Characterization of Clinical Isolates of Mycobacterium simiae Using Drug Susceptibility Tests and Molecular Analyses.

Mycobacterium simiae is an emerging nontuberculous mycobacterium (NTM) and an opportunistic pathogen which is described mainly in Asia and presents in the environment that can cause pulmonary infection. The objective of this study is to characterize M. simiae clinical isolates using mycobacterial interspersed repetitive unit variable-number tandem repeats (MIRU-VNTR) typing for the differentiation of the strains. A total of 169 clinical isolates of NTM were recovered from patients suspected of having tuberculosis (TB)-like and related infections. After isolation and identification of mycobacterial strains by conventional biochemical and PCR-based tests, M. simiae strains were confirmed using restriction fragment length polymorphism (RFLP)-based identification assay. Furthermore, drug susceptibility and MIRU-VNTR typing was performed using on the clinical isolates of M. simiae. Out of 169 NTM strains, 92 (54.4%) isolates were identified as M. simiae. Antibiotic susceptibility experiments indicated that all 92 M. simiae isolates were resistant to first line antimycobacterial agents. Moreover, 8 (8.6%) M. simiae isolates were resistant to ciprofloxacin; and 6 (6.5%) were resistant to both amikacin and kanamycin, while the remaining were susceptible to second line antimycobacterial agents. MIRU-VNTR analysis showed that the M. simiae isolates were classified in four distinct M. simiae clusters and two single types. The minimum spanning analysis revealed that the isolates were grouped in three complexes. The data suggested that MIRI-VNTR typing is useful for typing of M. simiae isolates, however, MIRU-16 locus was absolutely absent in M. simiae.

Bovis Bacillus Calmette-Guerin (BCG) infection induces exosomal miRNA release by human macrophages.

BACKGROUND: Tuberculosis (TB) remains a significant global health concern and its diagnosis is challenging due to the limitations in the specificity and sensitivity of the current diagnostic tests. Exosomes are bioactive 30-100 nm vesicles produced by most cell types and are found in almost all human body fluids. Exosomal microRNAs (miRNAs) can transfer biological information between cells and tissues and may act as potential biomarkers in many diseases. In this pilot study, we assessed the miRNA profile of exosomes released from human monocyte-derived macrophages upon infection with Mycobacterium bovis Bacillus Calmette-Guerin (BCG). METHODS: Human monocytes were obtained from the peripheral blood of three healthy subjects and driven to a monocyte-derived macrophage (MDM) phenotype using standard protocols. MDMs were infected with BCG or left uninfected as control. 72 h post-infection, exosomes were collected from the cell culture medium, RNA was isolated and RNA-seq performed. The raw reads were filtered to eliminate adaptor and primer sequences and the sequences were run against the mature human miRNA sequences available in miRBase. MicroRNAs were identified using an E value <0.01. miRNA network analysis was performed using the DIANA miRNA tool, miRDB and functional KEGG pathway analysis. RESULTS: Infection of MDMs with BCG leads to the release of several exosomal miRNAs. These included miR-1224, -1293, -425, -4467, -4732, -484, -5094, -6848-6849, -4488 and -96 all of which were predicted to target metabolism and energy production-related pathways. CONCLUSIONS: This study provides evidence for the release of specific exosomal miRNAs from BCG-infected MDMs. These exosomal miRNAs reflect host-pathogen interaction and subversion of host metabolic processes following infection.

Pharmacogenetic Study of Drugs Affecting Mycobacterium tuberculosis.

BACKGROUND: Pharmacogenetic research has led to significant progress in understanding how genetic factors influence drug response in tuberculosis (TB) treatment. One ongoing challenge is the variable occurrence of adverse drug reactions in some TB patients. Previous studies have indicated that genetic variations in the N-acetyltransferase 2 (NAT2) and solute carrier organic anion transporter family member 1B1 (SLCO1B1) genes can impact the blood concentrations of the first-line anti-TB drugs isoniazid (INH) and rifampicin (RIF), respectively. This study aimed to investigate the influence of pharmacogenetic markers in the NAT2 and SLCO1B1 genes on TB treatment outcomes using whole-exome sequencing (WES) analysis. METHODS: DNA samples were collected from 30 healthy Iranian adults aged 18-40 years. The allelic frequencies of single-nucleotide polymorphisms (SNPs) in the NAT2 and SLCO1B1 genes were determined through WES. RESULTS: Seven frequent SNPs were identified in the NAT2 gene (rs1041983, rs1801280, rs1799929, rs1799930, rs1208, rs1799931, rs2552), along with 16 frequent SNPs in the SLCO1B1 gene (rs2306283, rs11045818, rs11045819, rs4149056, rs4149057, rs2291075, rs201722521, rs11045852, rs11045854, rs756393362, rs11045859, rs74064211, rs201556175, rs34671512, rs71581985, rs4149085). CONCLUSION: Genetic variations in NAT2 and SLCO1B1 can affect the metabolism of INH and RIF, respectively. A better understanding of the pharmacogenetic profile in the study population may facilitate the design of more personalized and effective TB treatment strategies. Further research is needed to directly correlate these genetic markers with clinical outcomes in TB patients.

The Role of Efflux Pumps transporter in Multi-drug Resistant Tuberculosis: Mycobacterial memberane protein(MmpL5).

BACKGROUND: The overexpression of efflux pumps (Eps) was reported to contribute to multidrug resistant tuberculosis (MDR-TB). Increases in Eps that expel structurally unrelated drugs contribute to reduced susceptibility by decreasing the intracellular concentration of antibiotics. In the present study, an association of mycobacterial membrane protein (MmpS5-MmpL5) Ep and its gene regulator (Rv0678) was investigated in MDR-tuberculosis isolates. METHODS: MTB strains were isolated from patients at two different intervals, i.e., once when they had persistent symptoms despite 3-15 ≥ months of treatment and once when they had started new combination therapy ≥2-3 months. Sputum specimens were subjected to Xpert MTB/rifampicin test and then further susceptibility testing using proportional method and multiplex polymerase chain reaction (PCR) were performed on them. The isolates were characterized using both 16S-23S RNA and hsp65 genes spacer (PCR-restriction fragment length polymorphism). Whole-genome sequencing (WGS) was investigated on two isolates from culture-positive specimen per patient. The protein structure was simulated using the SWISS-MODEL. The input format used for this web server was FASTA (amino acid sequence). Protein structure was also analysis using Ramachandran plot. RESULTS: WGS documented deletion, insertion, and substitution in transmembrane transport protein MmpL5 (Rv0676) of Eps. Majority of the studied isolates (n = 12; 92.3%) showed a unique deletion mutation at three positions: (a) from amino acid number 771 (isoleucine) to 776 (valine), (b) from amino acid number 785 (valine) to 793 (histidine), and (c) from amino acid number 798 (leucine) to 806 (glycine)." One isolate (7.6%) had no deletion mutation. In all isolates (n = 13; 100%), a large insertion mutation consisting of 94 amino acid was observed "from amino acid number 846 (isoleucine) to amino acid number 939 (leucine)". Thirty-eight substitutions in Rv0676 were detected, of which 92.3% were identical in the studied isolates. WGS of mycobacterial membrane proteins (MmpS5; Rv0677) and its gene regulator (Rv0678) documented no deletion, insertion, and substitution. No differences were observed between MmpS5-MmpL5 and its gene regulator in isolates that were collected at different intervals. CONCLUSIONS: Significant genetic mutation like insertion, deletion, and substitution within transmembrane transport protein MmpL5 (Rv0676) can change the functional balance of Eps and cause a reduction in drug susceptibility. This is the first report documenting a unique amino acid mutation (insertion and deletion ≥4-94) in Rv0676 among drug-resistant MTB. We suggest the changes in Mmpl5 (Rv0676) might occurred due to in-vivo sub-therapeutic drug stress within the host cell. Changes in MmpL5 are stable and detected through subsequent culture-positive specimens.

Typing of Pneumocystis jirovecii isolates from Iranian immunosuppressed patients based on the Internal Transcribed Spacer (ITS) region of the rRNA gene.

Since there have been no published molecular studies of Pneumocystis jirovecii isolates from Iranian patients, we investigated the genotypes of such isolates recovered from HIV-infected patients, those undergoing cancer chemotherapy and patients with chronic obstructive pulmonary disease (COPD). P. jirovecii typing, based on ITS1 and ITS2 sequence analysis, was performed on 34 isolates from Iranian immunosuppressed patients. In total, 44 genotypes were detected of which relative to ITS1, eight known genotypes (A, B, C, E, G, H, N and O) and one novel sequence were noted. Eight known genotypes (b, c, e, g, h, i, j and n) were also found with ITS2. The most frequent ITS1 and ITS2 genotypes were E (21/44, 47.7%) and g (22/44, 50%), respectively. From determined haplotypes, the four most frequent ones were Eg (11/44, 25%), Gg (5/44, 11.3%), Gi (4/44, 9.1%), Ei (3/44, 6.8%), and Hg (3/44, 6.8%). Two novel haplotypes (Hb and Hi) were also identified, along with mixed infections as seven (20.5%) patients were found to have more than one haplotype. It is suggested that novel haplotypes in Iranian patients may be generated through sexual recombination within the host.

Identification of seven types of pili in Mycobacterium tuberculosis: Using atomic force microscopy.

Background: Pili are polymeric, hydrophobic, proteinaceous structures generally composed of a major repeating subunit called pilin and, in some cases, a minor tip-associated adhesin subunit. Pili are involved in many virulence-associated functions, such as biofilm formation, adherence, and colonization of mucosal surfaces. Methods: Mycobacterium tuberculosis (MTB) strains were isolated from clinically and laboratory-confirmed cases of tuberculosis (TB). The TB isolates were subjected to the Xpert MTB/rifampicin test and then, further susceptibility testing was performed on them against first- and second-line drugs using proportional methods. Thereafter, the selected isolates were subculture in Dubos Tween-albumin liquid culture medium, and at their exponential growth phase (OD600 = 0.05 (5 × 106 colony-forming unit/mL), cells were observed under atomic force microscopy (AFM). For each isolate, 15-20 steel sample packs were prepared and observed under AFM. Here, the data presented are the result of average observation. Results: Under AFM, seven different types of pili were detected, out of which four types, i.e., Type III, Type IV secretion pili, and Type IV-like pili, curli-like pili (MTP) were similar to reported pili in Gram-negative and Gram-positive bacteria. Whereas the other three forms, i.e., Type V (relief funnel pili), Type VI (adhesion tapering), and Type VII (adhesion flap pili), were newly identified and named according to their appearance. Both Types of IV pili were detected in all clinical isolates irrespective of their susceptibility patterns, although significant differences were observed from the side of their protruding. Type Curli pili is similar in appearance in all clinical isolates. Types VI and VII were detected only in extensively drug-resistant and totally drug-resistant-TB isolates (100%). The Type III pili (secretion needle pili) was present in both susceptible- and drug-resistant bacilli, although in drug-resistant strains, we found a considerable difference in their length (50 µ ±10 nm in length) and sometimes, they also had tapering at end. The Type V pili was seen in susceptible isolates but it was at the resting stage (100%; lying aside of cell wall) whereas in drug-resistant isolates, they were getting apart from the cell wall of bacilli with a clear tapering or funnel shape structure. Conclusion: The results of this study highlight the importance of new types of pili expressions in respect of susceptibility patterns in TB. The identified new types of pili would be promising approaches for the treatment and prevention of drug-resistant TB, which needs further investigation.

Molecular Dynamic Simulations and Molecular Docking as a Potential Way for Designed New Inhibitor Drug without Resistance.

Mycobacterium tuberculosis is the cause of tuberculosis in humans and is responsible for more than 2 million deaths per year. Despite the development of anti-tuberculosis drugs (Isoniazid, Rifampicin, Ethambutol, pyrazinamide, streptomycin, etc.) and the TB vaccine, this disease has claimed the lives of many people around the world. Drug resistance in this disease is increasing day by day. Conventional methods for discovering and developing drugs are usually time-consuming and expensive. Therefore, a better method is needed to identify, design, and manufacture TB drugs without drug resistance. Bioinformatics applications in obtaining new drugs at the structural level include studies of the mechanism of drug resistance, detection of drug interactions, and prediction of mutant protein structure. In the present study, computer-based approaches including molecular dynamics simulation and molecular docking as a novel and efficient method for the identification and investigation of new cases as well as the investigation of mutated proteins and compounds will be examined .

Effect of COVID-19 pandemic on incidence of mycobacterial diseases among suspected tuberculosis pulmonary patients in Tehran, Iran.

Background: Recent pandemic of coronavirus SARS-CoV-2 (COVID-19) caused limitations in the country's strategies to fight against mycobacterial infections. The aim of this study was to compare the suspected tuberculosis (TB) pulmonary patients before and during the COVID-19 pandemic (January 2018-December 2021) who were referred to the National Reference TB Laboratory (NRL TB), Tehran, Iran. The mycobacterial isolated strains were identified and compared with previous data. Methods: A total of 16,899 clinical samples collected from 7041 suspected pulmonary TB patients were received from 2018 to 2021. Primary isolation of Mycobacterium isolates was done on Löwenstein-Jensen medium. Then, the DNA was extracted from acid-fast bacillus culture-positive samples and identification was performed by IS6110, Hsp65, and 16S-23S rRNA genes using polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism, and nested PCR methods. Results: A total of 11679 specimens (69.1%) from 4866 suspected TB patients were collected in 2018-2019 and 5220 specimens (30.8%; from 2175 patients) in 2020-2021. Out of 11679 specimens, 2046 samples that belong to 852 patients were infected with Mycobacterium tuberculosis, and the remaining were non-TB Mycobacterium (NTM) species (n = 244) isolated from 102 patients. The cultures for 12894 specimens were either negative (76.3%) or contaminated (845/16899; 5%). A comparison of the total number of patients who were referred for diagnosis and treatment (954/666 patients, P > 0.05) showed a 30.1% reduction during the COVID-19 pandemic. Although, with these low number of patients, the significant increases of NTM species (P < 0.05) among suspected TB pulmonary patients were observed. Besides, new species of NTM, for example, Mycobacterium peregrinum and Mycobacterium montefiorense, were detected. For the past 20 years, these two species were not reported from pulmonary patients in Iran. Conclusions: During the pandemic of COVID-19, the TB diagnosis network became irregular, as a consequence, many patients could not reach the treatment center, and this could increase the circulation of mycobacterial diseases (TB and NTM). The study shows the emergence of new opportunistic NTM species also.

Correlation of single-nucleotide polymorphism at interferon-gamma R1 (at Position - 56) in positive purified protein derivative health workers with COVID-19 infection.

Background: The aim of the present study was to investigate the susceptibility of purified protein derivative (PPD) plus health-care workers to SARS-CoV-2 (COVID-19). For this reason, single-nucleotide polymorphism (SNP) of interferon-gamma (IFN-γ) gene at position +2109 and IFN-γ receptor 1 (R1) at position -56 was assessed in PPD plus group before and after COVID-19 infection (2017-2018; 2020-2021). Methods: The selected study cases (n = 100) that were working in tuberculosis (TB) unite (5-10 years) with PPD positivity >15 mm (16-20 mm) were included in this investigation. Sampling was done twice, once before and after the COVID-19 pandemic. Group A contains 50 samples collected from the GenBank TB laboratory that belong to TB staff before the pandemic (2017-2018). The other sample (Group B; 2021) was collected from the same unite during the COVID-19 pandemic. The SNP in the IFN-γ gene (+2109; 670 bp) and IFN-γ R 1 (-56; 366 bp) was performed using a specific primer and the polymerase chain reaction products were digested using restriction enzyme Fau I and Bts I, respectively. Statistical analyses were used to obtain the frequency of alleles among all studied cases. The confidence intervals (CIs) and t-test were calculated using the SPSS and GraphPad Prism software. Results: In overall, the most frequent genotype in Group A was AA (41/50; 82%) and Group B was 76% (38/50) in position + 2109 (odds ratio [OR] = 0.69, 95% CI, 0.26-1.83, and P = 0.46). Although in position -56, the most frequent genotype in Group A was TT (35/50; 70%) which significantly was than Group B TT (15/50; 30%) (OR = 0.184, 95% CI, 0.78-0.43, and P = 0.00). The frequency of allele A was more in both groups at position + 2109 (OR = 0.815, 95% CI, 0.23-2.86, and P = 0.75), whereas the dominate allele at position -56 was T in Group A (OR = 1.37, 95% CI, 0.62-3.02, and P = 0.42). Conclusion: No significant differences were observed in + 2109 in genotype among Group A and B. The main differences were seen in IFN-γ R1 at position (-56) between Group A and B. Hence, the IFN-γ R1 may play important role in COVID-19 infection. However, more study is needed to clear the IFN-γ correlation to COVID-19 infection.

Assessment of Cystic Fibrosis Distribution Based on Air Pollution by Geographical Information System (GIS).

Background: It is widely accepted that concerns have been recently raised regarding the impact of air pollution on the health of children with cystic fibrosis (CF). Air pollution probably affects the exacerbation of CF and its laboratory findings. On the other hand, the World Health Organization (WHO) has asked all countries to update their data and reports on the distribution and prevalence of CF in different areas. The purpose of the present study was to investigate the distribution and prevalence of CF based on the levels of atmospheric pollutants, such as PM10, PM2.5, SO2, NO2, CO, and O3 in 22 zones of Tehran, and to report the abnormal laboratory findings that might indicate the exacerbation of CF. Materials and Methods: The studied statistical population included children with CF referred to Masih Daneshvari Hospital from 2003 to 2020. Demographic data, location of living area, and laboratory findings were extracted from patient records. The geographic information system (GIS) was applied to indicate the distribution and dispersion of the disease. The information related to air pollutants was collected from all stations in Tehran during the studied period by the Department of Environment of Tehran Province, and the average levels were used for final reporting. Results: The analysis results on 287 CF patients demonstrated that the risk of disease exacerbation significantly increased by the presence of air pollutants. In areas with multiple air pollutants, more laboratory findings were observed to be abnormal, and the lower survival rate for patients with CF was recorded. Investigating the CF distribution pattern based on climatic layers and above mean sea level (AMSL) indicated that distribution of the disease was higher in dry areas with lower AMSL and the higher volume of the atmospheric pollutants, which were primarily centralized in southern and central Tehran. Conclusion: Environmental factors, such as air pollution, can be considered vital parameters, along with high-risk factors, such as pure and integrated race, migration, and mutation, influencing the prevalence and exacerbation of CF symptoms. Considering the higher prevalence of CF in deprived areas of Tehran, households' cultural and economic level appears to be a factor in the lack of diagnostic screening and prevention of CF in these areas. On the other hand, continuous monitoring of the air pollution caused by traffic and giving warnings to CF patients and their parents is particularly important.

The role of interferon-gamma and interferon-gamma receptor in tuberculosis and nontuberculous mycobacterial infections.

Mycobacterium tuberculosis (Mtb) and nontuberculous mycobacteria (NTM) remain the leading causes of lung disease and mortality worldwide. Interferon-gamma (IFN-γ) and its receptor (IFN-γR) play a key role in mediating immunity against Mtb and NTM. This study was conducted as a systematic review; all information was collected from databases such as: PubMed, Scopus, Medline, SID, and medical databases. Finally, all the collected data were reviewed, and all content was categorized briefly. There is growing evidence that IFN-γ plays an important role in host defense against these two intracellular pathogens by activating macrophages. In addition, IFN-γ has been shown to be an integral part of various antibacterial methods such as granuloma formation and phagosome-lysosome fusion, both of which lead to the death of intracellular Mycobacterium. As a result, its absence is associated with overgrowth of intracellular pathogens and disease caused by Mtb or Mycobacterium nontuberculosis. We also look at the role of IFN-γR in Mtb or NTM because IFN-γ acts through IFN-γR. Finally, we introduce new approaches to the treatment of M. tuberculosis complex (MTC) and NTM disease, such as cell and gene-based therapies that work by modulating IFN-γ and IFN-γR.

An Overview of Genetic Information of Latent Mycobacterium tuberculosis.

Mycobacterium tuberculosis has infected more than two billion individuals worldwide, of whom 5%-10% have clinically active disease and 90%-95% remain in the latent stage with a reservoir of viable bacteria in the macrophages for extended periods of time. The tubercle bacilli at this stage are usually called dormant, non-viable, and/or non-culturable microorganisms. The patients with latent bacilli will not have clinical pictures and are not infectious. The infections in about 2%-23% of the patients with latent status become reactivated for various reasons such as cancer, human immunodeficiency virus infection, diabetes, and/or aging. Many studies have examined the mechanisms involved in the latent state of Mycobacterium and showed that latency modified the expression of many genes. Therefore, several mechanisms will change in this bacterium. Hence, this study aimed to briefly examine the genes involved in the latent state as well as the changes that are caused by Mycobacterium tuberculosis. The study also evaluated the relationship between the functions of these genes.

The most predominant spoligopatterns of Mycobacterium tuberculosis isolates among Iranian, Afghan-immigrant, Pakistani and Turkish tuberculosis patients: a comparative analysis.

BACKGROUND: The aim of this study was to identify prevalent spoligopatterns of Mycobacterium tuberculosis (MTB) strains within Iran and its neighboring countries. METHODS: The spacer oligonucleotide typing (spoligotyping) was performed on 1,742 MTB strains isolated from Iranian and Afghan patients. Subsequently, these results were compared with published results of Pakistan and Turkey. RESULTS: Based on the international spoligotyping database T, Central Asian Strain (CAS), East African-Indian and Latin American and Mediterranean families were prevalent in the region. T was dominant amongIranian (32.3%) and Turkish (36.5%), whereas CAS was more prevalent among Pakistani (61.3%) and Afghan-immigrant (27.4%) TB patients. CONCLUSION: The majority of strains isolated from Pakistan and Afghanistan belonged to ancient MTB genotypes (principal genetic group, PGG, I). In contrast, in Turkey there was prevalence of modern TB (PGG II and III), whereas in Iran, the prevalence of ancient and modern TB was almost equal. Therefore, Iran may be considered as the connecting geographical location between ancient and modern TB.

Evaluating the role of vitamin D receptor polymorphisms on susceptibility to tuberculosis among iranian patients: a case-control study.

Many genetic studies recently have focused on HLA, VDR, NRAMP1, MBL, TNF-a, and their relationships with susceptibility to diseases such as pulmonary tuberculosis. Some studies showed predisposing and protective roles for VDR polymorphisms in pulmonary tuberculosis. Through a case-control study, blood samples were taken from tuberculosis case (n= 164) and control (n= 50) groups. DNA was extracted from white blood cells by PCR-RFLP technique using special primers and enzymes for each polymorphism. VDR polymorphisms are known as ApaI, BsmI, FokI, and Taq I which were evaluated within the two mentioned groups. Combined genotypes AbfT and AabbFfTT were the only statistically significant factors which protected people against pulmonary TB in this study. Two mentioned genotypes were protective factors against TB and this study could not find any predisposing genotype to TB. More study is requested on this matter.

Human genetic background in susceptibility to tuberculosis.

Tuberculosis (TB), especially in developing countries, is a major threat to human health. The pathogenesis of TB remains poorly understood, and <5%-10% of individuals infected with Mycobacterium tuberculosis (MTB) will develop clinical disease. The human genetic factors contributing to susceptibility or resistance to TB pathogenesis have been investigated by high-throughput and low-throughput association studies. Genetic polymorphisms of several genes including TLR, IGRM, VDR, ASAP1, AGMO, FOXP1, and UBLCP1 effect on the disease phenotype and also the outcome of TB treatment. Recently, microRNAs (miRNAs), which negatively regulated gene expression at the posttranscriptionally level, have gained increasing attention due to their altered expression in various human diseases, including some infections. They are crucial posttranscriptional regulators of immune response in both innate and adaptive immunity. It has been established in recent studies that the host immune response against MTB is regulated by many miRNAs, most of which are induced by MTB infection. Moreover, differential expression of miRNAs in TB patients may help distinguish between TB patients and healthy individuals or latent TB. In this review, we summarize and discuss the literature and highlight the role of selected single nucleotide polymorphisms and miRNAs that have been associated with TB infection.

Prevalence of Mycobacterium abscessus among the Patients with Nontuberculous Mycobacteria.

BACKGROUND: Considering the importance of the increasing incidence of non-tuberculous mycobacteria, especially Mycobacterium abscessus worldwide, we conducted a study to evaluate the incidence of these diseases in our area. The aim of this study was to evaluate the prevalence of M. abscessus in patients with non-tuberculous mycobacteria. METHODS: This descriptive study was performed on 18,083 samples isolated from patients with non-tuberculous mycobacteria during 2011-2017 at the Mycobacteriology Research Center (MRC), Tehran, Iran. To identify the Mycobacterium species, a 439 bp fragment of the IS6110 gene was first amplified using primers TB1 and TB2. Samples with a negative polymerase chain reaction (PCR) result were analyzed to investigate non-tuberculosis mycobacteria (NTM), especially M. abscessus using the RFLP method. RESULTS: Of the 18,083 samples, 5513 (30.49%, 95% CI, 12.95) strains of Complex Tuberculosis and 236 (1.31%, 95% CI, 1.84) strains of NTM were identified. The mean age of the patients with NTM was 18 years, and most of them were male. The most commonly identified species in this study were M. abscessus type Ι 32 (13.56%, 95% CI, 18.36) and M. abscessus type II 13 (5.51%, 95%CI, 20.04). CONCLUSION: In this study, we observed a high prevalence of Mycobacterium abscessus type 1 in patients. As the treatment protocol for non-TB mycobacteria is different from M. abscessus complex, the diagnosis of these species as soon as possible will be significant for physicians.

Differences in cell wall thickness between resistant and nonresistant strains of Mycobacterium tuberculosis: using transmission electron microscopy.

The ultrastructure of the cell wall of extensively drug-resistant (XDR), multidrug-resistant (MDR) and susceptible tuberculosis (TB) bacilli was viewed under transmission electron microscopy (TEM). Under the TEM, marked differences were observed in the thickness of their cell wall: 20.2 +/-1.5 and 17.1 +/-1.03 nm for the XDR and MDR TB bacilli, respectively, and 15.6 +/- 1.3 nm for the susceptible isolates (p < 0.05). In MDR bacilli, thickening of the cell wall was observed in the intermediate electron-transparent layer (ETL) and outer electron-opaque layer, whereas in XDR TB cells the basal peptidoglycan layer was denser and almost fused with the ETL. Five to seven percent of XDR TB bacilli had the appearance of the stationary phase with cell wall thickness ranging from 21 to 26 nm (p < 0.001). Information provided in this study is of significant importance in terms of drug selection for effective treatment of resistant strains.

Colorimetric detection of multidrug-resistant or extensively drug-resistant tuberculosis by use of malachite green indicator dye.

The malachite green microtube (MGMT) susceptibility assay was performed directly on sputum specimens (n = 80) and indirectly on Mycobacterium tuberculosis clinical isolates (n = 60). The technique is based on the malachite green dye, which changes color in response to M. tuberculosis growth. The MGMT assay is simple and rapid and does not require expensive instruments.

The recent-transmission of Mycobacterium tuberculosis strains among Iranian and Afghan relapse cases: a DNA-fingerprinting using RFLP and spoligotyping.

BACKGROUND: Relapse of tuberculosis (TB) may develop as the result of reactivation of the endogenous primary infection, or as a result of a exogenous reinfection. This survey evaluated the rate of reactivation versus recent transmission among Iranian and Afghan relapse cases. METHODS: The sputum specimens were digested, examined microscopically for acid-fast bacilli, and inoculated into Löwenstein-Jensen slants by standard procedures. Thereafter, the susceptibility and identification tests were performed on culture positive specimens. Subsequently, the strains that were identified as Mycobacterium tuberculosis (258 isolates) were subjected to IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping. Additional patient's information was collected for further epidemiological analysis. Patients whose isolates had identical genotyping patterns were considered a cluster with recent transmission episode. RESULTS: Out of 258 available isolates, 72(28%) had multi-drug resistant (MDR-TB) in ratio and 42 (16.2%) had other resistant. Notably, 38 of MDR-TB cases (52%) were isolated from Afghan patients. By IS6110-RFLP typing method, 65 patients (25%) were clustered in 29 clusters. In cluster cases, the intra-community transmissions between Iranian and Afghan patients were 41%. All MDR-TB patients in clusters had either Haarlem I or Beijing characteristic. The risk factors like sex, family history, close contact, living condition, PPD test result and site of TB infection were not associated with clustering. Although, the MDR-TB strains were more frequent in non-cluster cases (31%) than cluster one(18%) (P < 0.05). Majority of M. tuberculosis strains isolated from non-cluster cases were belong to EAI3 (51; 30%) and CASI(32;18.6%) superfamilies. CONCLUSION: During the studied period, reactivation of a previous infection remain the more probable cause of recurrence. Although, the evidence of intra- community transmission between Iranian and Afghan TB cases, highlighted the impact of afghan immigrants in national tuberculosis control program (NTP) of Iran.

Drug-resistant Mycobacterium tuberculosis: Epidemiology and role of morphological alterations.

In this brief review, various forms of drug-resistant tuberculosis (DR-TB) are discussed. The focus of the study is to highlight morphological alterations of DR-TB bacilli at the cellular level.