Evaluation of miR-let-7f, miR-125a, and miR-125b expression levels in sputum and serum samples of Iranians and Afghans with pulmonary tuberculosis.

Background and Objectives: The role of microRNAs (miRNAs) in tuberculosis infection is well established. As microRNAs are able to change expression profiles according to different conditions, they can be useful biomarkers. Iranians and Afghans with tuberculosis were studied for three immune-related miRNAs (miR-let-7f, miR-125a, and miR-125b). Materials and Methods: A total of 60 Iranian and Afghan patients with active pulmonary TB were enrolled in the Pulmonary Department of the Pasteur Institute of Iran. Serum and sputum samples were collected simultaneously from all participants. A Real-time PCR was conducted to detect differentially expressed miRNAs. Results: Iranian (P<0.0001) and Afghan (P<0.0001) serum samples and Afghan (P<0.0001) sputum samples overexpressed miR-125a, whereas Iranian sputum samples showed downregulation (P=0.0039). In both Iranian (P<0.0001; P=0.0007) and Afghan (P<0.0001; P<0.0001) serum and sputum samples, miR-125b was overexpressed. Furthermore, miR-let-7f down-regulation was observed in serum and sputum samples (P<0.0001), whereas Iranian sputum samples had no statistically significant differences (P=0.348). Conclusion: Overexpression of miR-125a and miR-125b has been detected in Iranian and Afghan samples. In both races, miR-let-7f downregulation has been confirmed. Identification of miRNA profiles under different conditions opens the door to evaluating potential new biomarkers for diagnosis, disease monitoring, and therapeutic markers in TB infection.

An effective nano drug delivery and combination therapy for the treatment of Tuberculosis.

Drug resistance in tuberculosis is exacerbating the threat this disease is posing to human beings. Antibiotics that were once effective against the causative agent, Mycobacterium tuberculosis (Mtb), are now no longer usable against multi- and extensively drug-resistant strains of this pathogen. To address this issue, new drug combinations and novel methods for targeted drug delivery could be of considerable value. In addition, studies have shown that the use of the antidepressant drug fluoxetine, a serotonin reuptake inhibitor, can be useful in the treatment of infectious diseases, including bacterial infections. In this study, an isoniazid and fluoxetine-conjugated multi-walled carbon nanotube nanofluid were designed to increase drug delivery efficiency alongside eliminating drug resistance in vitro. The prepared nanofluid was tested against Mtb. Expression levels of inhA and katG mRNAs were detected by Real-time PCR. ELISA was applied to measure levels of cytokine secretion (TNF-α, and IL-6) from infected macrophages treated with the nano delivery system. The results showed that these nano-drug delivery systems are effective for fluoxetine at far lower doses than for free drugs. Fluoxetine also has an additive effect on the effect of isoniazid, and their concomitant use in the delivery system can have significant effects in treating infection of all clinical strains of Mtb. In addition, it was found that the expression of isoniazid resistance genes, including inhA, katG, and the secretion of cytokines TNFα and IL6 under the influence of this drug delivery system is well regulated. It was shown that the drug conjugation can improve the antibacterial activity of them in all strains and these two drugs have an additive effect on each other both in free and conjugated forms. This nano-drug delivery method combined with host targeted molecules could be a game-changer in the development of a new generation of antibiotics that have high therapeutic efficiencies, low side effects, and the potential to overcome the problem of drug resistance.

Gene Expression and In Vitro Pharmacogenetic Studies of Dopamine and Serotonin Gene Receptors in Tuberculosis.

BACKGROUND: Dopamine and serotonin receptors are present in lymphocytes, macrophages, and neutrophils, and have a mediating role in the immune system to respond to infections, including bacterial tuberculosis. MATERIALS AND METHODS: In this study, at first, the changes in the expression pattern of 5 dopamine and 2 serotonin (5HTR2B & 5HTR2C) gene receptors were examined in the two groups of healthy and Tuberculosis patients using Real-Time PCR. Then pharmacogenetic studies aimed to induce autophagy on a lung monocyte cell line (THP1) infected with the standard strain of Mycobacterium tuberculosis (H37RV) were performed. Stimulation of the pro-inflammatory pathway by secreting cytokines before and after drug efficacy was investigated. RESULTS: According to the result, dopamine receptor 2 genes showed decreased expression in patients with tuberculosis compared to normal individuals, and serotonin receptor genes showed increased expression. Additionally, with the effects of Bromocriptine and Fluoxetine, pro-inflammatory pathways were activated in macrophages infected with H37RV, and ELISA results showed that the levels of IL6 and TNFα secreted in these cells were significantly increased. CONCLUSION: According to the results, these receptors agonists or antagonists can activate the autophagy pathway to kill TB bacteria.

The regulation of Niemann-Pick C1-Like 1 (NPC1L1) gene expression in opposite direction by Bacteroides spp. and related outer membrane vesicles in Caco-2 cell line.

PURPOSE: The intestine has substantial role in cholesterol homeostasis due to the presence of various cholesterol transporters and gut microbiota. Bacteroides spp. are important members of gut microbiota that employ outer membrane vesicles (OMVs) to interact with host. In this regard, we evaluated the effect of Bacteroides fragilis, Bacteroides thetaiotaomicron and related OMVs on the gene expression of important cholesterol transporters, Niemann-Pick C1-Like 1 (NPC1L1), ATP-binding cassette (ABCA1), and liver X receptors (LXRs) in Caco-2 cells. METHODS: OMVs were isolated from overnight brain heart infusion (BHI) broth of bacterial standard strains using deoxycholate and assessed by Scanning electron microscopy (SEM). The relative change in genes expression was assessed by Quantitative reverse transcription PCR (RT-qPCR) based on SYBR Green and 2-∆∆ct method in Caco-2 cells that were treated with bacteria and OMVs. Data were statistically analyzed with GraphPad Prism software. Finally, pathway enrichment based on the studied genes was performed using Cytoscape plugin ClueGO. RESULTS: B. fragilis (P value = 0.002) and B. thetaiotaomicron (P value = 0.001) significantly reduced NPC1L1 gene expression in Caco-2 cells. Interestingly, NPC1L1 transcripts were significantly increased by both OMVs(P value = 0.04) (P value = 0.01). Also, LXRß was significantly down regulated by B. thetaiotaomicron (P value = 0.02). ClueGO analysis on the studied genes demonstrated several functional groups which involve in lipid and cholesterol metabolism. CONCLUSION: The opposite effect of B. fragilis, B. thetaiotaomicron and related OMVs on the NPC1L1 gene expression was observed in Caco-2 cells. Interestingly, these effects partially were in line with the alternation of LXRs expression. However, based on pathway enrichment analysis, further molecular investigations are required to elaborate in details the specific association between Bacteroides spp. and OMVs with regulation of cholesterol signaling pathways including cholesterol transport, lipid storage, lipid homeostasis and cholesterol homeostasis.

The inter-talk between Mycobacterium tuberculosis and the epigenetic mechanisms.

Epigenetics regulate gene function without any alteration in the DNA sequence. The epigenetics represent one of the most important regulators in different cellular processes and have initially been developed in microorganisms as a protective strategy. The evaluation of the epigenetic mechanisms is also important in achieving an efficient control strategy in tuberculosis (TB). TB is one of the most significant epidemiological concerns in human history. Despite several in vivo and in vitro studies that have evaluated different epigenetic modifications in TB, many aspects of the association between epigenetics and TB are not fully understood. The current paper is aimed at reviewing our knowledge on histone modifications and DNA methylation modifications, as well as miRNAs regulation in TB.

A new diagnostic tool for rapid and accurate detection of Mycobacterium tuberculosis.

Mycobacterium tuberculosis, acid fast bacilli from the family of Mycobacteriaceae, is the causative agent of most cases of tuberculosis. Tuberculosis, as a communicable disease, remains a serious public health threat, killing more than one million people globally every year. Primary diagnosis of tuberculosis bacilli (TB) relies mainly on microscopic detection of acid fast bacilli (AFB), but the method suffers from low sensitivity and the results largely depend on the technician's skill. New diagnostic tools are necessary to be introduced for rapid and accurate detection of the bacilli in sputum samples. We, in collaboration with Anda Biologicals, have developed a new platform, named as "Patho-tb", for rapid detection of AFB with high sensitivity and with low dependence on human skills. Evaluation of Patho-tb test performance was done in two settings: (1) primary field study conducted using 38 sputa from high TB prevalence area of Iran (Zabol city near to the Afghanistan border), and (2) main study conducted using 476 sputa from Tehran, capital of Iran. Patho-tb was applied for processed sputum samples in parallel with routine diagnostic methods (including AFB microscopy, culture and PCR). All test results were compared to final clinical diagnostic state of an individual and diagnostic sensitivity (DSe), specificity, positive predictive value, negative predictive value and accuracy of each test results were calculated using standard formulations. Analytical sensitivity and specificity of the Patho-tb test were also determined. Calculated values for five above mentioned parameters are as follows: for field study: AFB (DSe: 29.6, DSp: 81.8, PPV: 80, NPV: 23.1, AC: 44.7), Patho-tb (DSe: 63, DSp: 72.7, PPV: 85, NPV: 44.4, AC: 65.8), and for main study: AFB (DSe: 86.1, DSp: 99.4, PPV: 98.5, NPV: 93.9, AC: 95.2), Patho-tb (DSe: 97.4, DSp: 92.9, PPV: 86.5, NPV: 98.7, AC: 94.3). Reproducibility of Patho-tb test results were near to 100% (Cohen's kappa value between 0.85 and 1). The detection limit of Patho-tb test with 100% positivity rate was 3 × 103 cells/ml of sputum. In the field study, Patho-tb test was 33.4% more sensitive than AFB microscopy, while the improvement was only 11.3% during the main study. Patho-tb results are easy to interpret and the test can be merged with other screening tests, like AFB. Totally, Patho-tb test alone or in conjunction with AFB microscopy is a useful screening tool for TB detection especially in poor geographical lab conditions.

Sub-minimum inhibitory concentration of rifampin: a potential risk factor for resuscitation of Mycobacterium tuberculosis.

BACKGROUND: Mycobacterium tuberculosis possesses five resuscitation-promoting factors, Rpf A to E, which are required for the resuscitation of dormancy in mycobacteria. This study explores the transcriptional profile of all five rpfs of M. tuberculosis, in response to sub-MIC concentration of rifampin, in multidrug and mono-rifampin resistant clinical isolates. METHODS: Thirteen multidrug and two rifampin mono resistant clinical isolates were analyzed. Drug susceptibility testing and determination of MIC were performed. The relative expression of rpfs was measured, by real-time quantitative PCR. RESULTS: A significant upregulation of relative expression (p < 0.05) was observed, as follows: 7/15(46.66%); 5/15(33.33%); 9/15(60%); 10/15(66.66%) and 9/15(60%) in rpfA, rpfB, rpfC, rpfD and rpfE, respectively. CONCLUSION: Our results showed that the rpfs could be overexpressed in some extent in the presence of sub-MIC concentration of rifampin in multidrug and mono drug resistant M. tuberculosis. These results highlight the potential risk of sub-MIC rifampin concentrations, as a risk factor for tuberculosis reactivation.

Distribution of non-tuberculosis mycobacteria strains from suspected tuberculosis patients by heat shock protein 65 PCR-RFLP.

The genus Mycobacterium contains more than 150 species. Non-tuberculosis mycobacteria (NTM) often cause extrapulmonary and pulmonary disease. Mycobacteria detection at species level is necessary and provides useful information on epidemiology and facilitates successful treatment of patients. This retrospective study aimed to determine the incidence of the NTM isolates and Mycobacterium tuberculosis (Mtb) in clinical specimens collected from Iranian patients during February 2011-December 2013, by PCR-restriction fragment length polymorphism analysis (PRA) of the hsp65 gene. We applied conventional biochemical test and hsp65-PRA identification assay to identify species of mycobacteria in specimens from patients suspected of having mycobacterial isolates. This method was a sensitive, specific and effective assay for detecting mycobacterial species and had a 100% sensitivity and specificity for Mtb and Mycobacterium avium complex (MAC) species. Using PRA for 380 mycobacterial selected isolates, including 317 Mtb, four Mycobacterium bovis and of the 59 clinical isolates, the most commonly identified organism was Mycobacterium kansasii (35.6%), followed by Mycobacterium simiae (16.9%), Mycobacterium gordonae (16.9%), Mycobacterium fortuitum (5.1%), Mycobacterium intracellulare (5.1%), Mycobacterium avium (5.1%), Mycobacterium scrofulaceum (3.4%), Mycobacterium gastri (3.4%), Mycobacterium flavescens (3.4%), Mycobacterium chelonae (3.4%) and Mycobacterium nonchromogenicum (1.7%). PRA method, in comparison with classical methods, is rapid, useful and sensitive for the phylogenetic analysis and species detection of mycobacterial strains. Mycobacterium kansasii is the most common cause of infection by NTM in patients with non-HIV and HIV which demonstrated a high outbreak and diversity of NTM strains in our laboratory.

A comparative study of phenotypic and genotypic first- and second-line drug resistance testing of Mycobacterium tuberculosis.

This study aimed to evaluate the frequency of resistance to first- and second-line drugs using phenotypic and genotypic methods and its correlation with resistance-linked mutations in Mycobacterium tuberculosis (M. tb) isolated in Iran. Three different methods, including the indirect proportion method(PM), direct and indirect nitrate reductase assay(NRA), and direct sequencing were used to assess drug resistance. In this study, sensitivity, specificity, agreement, costs, and turnaround time of these methods were compared in 395 smear positive isolates. Compared to the PM, the NRA and the direct sequencing methods demonstrated higher specificity, sensitivity, and agreement for detection of all anti-tuberculosis drugs. The NRA had a short turnaround time and was more cost-effective than the other methods. Mutations in codon 531 in rpoB, 315 in katG, 18 in rpsL, and 306 in embB were associated with high-level resistance to the first-line drugs, and mutations in codon 94 in gyrA, and A1401G in rrs were correlated with resistance to the second-line drugs. We found that the NRA is a highly sensitive, specific, inexpensive, and rapid test with strong potential to be a useful and interesting alternative tool, particularly in low-income countries. In addition, these molecular data will be helpful for developing new molecular methods for detecting first- and second-line drug-resistant M. tb.

Bias in detection of Mycobacterium tuberculosis polyclonal infection: Use clinical samples or cultures?

The application of MIRU-VNTR has unveiled that infection by Mycobacterium tuberculosis can be polyclonal. Our comparative study demonstrated that based on the studied samples (clinical specimen or culture) detection of polyclonal M. tuberculosis infection can be significantly different.

Pros and cons of direct genotyping on tuberculosis clinical samples.

OBJECTIVE: Prompt genotyping of Mycobacterium tuberculosis (M. tuberculosis) is crucial for improving molecular epidemiological investigation of tuberculosis (TB). METHODS: We performed a retrospective study to evaluate the use of 24 loci MIRU-VNTR (mycobacterial interspersed repetitive unit-variable number of tandem-repeat) directly on 135 clinical samples from 84 TB patients. RESULTS: There was a direct correlation between genotyping on clinical samples by MIRU-VNTR and bacterial load (P = 0.001). VNTR loci were amplified successfully for 41.5% of the clinical samples (19-24 loci), 32.6% (13-18 loci), 23.7% (7-12 loci) and 2.2% (1-6 loci). Loci of 2401, 577, 2996 and 154 had the highest power to show the mixed strains infection in clinical samples. CONCLUSIONS: Direct MIRU-VNTR is partially successful in complete genotyping of M. tuberculosis strains. On the other hand, detection of polyclonal infection is undoubtedly reliable based on the direct MIRU-VNTR.

Haarlem 3 is the predominant genotype family in multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis in the capital of Iran: A 5-year survey.

The objective of this study was to further understand the genetic diversity of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis isolates prevalent in Tehran, the capital city of Iran. From January 2010 to March 2015, a total of 723 M. tuberculosis strains were isolated from patients with pulmonary tuberculosis (TB). A total of 23 MDR, pre-XDR and XDR M. tuberculosis isolates were genotyped by spoligotyping and 24-loci mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR) typing. The results showed that the MDR, pre-XDR and XDR M. tuberculosis strains mainly belonged to the Haarlem 3 genotype (11/23; 47.8%), followed by the Beijing family (9/23; 39.1%). In addition, the 23 strains were clustered into 21 genotypes using a 24-loci MIRU-VNTR. In conclusion, Haarlem 3 genotype was the predominant genotype among the isolates from MDR-TB cases in this study, which could be of special concern.

High prevalence of Mycobacterium tuberculosis mixed infection in the capital of moderate tuberculosis incidence country.

OBJECTIVE: Recent studies using molecular epidemiological techniques have demonstrated mixed infection with multiple strains of Mycobacterium tuberculosis especially in countries with high tuberculosis (TB) burden. We aimed to determine the prevalence of mixed infection among patients with TB in the capital of Iran as a country with moderate incidence rate. METHODS: Samples were collected randomly from January 2011 to December 2013 in Tehran, capital of Iran. A total of 75 M. tuberculosis isolates were genotyped by 24 loci mycobacterial interspersed repetitive unit-variable number tandem repeat typing (MIRU-VNTR) for screening the mixed infection. RESULTS: Twenty patients (20/75) were identified with mixed infection, and the estimated rate of mixed infection was 26.6%. Thirteen out of the 24 loci were able to detect the mixed infection in our study. CONCLUSIONS: Mixed infections occur at high prevalence among studied Iranian TB patients. Further research is inevitable to evaluate the association of mixed infection and disease progression and treatment.