treat tuberculosis. The most common mutations in resistance to Rifampin occur due to the displacements in Codons 531, 526, and 516 in rpoB gene. This study was carried out with the aim of introducing the method [Multiplex Allele Specific] PCR in order to identify tuberculosis patients resistant to rifampin through detecting mutations in the rpoB gene. In this study, three cases of mutation were evaluated in three Codons of rpoB gene in 90 samples of the positive cases of culture from pulmonary TB patients who had referred to Research Center of Mycobacteriology located in Masih-Daneshvari Hospital of Tehran from 1385 to 1387 after the drug sensitivity test. To examine three codons 531, 526 and 516, MAS PCR method was used. The results of the culture showed that 33.3% of the samples were sensitive and 66.6% were resistant to drugs of which 44.4% were resistant to Rifampin. By using MAS PCR method, 32.2% of these cases of resistance were identified. Among these cases, 43.4% had mutation in codon rpoB 531, 34.5% in rpoB 526 codon and 31% in rpoB 516 codon. Based on the results of this study, MAS PCR method used in this research is an accurate and appropriate method to rapidly diagnose resistance to Rifampin in the clinical samples of Mycobacterium tuberculosis

[Comparison of the genetic convergence degree of mycobacterium tuberculosis [TB] strains isolated from patients infected with TB by MIRU-VNTR technique]

In recent decades, epidemiology has significantly been considered in hygienic studies and disease control, and has made a way into all the programs and hygiene policies. By examining the convergence of harmful lineage genetic patterns, the common infectious resources among the patients can be inferred. The purpose of this study was to compare the Mycobacterium Tuberculosis genetic patterns convergence isolated from patients infected with Mycobacterium Tuberculosis by MIRU-VNTR technique. After isolation the samples from Lowenstein Jensen culture environment and taking segregate tests and drug susceptibility, the DNA was extracted using CTAB/Nacl technique. The genetic patterns of lineages were calculated according to 12 loci format with MIRU-VNTR technique. Demographic and molecular information of patients was used for epidemiological purposes. After performing drug sensitivity test, 65/140 [64/4%] samples fall into MDR, 29 [20/7%] samples in non MDR category, and the rest of them were among drug. sensitive lineages. Lineage genetic pattern analysis indicated that 49 [35%] of samples related to Delhi/CAS, 28 [20%] to Uganda I, 16 [11/4%] to New I, 1 [0.7%] to EAI, 3[2/1%] to Haarlem, and 5[3/5%] to H37RV families. The genetic pattern convergence comparison exhibited that the most common and variant genetic patterns was seen in Tehran province which were mostly connected to south [from the South of Tehran to Azadi Square] and to the border cities neighboring Afghanistan, Iraq, Turkmenistan and cities with extreme percentage of immigration, all of which signified shared polluted resources among patients

Comparison of the genetic convergence degree of Mycobacterium tuberculosis [TB] strains isolated from patients infected with TB by MIRU-VNTR technique

In recent decades, epidemiology has significantly been considered in hygienic studies and disease control, and has made a way into all the programs and hygiene policies. By examining the convergence of harmful lineage genetic patterns, the common infectious resources among the patients can be inferred. The purpose of this study was to compare the Mycobacterium tuberculosis genetic patterns convergence isolated from patients infected with Mycobacterium Tuberculosis by MIRU-VNTR technique. After isolation the samples from Lowenstein Jensen culture environment and taking segregate tests and drug susceptibility, the DNA was extracted using CTAB/NaCl technique. The genetic patterns of lineages were calculated according to 12 loci format with MIRU-VNTR technique. Demographic and molecular information of patients was used for epidemiological purposes. After performing drug sensitivity test, 65/140 [64/4%] samples fall into MDR, 29 [20/7%] samples in non MDR category, and the rest of them were among drug - sensitive lineages. Lineage genetic pattern analysis indicated that 49 [35%] of samples related to Delhi/CAS, 28 [20%] to Uganda I, 16 [11/4%] to New I, 1 [0.7%] to EAI, 3[2/1%] to Haarlem, and 5[3/5%] to H37RV families. The genetic pattern convergence comparison exhibited that the most common and variant genetic patterns was seen in Tehran province which were mostly connected to south [from the South of Tehran to Azadi Square] and to the border cities neighboring Afghanistan, Iraq, Turkmenistan and cities with extreme percentage of immigration, all of which signified shared polluted resources among patients

Phospholipase C in Beijing strains of mycobacterium tuberculosis

Phospholipase of Mycobacterium tuberculosis plays an important role in pathogenesis through breaking up phospholipids and production of diacylglycerol. In this study, we examined the Beijing strains of Mycobacterium tuberculosis isolated from Iranian patients for the genes encoding this enzyme. DNA extraction was performed using CTAB [cetyltrimethylammonium bromide] from positive culture specimens in tuberculosis patients. PCR was then used to amplify the plcA, plcB, plcC genes of Beijing strain, and non-Beijing strains were identified by spoligotyping. Of 200 specimens, 19 [9.5%] were Beijing strain and 181 [90.5%] were non-Beijing strains. The results of PCR for Beijing strains were as follows: 16 strains [84.2%] were positive for plcA, 17 [89.4%] were positive for plcB and 17 [89.4%] were positive for plcC genes. The standard strain [H37RV] was used as control. The majority of Beijing strains have phospholipase C genes which can contribute to their pathogenesis but we need complementary studies to confirm the role of phospholipase C in pathogenecity of Mycobacterium tuberculosis

[Relationship between TNF-alpha gene polymorphisms and susceptibility to pulmonary tuberculosis]

Tuberculosis [TB] caused by Mycobacterium tuberculosis, is an infectious disease in human which kills nearly three millions of people annually. Approximately, one - third of the world populations are infected with this bacteria and 5 - 10% of them develop the active form of the disease. Individuals are different in susceptibility to TB infection. These differences might be due to the host characteristics especially genetic factors. TNF- alpha as a pro-inflammatory cytokine, plays a key role in host defense against tuberculosis. Presence of mutation in this gene can influence the effectiveness, performance and capability of immune responses against TB infection. The Aim of this study was to investigate the frequency of TNF- alpha gene polymorphisms and its relation with susceptibility to the pulmonary TB. Sixty healthy controls and 60 TB patients were enrolled. Genotype of TNF[-238], TNF -244, TNF[-308], TNF[-857] and TNF[-863] were determined using PCR-RFLP method. The results were analyzed by Fisher Exact and kappa[2] tests using SPSS v.14 and evaluated with Hardy-Weinberg equilibrium. The results of this study showed a significant difference in TNF-308 and TNF [-857] regions between the control and study groups [P < 0.05]. Presence of mutation in TNF[-308] and TNF [-857] regions may increase the host susceptibility to mycobacterium tuberculosis and genotyping of these regions can be used for screening of the high risk individuals

[ rapid identification of atypical mycobacterium in pulmonary tuberculosis [PTB] patients: evaluation of QUB3232 locus using the VNTR method]

Identification of atypical mycobacterium [Non tuberculosis Mycobacterium; NTM] is important because of the worldwide propagation of these organisms. Recently, molecular studies have identified the specific loci for mycobacterium species by DNA - finger printing methods, but these methods are time-consuming and expensive. In this study, in addition to hsp65 PCR-RFLP method, QUB3232 locus was evaluated for differentiation of atypical mycobacterium from mycobacterium tuberculosis complex. This study was performed on 371 pulmonary and non pulmonary specimens separated from patients with the symptoms of pulmonary tuberculosis [PTB]. After the isolation and culturing of mycobacterium strains using the Lowenstein Jensen media, biochemical tests including production of Niacin, Catalase activity, Nitrate reduction, pigment production and growth rate were performed. Drug susceptibility testing was performed by proportional method. DNA extraction was performed by phenol-chloroform method. hsp65 gene was amplified by PCR. Subsequently the amplicons were digested with three restriction enzymes namely AvaII, HphI and HpaII and electrophoresed on 3% agarose gel. QUB3232 locus was also evaluated for differentiation of atypical mycobacterium and mycobacterium tuberculosis complex. Out of 371 isolates, 32 [8.6%] were multi-drug resistant TB [MDR-TB], 184 [49.5%] were susceptible and 155 [42.5%] were non MDR [combined resistance] that 15% of MDR cases and 25% of non MDR cases were non tuberculosis mycobacterium. Out of 31 slow growing isolates, 58% were M. simiae and 19% were M. kansasii. The sensitivity of QUB3232 locus for differentiation of the atypical mycobacterium from mycobacterium tuberculosis complex was 80%. From the total of 43 NTM samples, 12 [27.9%] were rapid growing and 72% were slow growing. QUB3232 locus has the high discriminative power for differentiation of atypical mycobacterium from the mycobacterium tuberculosis complex, therefore, it can be used as a substitute for PCR-RFLP method

[Mycobacterium tuberculosis complex strains identification with spoligotyping method in patients attending to Masih Daneshvari Hospital]

Spoligotyping is a method based on 36bp Direct Repeat [DR] chromosomal loci polymorphism which is connected to one or two 35-41 bp spacer sequences. There are 94 different intra DR spacer sequences which are identified so far and only 43 of them are used as usual. Mycobacterium tuberculosis complex strains can be identified based on lacking or having these sequences. Spoligotyping test was carried out on 238 TB smear positive patients. Primary separation of mycobacterium strains was done through Petrof 4% method and Lowenstein Jensen [LJ] media. Biochemical tests such as Niacin test/Catalase activity/Nitrate reduction were done in order to identify the strains. Drug sensitivity to INH [0.2Mg/ml]/ RIF [40Mg/ml]/ STM [10Mg/ml] and ETBl [2Mg/ml] identified by proportional method and according to that, the strains were divided into three groups: sensitive, multi drug resistance [MDR] and non MDR. Then DNA was extracted by CTAB method from the positive colonies. Sequences were amplified by PCR and after denaturizing, hybridization with Streptavidine peroxidase enzyme was performed by Line reverse blot method. Radiography was done after adding the Luminoscense and membrane onto the X-ray films. Serotypes were divided into 9 groups [Beijing/ CAS1/ Haarlem / U/ T2/ T1/ EAI3/ EAI2 and CAS2]. Most of the strains were from Haarlem [27%] and CAS1 [25%] groups. Two strains were also identified in this method that belonged to Mycobacterium bovis. Spoligotyping method is an easy, rapid and sensitive test in order to identify Mycobacterium tuberculosis complex strains

[ frequency of vitamin D receptor gene polymorphisms in pulmonary tuberculosis [TB] patients and its relation with susceptibility to TB]

Many genetic studies on predisposing factors for active tuberculosis have been conducted. Study on human leukocyte antigens [HLA], vitamin D receptor [VDR], NRAMP1, mannose binding lectin [MBL], and tumor necrotizing factor [TNF] are the most studies in this field. This study was planned to identify any relationship between VDR polymorphisms [Apa I, Bsm I, Fok I and Taq I] and susceptibility to TB. This case-control study was performed on blood samples from tuberculosis cases [n=164] and controls [n=50]. DNA was extracted from white blood cells and the sequences were amplified by PCR followed by restriction digestion [PCR-RFLP technique] using specific primers and enzymes for each polymorphism. VDR polymorphisms were evaluated for two mentioned groups. Two genotypes of AbfT and AabbFfTT were the only statistically significant genotypes which had adfferent frequency between the study and control groups. Results of this study showed that genotypes of AbfT and AabbFfTT are protective factors against TB in our patients. We could not find any genotype as a predisposing factor for TB in our study group. However, other studies with larger group of samples are needed to find such a relationship

Assessment of the diagnostic value of PCR method in detection of the mycobacterium tuberculosis in urine samples

Despite health improvement, the incidence of tuberculosis has increased during recent years. In this study, the diagnostic value of PCR method was evaluated in the detection of mycobacterium tuberculosis in urine samples. In this diagnostic test study, we evaluated all urine samples gathered from inpatients and outpatients visited in Massih Daneshvari hospital between 1999 and 2005. PCR results were compared to the findings of direct smear and culture of urine samples. Totally, 509 urine samples were studied. In 2 samples, every three tests [culture, bacterioscopy and PCR] were positive; in 5 samples bacterioscopy was positive, but PCR was negative. In 19 samples, PCR was the only positive test. In 471 samples every three tests were negative. Sensitivity, specificity, positive predictive value and negative predictive value of PCR method in detecting of mycobacterium tuberculosis in urine sample was 31%, 96%, 31% and 96%, respectively. As sensitivity of PCR test is low, it is not considered to be a useful method for detecting mycobacterium tuberculosis in urine, but according to high specificity, it can be used for identifying healthy people and following up patients' response to treatment

[Study on epidemiological patterns of Mycobacterium tuberculosis by fingerprinting]

Despite the availability of effective anti tubercle chemotherapy for more than 50 years and major advances in the biology of M. tuberculosis, TB remains the leading cause of adult mortality attributable to a single pathogen. The analysis of tuberculosis and tracing of the source of infection require the ability to discriminate among Mycobacterium tuberculosis strain. In present study Restriction Fragment Length Polymorphism [RFLP] analysis was used to study the molecular epidemiology of tuberculosis in Tehran. Mycobacterium tuberculosis isolates from 292 patients with culture positive tuberculosis during 2002-2003. Extraction of bacterial DNA and DNA fingerprinting with RFLP using lS6110 as probe, was performed by standard protocols. The digested DNA is separated according to fragment size on agarose gel by electrophoresis and southern transferred on to a membrane. The DNA probe was labeled with [HRP] and hybridizing to the genomic targets. Among 292 culture M. tuberculosis, 232 [79.4%] belonged to clusters and 60 [21.6%] did not. 39 Drugs resistant M. tuberculosis isolates were examined 33%of these were identical pattern lS6110. 4.9% of the isolates represented the Beijing genotype. Based on the obtained data it appears that tuberculosis among the study population in Tehran mainly from reactivation of latent infection

Characterization of rpoB mutations in rifampin-resistant isolates of mycobacterium tuberculosis cultured from the Iranian patients

In this project we investigated the frequency of mutations within the rifampin resistance-determining region [RRDR] of rpoB gene to determine whether this region is useful for molecular detection of rifampin resistant isolates of Mycobacterium tuberculosis from Iranian patients. A set of 25 rifampin resistant and 5 randomly chosen fully susceptible M. tuberculosis complex strains obtained from sputum samples of individual patients were investigated. The M. tuberculosis H37RvT and CDC 1551 standard strains were used as controls. Using the specific primers, the entire RRDR of rpoB gene of selected samples was amplified and sequenced directly. Genetic alterations in the RRDR were present among 96.0% of isolates. The majority of rifampin resistant isolates [72.0%] showed missense mutations in the core region of rpoB that led to substitutions of amino acids at Ser-531 [60.0%], His-526 [16.0%] or Asp-516 [8.0%]. While the codon 531 has been the most common site of nucleotide substitutions worldwide, the frequencies of mutations at the codons 526 and 516 among the Iranian isolates were different from other geographical regions. Mutation at codon 533 was found at higher frequency [8%] comparing to the report from other countries. The high rate of mutations within the RRDR of the rpoB gene suggests that targeted screening of the RRDR may be feasible for the determination of rifampin resistance in clinical isolates of Mycobacterium tuberculosis from Iran

[Frequency of beijing genotype in mycobacterium tuberculosis strains isolated from tuberculosis patients in city of Mashhad]

The Beijing genotype is one of the most important strains of Mycobacterium tuberculosis involving outbreaks of tuberculosis in various parts of the world. Unsought regarding Beijing genotype in Iran, is the reason this study is undertaken in order to evaluate the frequency of this genotype in Mashhad. This descriptive study was carried out on 113 M.tuberculosis Specimens isolated from patient with pulmonary tuberculosis in hygienic centers, located at Ghaem and Imam Reza hospitals in the City of Mashhad. In this study, Beijing genotypes were detected with PCR-based method, and spoligotyping. Results were processed with descriptive statistics and CI was evaluated. Beijing genotype was detected in 8 specimens of all the 113 isolated M.tuberculosis strains [7.1%, CI 95%, 2.36-11.84]. Out of 8 isolated specimens, 5 isolates belonged to Afghan patients and 3 specimens were isolated from Iranian patients. Out of 8 patients that were infected with Beijing genotype, 2 patients were male and 6 patients were female. Although the rate of Beijing family is low in Iran, in comparison to other Asian countries, however, one needs to adopt a suitable policy in order to prevent its spread

Primary and acquired drug resistance in childhood tuberculosis

This study determined the resistance pattern of Mycobacterium tuberculosis to 4 first-line anti-tuberculosis drugs in children with pulmonary tuberculosis at the Iranian National Research Institute of Tuberculosis and Lung Diseases from 1999 to 2004. There were 350 children with positive cultures over the study period: 7 [2%] were resistant to at least one of the 4 anti-tuberculosis drugs. Primary resistance was detected in 4 cases and secondary resistance in 3 cases. Most cases [6] were among Afghan refugees. Resistance to rifampicin both in primary and secondary resistances was high, showing that children in the Islamic Republic of Iran face the threat of drug-resistant tuberculosis transmission

Genotyping of Mycobacterium tuberculosis isolates from TB patients with spoligotyping

Spoligotyping was applied to investigate the prevalence of all genotype in M. tuberculosis isolates. The associated risk factors among patients with different nationalities residing in Iran were also determined. In this analytic cross-sectional study a total of 439 patients that referred to the NRITLD, the referral tuberculosis center in Iran; have been registered during March 21[st]' 2003 to March 21[st] 2004. The isolated Mycobacterium tuberculosis strains have been characterized by performing susceptibility tests against four first-line antituberculosis drugs and were then subjected to spoligotyping characterization. T-test and chi-square were used for analysis of the data. Spoligotyping of M. tuberculosis strains resulted in 140 different patterns that divided into 9 clades. One hundred twenty two [87.1%] of these spoligotype isolates were unique and reported for the first time. The remaining 18 [12.8%] spoligotype patterns were previously reported from other geographical regions of the world. Haarlem family was most prevalent than other genotype. Interestingly, 6.3% of the strains belonged to the Beijing family. The MDR [multi drug resistance], double and triple resistance were seen in group I of evolutionary scenario. Antibiotic resistances were higher in those isolated from the Afghani patients [p<0.001]. The other risk factors such as sex and age were also contributing factors to the diseases state. The results showed that multi drug-resistance was more prevalent in bacteria isolated from Afghani TB patients residing in Iran. In addition, spread of M. tuberculosis strains belonging to the Beijing family among Iranian patients has to be considered seriously. It is also important to undertake studies to identify which factors are the most significant to consider in tuberculosis control program