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Objective/background: Whole Genome Sequencing (WGS) is becoming affordable with overall costs comparable to other tests currently in use to perform the diagnosis of drug-resistant tuberculosis (TB) and cluster analysis. The WGS approach allows an “all-in-one” approach providing results on expected sensitivity of the strains, genetic background, epidemiological data, and indication of risk of laboratory cross-contamination. Methods: Although ideal, WGS from the direct diagnostic specimen is not yet standardized, and to date the two most promising approaches are WGS from early positive liquid culture and targeted sequencing from diagnostic specimens using Next-Generation Technology. Both have advantages and disadvantages. Sequencing from early MGIT requires positive cultures, whereas targeted sequencing can be performed from a specimen positive for Mycobacterium tuberculosis with a consistent gain in time to information. The aim of this study is to evaluate the feasibility and cost of using WGS with a centralized approach to speed up diagnosis of TB in a low-incidence country. Methods: From March 2016 to September 2016, we collected and processed by WGS 89 early positive routine MGIT960 tubes. Time to diagnosis and accuracy of this technique were compared with those of standard testing performed in a regular laboratory. A 2-mL aliquot of early positive MGIT was processed, starting with heat inactivation. DNA was then isolated by using the Maxwell 16 Cell DNA Purification Kit and Maxwell 16 MDx for automated extraction. Paired-end libraries of read-length 75–151bp were prepared using the Nextera XT DNA Sample Preparation kit, and sequenced on Illumina Miseq/Miniseq platform (based on the 1st available run). Total variant calling was performed according to the pipeline of the Phyresse web-tool. The DNA isolation step required 30min for inactivation plus 30min for extraction. The concentration obtained ranged from 0.1 to 1 ng/ μL, suitable for library preparation. Samples were sequenced with a turnaround time of 24–48h. The percentage of reads mapped to the H37Rv reference genome was 83% on average. The mean read coverage was 65×. The main challenge was the presence of nonmycobacterial DNA contamination in a variable amount. Lineage detection was possible for all cases, and mutations associated with drug resistance to antitubercular drugs were examined. We observed high diagnostic accuracy for species identification and detection of full drug resistance profile compared to standard DST testing performed in MGIT. Results: Two events of recent transmissions including respectively three and two patients were identified, and two laboratory cross-contaminations were investigated and confirmed based on the analysis. Time to availability of report was about 72h from MGIT positivity compared to up to 6–9 weeks for XDR-TB diagnosis with standard testing. In addition to speed, the main advantages were the availability of a full prediction of resistance determinants for rifampicin-resistant cases, and the fast detection of potential cross-contaminations and clusters to guide epidemiological investigation and cross-border tracing. Cost analysis showed that the cost per strain was approximately €150 inclusive of staff cost, reagents, and machine cost. Conclusion: WGS is a rapid, cost-effective technique that promises to integrate and replace the other tests in routine laboratories for an accurate diagnosis of DR-TB, although it is suitable nowadays for cultured samples only.

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Background/objective: Outcomes of treatment for multidrug-resistant tuberculosis (MDR-TB) remain poor worldwide. Among patients with MDR-TB in Belarus who started treatment in 2012, only 54% completed it successfully, with treatment failure reported in 22% of the patients; additionally, 11% died and 13% were lost to follow-up or remained unevaluated. In Belarus, to improve outcomes, bedaquiline was introduced in MDR-TB treatment in June 2015. The national TB program developed measures to monitor safety and effectiveness of bedaquiline-containing regimens in line with the World Health Organization recommendations. Methods: After enrollment of patients, clinical, radiological, laboratory, and microbiological data were carefully collected at start, during treatment, and at follow-up. A total of 197 patients were enrolled: male, 140 (71%); female, 57 (29%); new TB cases, 83 (42%); previously treated, 114 (58%); extensively drug-resistant-TB (XDR-TB), 128 (65%), pre-XDR-TB (fluoroquinolone resistant), 34 (17%), pre-XDR-TB (injectables resistant), 25 (13%), and other MDR-TB cases, 10 (5%). Results: According to the intermediate analysis, 186 patients currently are continuing with the treatment, two patients died, and nine patients were lost to follow-up. Sputum culture conversion were observed in 186 patients (94%) at 6 months and one (0.5%) of these 197 patients started treatment; six patients (3%) remain sputum culture positive. The safety data were as follows: 135 patients (68%) experienced metabolism and nutrition disorders (hyperuricemia being the most common), 127 patients (64%) experienced hepatobiliary disorders (hepatic functions abnormality being the most common), 93 patients (47%) experienced electrolyte disorders (hypomagnesemia being the most common), 80 patients (41%) experienced cardiac disorders (abnormal electrocardiogram and arrhythmia being the most common), 68 patients (35%) experienced gastrointestinal system disorders (nausea, vomiting, and abdominal pain being the most common disorders), 54 patients (27%) experienced blood and the lymphatic system disorders (low platelet count being the most common), 42 patients (21%) experienced renal and urinary disorders (creatinine clearance decrease being the most common), 40 patients (20%) experienced nervous system disorders (headache, dizziness, and paresthesia being the most common ones), 36 patients (18%) experienced skin and subcutaneous tissue disorders (rush and pruritus being the most common), 35 patients (17%) experienced ear and labyrinth disorders (tinnitus and decreased hearing being the most common ones), 32 patients (15%) experienced psychiatric disorders (insomnia being the most common disorder), and 30 patients (14%) experienced infections and infestations (candidiasis being the most common). The most adverse events were mild or moderate in severity and reversible. One death was possibly related to MDR-TB therapy. Conclusion: Our interim results on safety and effectiveness of bedaquiline-containing regimens in multidrug and extensively drug-resistant tuberculosis (M/XDR-TB) patients are encouraging. They will add value to understanding role and place of this new anti-TB drug in M/XDR-TB treatment.

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Background: Although the prevalence of pulmonary tuberculosis (TB) and HIV infection in Japan is low, careful monitoring of these two diseases is necessary. We conducted a nationwide survey on multidrug resistant (MDR)-TB (2011–2013) and HIV-TB (2007–2014) to understand the mode of prevention and the effect of therapy. A study on MDR-TB and HIV in San Lazaro Hospital (SLH) in the Philippines was also conducted. These studies introduced an international collaborative study against the global epidemics of HIV-TB/MDR-TB. Methods: The nationwide survey of MDR-TB was done in hospitals that treat TB patients in Japan from 2011 to 2013. The HIV-TB survey has been done every year since 2007. Classic information such as chest X-ray (CXR) as well as computed tomography (CT) results for each patient were analyzed. Likewise, the presence of a cavity, involved segments, and patterns of parenchymal lesion were assessed. Finally, tentative diagnosis and disease activity, bronchogenic spread of the lesion with CT, and bronchiectasis were recorded. At SLH, sputa of suspected cases were subjected to GeneXpert testing and HIV testing was performed on all TB patients. Results: In the nationwide MDR survey in Japan, 171 patients were diagnosed as pulmonary MDR-TB (0.2% of total Mycobacterium tuberculosis (MTB) in Japan). Among them, 48 (28%) were foreigners and most were living in big cities. In Tokyo metropolitan areas, 27 out of 53 MDR-TB patients were foreigners: 13 were from China, 4 from the Philippines, and 3 from Myanmar. Thirty nine among 53 MDR-TB patients were cured or treatment was completed with favorable prognosis. Five deaths (9.4%) and six departures from Japan (11.3%) were noted. In the HIV-TB survey in National Hospitals, the HIV-positive rates on MTB were constantly low (0.23–0.46%) from 2007 to 2014. Among the reported 114 HIV-TB patients (0.37% of total MTB in National Hospitals), 17 were foreigners and 3 (2.6%) were MDR-TB cases (2 Chinese, 1 Japanese). Half of the HIV-TB patients have low CD4 cell numbers (<100/ μL). Two out of the 3 MDR-TB patients were cured. Imme reconsitution inflammatory syndrome (IRIS) was found in 48% of all HIV-TB cases. At SLH in the Philippines, total numbers of GeneXpert examined cases were 1052 cases in 2015, and 122 Rifampicin resistant (RR)-TB cases (11.6%) were found and among the enrolled 96 cases 14 (15%) were found to be HIV positive. Conclusion: Very low numbers of TB or HIV patients have been observed in Japan. An increasing number of foreign-born MDR-TB patients in the country was found. To combat against these global epidemics, an international collaboration against HIV-TB/MDR-TB is needed.

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Objective/background: Mycobacterium tuberculosis pyrazinamidase (PZase) is known an enzyme that is involved in degradation of pyrazinamide to ammonia and pyrazinoic acid. Pyrazinamide is an important first-line drug used in the short-course treatment of tuberculosis. Previous investigations have indicated that the pyrazinamide (PZA)-resistant M. tuberculosis strains are caused by point mutations in the PZase enzyme which is the activator of the prodrug PZA. Although the general fold of PZase was determined, the structural and functional properties of the enzyme in solution were not understood very well. In this study, the PZase enzyme was overexpressed and purified. In addition, two polyols, namely sorbitol and glycerol, were chosen to study their effects on the structure, dynamics, and stability of the enzyme. To gain a deeper insight, molecular dynamics simulation and spectroscopic methods, such as fluorescence spectroscopy and circular dichroism (CD), were used. Methods: The genes were cloned in Escherichia coli BL21 (DE3), harboring the recombinant pET-28a (+) plasmid, overexpressed and purified by Ni-NTA Sepharose. The far UV–visible CD spectra were measured by a Jasco-810 spectropolarimeter. The intrinsic fluorescence spectra were measured on a Cary Varian Eclipse spectrofluorometer. For molecular dynamics (MD) simulations, we have applied GROMACS4.6.5. Results: The results showed that glycerol and sorbitol increased the enzyme activity up to 130% and 110%, respectively, at 37°C. The stability of PZase was decreased and the half-life was 20 min. Glycerol and sorbitol increased the PZase half-life to 99 min and 23 min, respectively. The far UV CD measurements of PZase indicated that the CD spectra in glycerol and sorbitol give rise to an increase in the content of α-helix and β-sheets elements. The average enzyme root mean square deviation (RMSD) in sorbitol solution was about 0.416nm, a value that is higher than the enzyme RMSD in the pure water (0.316). In dictionary of protein secondary structure (DSSP) results, we observed that the secondary structures of the protein are partially increased as compared to the native state in water. The experimental and simulation data clearly indicated that the polyols increased the PZase stabilization in the order: glycerol>sorbitol. Conclusion: It can be concluded that the native conformation of the enzyme was stabilized in the sorbitol and glycerol and tend to exclude from the PZase surface, forcing the enzyme to keep it in the compactly folded conformation. The glycerol molecules stabilized PZase by decreasing the loops flexibility and then compacting the enzyme structure. It appears that more stability of PZase in glycerol solution correlates with its amphiphilic orientation, which decreases the unfavorable interactions of hydrophobic regions.

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Objective/background: Mycobacterium africanum (MAF) remains an important TB causing pathogen in West Africa; however, little is known about its population structure and actual diversity which may have implications for diagnostics and vaccines. We carried out comparative genomics analysis of candidate Mycobacterium tuberculosis (MTB) and MAF using whole genome sequencing. Methods: Clinical MTB complex strains (n = 187) comprising L4 (n = 22), L5 (n = 126), and L6 (n = 39) isolated over 8 years from Ghana were whole genome sequenced. The reads were mapped onto a reference genome for phylogenetic and functional genomics analysis. A maximum likelihood tree with 100 bootstraps was constructed from the single nucleotide polymorphisms (SNPs) found using RAxML and clustered with hierBAPS. A total of 147 (18 L4, 36 L6, and 93 L5) of the genomes were de novo assembled and annotated for comparative pangenome analysis using Roary. Results: The population structure of MAF revealed at least five clusters of L5 as compared to three for L6. We also identified a group of three multi-drug-resistants (MDRs) within a single cluster of L5 strains from Southern Ghana isolated in 2013. Among the global collection of MTB complex, there were four Ghana-specific L5 clusters of which one (L5.1.1) had traits of clonal expansion. From the 5947pan genes extracted from the collection, 3215 (54.1%) were core to all the 147 genomes whereas 719 (12.1%) were found in single genomes. Most of the variable genes were PE-PGRS/PPE (1,281) duplicates of other genes (431). The genome degradation was more pronounced in Lineages 4 and 6 as compared to Lineage 5. We identified the absence of some unique genes among specific lineages and/or clades with possible clinical implications. For example, mpt64 and mlaD encoding respectively an immunogenic protein and a mammalian cell entry protein were missing from all L6 genomes. In addition, all L5 strains had an amino acid substitution I43N within the mpt64 gene. Analysis of SNPs within some genes encoding proteins for substrate metabolism, ion transport and secretory systems showed higher proportion of SNPs among L6 compared to L5 and L4. We also identified a number of lineage/sublineage specific SNPs and indels that may be utilized in rapid PCR based genotyping of MTB complex. Conclusion: This work emphasizes on the possibility that the mpt64-based rapid diagnostic kit would not be effective in MAF endemic settings. More mutations in ESAT-6 secretory system of MAF compared to MTB sensu stricto can affect efficacy of ESAT-6-based vaccines in the future.

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Objective/background: Ethionamide (ETH) and isoniazid (INH) are part of the backbone regimen used for the treatment of multidrug-resistant tuberculosis (MDR-TB). Both ETH and INH are structurally similar and are activated by ethA and katG gene products. Resistance to INH among MDR-TB patients may cause ETH to be ineffective, as both target nicotinamide adenine dinucleotide-dependent enoyl-acyl carrier protein reductase inhA protein and mutations within inhA gene may lead to their cross-resistance. Furthermore, ETH resistance is caused by mutations within ethA and ethR genes forming part of the ETH drug activation pathway. Nicotinamide adenine dinucleotide is coded by the ndh gene, and its overexpresion may lead cross-resistance between INH and ETH drugs. Phenotypic drug susceptibility testing of ETH is difficult and often unreliable. We used whole genome sequencing to compare inhA, inhA promoter, ethA, ethR ndh, and katG genetic regions in serial isolates (baseline and follow-up) with treatment outcomes. Methods: MDR-TB strains were collected from 46 patients before and during second-line drug treatment in KwaZulu-Natal and Eastern Cape between 2005 and 2009. All patients had phenotypically determined MDR-TB at baseline and had treatment outcomes documented. Unfavorable treatment outcomes were defined as death, default, and failure, while favorable outcomes were cure and treatment completion. Each strain had baseline and at least one strain collected on follow-up. From each strain, DNA was extracted from colonies grown on Löwenstein–Jensen slants, and fragment and jumping paired-end Illumina DNA libraries were constructed and sequenced on the Illumina HiSeq 2000 (Broad Institute, Cambridge, MA, USA). Sequences were aligned to H37Rv genome and Pilon was run to generate a list of SNPs. In silico spoligotyping was performed to a database 43 unique spacer sequences. Cross-resistance was defined as the presence of both inhA and either ethA or ethR mutations in clinical isolates. Results: A total of 92 sequences from 46 serial isolates of MDR-TB patients from KwaZulu-Natal (29 isolates) and Eastern Cape (17 isolates) were analyzed. Most patients (29/46; 63.0%) had unfavorable outcomes, 13 (28.3%) had favorable outcomes, while four (8.7%) had unknown outcomes. Phylogenetic reconstruction revealed that primary genotype differed by province. The Beijing genotype was predominant in Eastern Cape, while EuroAmerican lineage (S, T, LAM, X) was found in KwaZulu-Natal. Whole genome analysis revealed nonsynonymous insertions and deletions within katG, ethA, ethR, ndh, and inhA and its promoter region. Among patients with treatment outcome data, mutations were detected in 92.8% in katG, 50% in inhA, 53.6% in ethA, 2.4% in ethR, and 19% in ndh. The majority of mutations causing ETH (20/29; 68.9%) and INH (18/29; 62.1%) resistance occurred among patients with unfavorable outcomes. Both inhA and either ethA or ethR mutations were detected in 16/29 (55.2%) patients with unfavorable outcomes. Cross-resistance of both INH and ETH drugs was associated with unfavorable treatment outcomes (p = 0.021) in 16/29 (55.2%) patients compared with favorable treatment outcomes in 2/13 (15.4%) patients. Conclusion: Baseline ETH molecular resistance before second-line treatment is a concern. Unfavorable treatment outcomes of patients with ethA, ethR, and inhA mutations highlight the importance of genotypic testing before initiation of treatment containing ETH. The clinical significance of whole genome analysis for early detection of mutations predictive of treatment failure needs further investigation.

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Tuberculosis (TB) is the main cause of infection-related death in the world. Children make up ˜5–15% of all TB cases. Severe forms of disease such as meningitis and disseminated form are more common in children. Due to current estimates of World Health Organization (WHO) in 2015, one million children suffer from TB around the world; of these, it is estimated that more than 136,000 die a year. One study estimated 67 million children with latent TB which develops to active form with the rate of ˜850,000 each year. The main way of entrance of tuberculosis in children is the respiratory tract. By contrast to adults, the majority of children with tuberculosis are not infectious to others. Usually children with TB do not have dominant signs or symptoms. Most common primary symptoms in children are cough and low grade fever. In some rare situations children present with a flu-like syndrome which recover within a week. The most common type of TB disease in children is pulmonary form. A total of 25–35% of cases of TB have an extra pulmonary manifestation. Disseminated TB (Miliary TB and TB meningitis) particularly occurs in young children <3 years old. Depending on the age of onset, physical and clinical manifestations would be different in children. In infants due to small airways we can see clinical manifestations such as nonproductive cough and mild dyspnea. In some cases failure to thrive is represented. Significant signs and symptoms are most common in preschool and adult patients. Half of school-age close contact children with radiographically moderate to severe pulmonary TB, have no symptoms or physical findings. In tuberculosis infection, the child has a positive tuberculin test but no clinical and radiological signs or symptoms. However, enlarged lymph nodes can be seen in rare cases. Depending on the position of the node these findings can be detected: partial or complete obstruction, segmental atelectasis, endobronchial tuberculosis and fistulous tract, collapse consolidation, segmental lesion, pericarditis, or tracheoesophageal fistula. Early morning gastric lavage is the best sample for diagnosis of pulmonary TB in a child. Gastric aspirates are used in children younger than 6 years instead of sputum. In <50% of cases Mycobacterium tuberculosis are detected in three gastric aspirates. The aim of the BCG vaccination is to prevent life threatening tuberculosis in children such as meningitis and disseminated TB. The rate of miliary TB and meningitis are higher in neonates. They are also more disposed to progression to disease. Development to disease between 5 years and 10 years is rare. Atypical forms of TB present in children with AIDS. In conclusion most cases of tuberculosis in children occur within 1year of the infection, tuberculosis disease in a child shows recent transmission of the organism.

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Objective/background: Tuberculosis (TB) continues to be a major public health problem worldwide. This is especially true in Northern Iran, which has high TB prevalence. The chronic nature of this disease is further exacerbated if it is accompanied by fungal infection, which usually remains undiagnosed and thus untreated. Thus, mycotic infections add fatal dimensions to pulmonary TB. Our objective was to determine the prevalence of invasive forms of fungal elements in sputum samples collected from patients with pulmonary TB at a reference laboratory in Ghaemshahr, Northern Iran, during the past 10 years. Methods: In this retrospective study, sputum samples collected from 430 patients were examined. Pulmonary TB in patients was confirmed in our laboratory, and samples obtained during the period from March 2006 to February 2016 were analyzed. The sputum samples were subjected to biological (bacterial) staining (Ziehl–Neelsen and fluorochrome) and mycological investigation using KOH+ Calcofluor White (Sigma-Aldrich, India) by fluorescent microscopy and fungal culture on Sabouraud dextrose agar (Sigma-Aldrich, India) and CHROMagar (Paris, France). Results: Invasive forms of fungal pathogens were observed as co-infection with Mycobacterium tuberculosis in 28/430 cases (6.51%). The frequency of Aspergillus, both branching and dichotomous infection, accounted for 3.72% (16/430): Aspergillus flavus, 1.63%; Aspergillus fumigatus, 1.16%; Aspergillus niger, 0.69%; and Aspergillus oryzae, 0.23%, respectively. Blastoconidia and pseudohyphae forms of yeast were observed as co-infection with M. tuberculosis in 2.79% (12/430) of the cases: Candida albicans, 1.86%; Candida krusei, 0.46%; and other Candida species, 0.46%, respectively. Conclusion: Northern Iran is a critical region in the TB world and multidrug-resistant TB is a serious problem in this region. Although it is believed that there exists a commensal relationship between fungus and TB infections, the invasive forms of fungal pathogens and their co-infection can be caused by increasing disability and failure of treatment. Diagnosis of secondary or co-existing fungal infections in TB is most important for reducing the mortality and morbidity of these patients.

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Objective/background: QuantiFERON-TB Gold In-Tube (QFT-GIT, Qiagen, Hilden, Germany) is an interferon-γ (IFN-γ) release assay designed to detect latent tuberculosis infection (LTBI). Although QFT-GIT has several advantages (mainly that it is not affected by the Bacille Calmette–Guérin vaccination), it has a poor sensitivity in immune-compromised individuals as it involves an immune response-based detection. Recently, QuantiFERON-TB Gold Plus (QFT-Plus) assay has been proposed as a new generation of QFT-GIT. QFT-Plus includes two tubes, TB1 and TB2 with Mycobacterium tuberculosis antigens to elicit a specific immune response. TB1 contains peptides derived from the antigens 6 kDa early secretory antigenic target (ESAT-6) and 10 kDa culture filtrate protein (CFP-10) (TB-7.7, present in QFT-GIT, has been removed), and it is designed to induce a specific CD4 T-cell response. TB2 contains newly designed peptides stimulating IFN-γ production by both CD4 and CD8 T cells. The additional peptides for eliciting CD8 T-cell responses have been included to increase the sensitivity of the test for LTBI detection. The aim of the study was to evaluate specific CD4 and CD8 T-cell responses to the M. tuberculosis antigens contained within the QFT-Plus test by flow cytometry in individuals with active TB and LTBI. Methods: We enrolled 23 individuals with active TB and 30 individuals with LTBI. QFT-Plus assay and intracellular staining were performed. One million of peripheral blood mononuclear cells in 1 ml of complete medium (RPMI 1640) were dispensed in QFT-Plus tubes. Following 16–24 h stimulation, antigen-specific T cells were characterized by flow cytometry evaluating CD4, CD8, CD3 markers, and IFN-γ production. For statistical analysis, nonparametric tests were performed. Results: We found that CD4 T-cell responses were induced by both TB1 and TB2. Differently, the CD8 T-cell response was mainly induced by TB2 and was significantly higher than that induced by TB1 (p = 0.01). The frequency of Mtb specific T-cells observed in individuals with active TB was significantly higher than in those with LTBI (p = 0.04). Finally, TB2-specific CD8 T-cell responses in individuals with active TB were associated with high radiological severity of lung lesions and microbiological diagnosis (based on M. tuberculosis isolation in sputum culture). Conclusion: This is the first characterization of CD4 and CD8 T-cell responses to QFT-Plus TB1 and TB2 tubes in individuals with active TB and LTBI enrolled in a low TB-endemic country such as Italy. We demonstrated that the increased sensitivity is a consequence of the ability of TB2 to induce a CD8 T-cell response which is mainly associated with active TB. This assay has the potential to be very useful in conditions of immune depression due to CD4 T-cell impairments.

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Objective/background: World Health Organization tuberculosis (TB) indices from 2014 to 2016 showed that Nigeria had the 6th highest prevalence, 4th highest incidence, and the highest mortality rate globally. In efforts to improve TB care, the XpertMTB/Rif (GeneXpert) technology, Cepheid, Sunnyvale, California, USA, which has revolutionized TB detection with concomitant rifampicin-resistance molecular detection, was introduced in Cross River State, South–South Nigeria, in 2014. The GeneXpert uses molecular beacons to detect five overlapping 81-bp regions in the rpoB gene known as the Rifampicin Resistant Determinant Region (RRDR). These probes are represented as Probe A (507–511), Probe B (512–518), Probe C (518–523), Probe D (523–529), and Probe E (529–533). Mutations in this region have been shown to account for about 93% of resistance to rifampicin, which is the most important drug in tuberculosis treatment. The objective of this study was to determine the frequency of rifampicin resistance and the commonly associated probes for various rpoB gene mutations within the 81-bp RRDR of Mycobacterium tuberculosis in Cross River State, Nigeria. Method: We collated and analyzed data from the 10 Xpert MTB/Rif sites in Cross River State from June 2014 to June 2016 and determined the frequency of mutations associated with different probes designated A–E, which represent the RRDR of rpoB gene. All centers use XpertMTB/Rif version G4. Result: In total, 973 tuberculosis cases were detected from 4671 cases tested. Rif resistance was detected in 6.0% (58/973) of cases. Probe E mutations were the most common, seen in 60.3% (35/58); followed by Probe D, 17.2% (10/58); and Probe B, 13.8% (8/58). Probe A occurred in 3.4% (2/58). No Probe C mutation was seen. Multiple mutation combinations involving probes B and D occurred in 3.4% (2/58), while one isolate had triple site mutations involving A, D, and E. One isolate that at initial testing showed a Probe A mutation displayed a Probe D mutation when tested in another site prior to treatment enrollment. Conclusion: In our setting, 6.0% of tuberculosis isolates are rifampicin resistant. Mutations associated with probe E commonly due to codon 531 are the most predominant cause of rifampicin resistance. Mutations at probe C (codons 518–523) were uncommon. A change in mutation may have occurred in one of the patients.

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Tuberculosis (TB) causes 1.3 million deaths annually. There are 0.5 million cases of multidrug resistant TB (MDR-TB) and the number of cases is rising globally. The current status quo of the lengthy treatment duration and poor treatment outcomes associated with MDR/extensively drug-resistant TB, and those with comorbidity of TB with human immunodeficiency virus and noncommunicable diseases in sub-Saharan Africa is unacceptable. The TB drug pipeline remains sparse. New innovations for shortening the duration of therapy and improving treatment outcomes (cure and long-term functional disability due to lung damage) are urgently required. A wide range of host-directed therapies (HDT) are now available which require evaluation as adjuncts to current TB drug treatment. Examples are:

1. Repurposed drugs:
• Analgesics/nonsteroidal anti-inflammatory drugs (cyclooxygenase-2 inhibitors, e.g., ibupofen).
• Cholesterol-lowering drugs (3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, e.g., simvastatin).
• Asthma drugs (leukotriene synthesis inhibitors, e.g., zileuton).
• Diabetes drugs (reactive oxygen species removal and increased CD8+ T-cell responses (e.g., metformin).
• Anticonvulsants (inhibition of histone deacylation, e.g., valproic acid).
2. Cellular therapy: using the patient's own bone marrow-derived stromal cells.
3. Immune therapies: for example, anti-interleukin-6/interleulin-6 receptor monoclonal antibody.
4. Therapeutic vaccines: protein vaccines (e.g., granulysin), DNA vaccines, environmental mycobacteria vaccines (e.g., Mycobacterium vaccae, Mycobacterium indicus pranii).
5. Micronutrients: for example, Vitamin D, zinc, probiotics, and so forth. The Host-directed Therapies Network consortium of 64 partners was launched in Cape Town after a meeting hosted by the South African Medical Research Council in April 2015. This network (which is open to anyone interested) plans to take forward a wide range of HDTs in randomized, placebo-controlled clinical trials as adjuncts to current TB treatment regimens with the aims of:

1. Shortening the duration of treatment for drug-sensitive TB and MDR-TB.
2. Improving treatment outcomes (mortality/morbidity) for MDR/extensively drug-resistant TB patients.
3. Improving lung function and preventing lung damage so that the patient can return to gainful employment after treatment.
4. Improving treatment outcomes for clinical presentations associated with tissue injury:
1. Miliary TB (including TB meningitis and TB pericarditis).
2. Immune reconstitution inflammatory syndrome.
5. Improving treatment outcome of TB and/or human immunodeficiency virus-positive individuals with comorbidities such as noncommunicable diseases (e.g., diabetes, liver disease, and cardiac disease), and cancers.

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Background: India accounts for more than one-fifth of the world's tuberculosis (TB) burden. In spite of efforts taken by the Revised National Tuberculosis Control Programme, tribal areas of the state of Odisha report a high TB incidence over the years. One of the reasons could be delay in reporting to health facilities by the symptomatic patients. During such delays an active case may infect numerous susceptible people thereby contributing to the perpetuation of the infection. The delay in diagnosis may be as long as 2–3 months and even more in hard-to-reach areas. Objective The present study aims to find out the extent of delay in diagnosis among pulmonary TB patients of a tribal dominated district that may help in planning effective control strategies for similar situations. Methods: The information on delay in diagnosis is part of a cross-sectional drug resistance study carried out from June 2011 to May, 2013 in 20 Designated Microscopy Centres (DMCs) of Rayagada district of Odisha, India. Out of 634 smear positive pulmonary TB patients enrolled in this study, information on health seeking by the patients were available for 580 patients. The patients included had clinical signs and symptoms suggestive of pulmonary TB (cough, chest pain, and hemoptysis), with/without radiological evidence. Patients found smear positive by Ziehl–Neelsen microscopy were requested to take part in the study and accordingly a written questionnaire including history of: TB treatment; symptoms experienced by the patient and duration of suffering; and radiological examination was completed. The delay in diagnosis at the DMCs due to delay in health seeking by the symptomatic TB patients was evident as only 5.2% patients reported within 2 weeks and 62.6% up to 1 month of onset of symptoms. Results: The delay in health seeking by the patients was not differentiated by sex or resistance profile, although more men attended the DMC for diagnosis. The present study is the first of its kind to report diagnostic delay of TB among smear-positive TB patients of Rayagada, a tribal-dominated district of Odisha, India and it reveals an extremely long diagnostic delay of TB in this area. We found that 12.9% of patients had a delay exceeding 2 months, and 50% of them had high sputum grade. This is a serious concern due to the fact that each of these patients dispenses up to 3500 bacilli in each cough, and may infect 10–15 people each year, eventually creating a public health problem. Conclusion: Poor awareness of patients about the disease and limited access to health care are the bottom-line in apparent diagnostic delay of TB patients. This substantial patient delay to diagnosis is a major contributing factor for increasing transmission of TB in tribal districts of Odisha. Increased awareness of the disease is crucial in improving health-seeking behavior in these areas.

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Background: Surgery in DR-TB is a highly contested intervention. However, in suitable selected cases, it has a great role in improving outcomes of treatment as well as symptomatic improvement in the quality of life of the patient. Indications of surgery in this setting will be localized disease with high likelihood of persistent progression or sputum positivity despite adequate therapy. Recurrent hemoptysis, intolerance to drugs or absence of radiological and bacteriological improvement during initial 3–4 months of therapy becomes additional indications for surgical intervention. A review of 11 studies published in a period from 1993 to 2013 provides enough justification for the role of surgical intervention in pulmonary tuberculosis. Interventions: At the NITRD, in the last 20 years a total of 107 cases have been operated upon for DR-TB. Procedures done were 70 pneumonectomies, 20 lobectomies, 5 bilobectomies, 4 nonanatomical resections and 7 thoracoplasties. Results: Sputum negativity was achieved in 93 cases after surgery and 62 were declared cured after 4 years of follow up. 6 cases of DR TB were also operated upon in March 2013 in one MSF TB surgery mission and all of them are sputum negative till March 2015. Conclusions: In conclusion, surgical intervention should be offered and made available for greater number of DR-TB patients.

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Objective/Background: To evaluate the performance of Lowenstein–Jensen medium (LJ) supplemented with pyruvate and glycerol (LJPG), compared with LJ supplemented with pyruvate (LJP) or glycerol (LJG) for the support of mycobacterial growth. Method: This study used 100 Ziehl-Neelsen-confirmed positive mycobacterium growth indicator tube 960 culture samples that were obtained from clinical samples during routine diagnosis. All cultures were inoculated in parallel on LJG/LJP and on LJGP, which were incubated and read weekly for the evidence of growth. The mycobacterial recovery rate, contamination rate, and time to detection were compared. Result: The recovery rate for LJG/LJP and for LJPG was 90% (90 samples) and 88% (88 samples), respectively (kappa p-value, 0.9). There was no significant difference in the contamination rate, which was 8% (8 samples) for LJG/LJP and 9% (9 samples) for LJPG. Mycobacterial growth was faster in LJPG (1.6 weeks) than in LJG/LJP (2 weeks). Conclusion: A single LJPG slope was not significantly different, compared with the usual pair of LJG or LJP slopes. This is a promising new culturing approach that could be used in Mycobacterium africanum-endemic in West African countries. It significantly reduces labor time and consumable costs and more quickly detects the M. tuberculosis complex.

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In the past two decades, DNA techniques have been increasingly used in the laboratory diagnosis of tuberculosis (TB). The (sub) species of the Mycobacterium tuberculosis complex are usually identified using reverse line blot techniques. The resistance is predicted by the detection of mutations in genes associated with resistance. Nevertheless, all cases are still subjected to cumbersome phenotypic resistance testing. The production of a strain-characteristic DNA fingerprint, to investigate the epidemiology of TB, is done by the 24-locus variable number tandem repeat (VNTR) typing. However, most of the molecular techniques in the diagnosis of TB can eventually be replaced by whole genome sequencing (WGS). Many international TB reference laboratories are currently working on the introduction of WGS; however, standardization in the international context is lacking. The European Centre for Infectious Disease Prevention and Control in Stockholm, Sweden organizes a yearly round of quality control on VNTR typing and in 2015 for the first time also WGS. In this first proficiency study, only three out of eight international TB laboratories produced WGS results in line with those of the reference laboratory. The whole process of DNA isolation, purification, quantification, sequencing, and analysis/interpretation of data is still under development. In this presentation, many aspects will be covered that influence the quality and interpretation of WGS results. The turn-around-time, analysis, and utility of WGS will be discussed. Moreover, the experiences in the use of WGS in the molecular epidemiology of TB in The Netherlands are detailed. It can be concluded that many difficulties still have to be conquered. The state of the art is that bacteria still have to be cultured to have sufficient quality and quantity of DNA for succesful WGS. The quality of sequencing has improved significantly over the past 7 years, and the detection of mutations has, therefore, become more reliable. The resistance mutations detected in WGS are in line with the ones visualized in reverse line blot techniques. The turnover in the genome of M. tuberculosis is very low, ˜0.3–0.5 mutations per genome per year. However, there is a wide variation in the occurrence of mutations per strain and genotype. Still, the resolution of WGS in epidemiological typing is higher than that in VNTR typing; previously suggested epidemiological links by VNTR typing are sometimes refuted on the basis of WGS. Although WGS offers the highest resolution in typing, in a country like The Netherlands, there are many strains with a limited genetic distance up to 100 mutations, without an apparent epidemiological link between the respective cases. These lookalikes are presumably even more prevalent in settings where predominant genotypes of M. tuberculosis are circulating. In summary, WGS seems to yield a more reliable prediction of resistance by the (lack of) detection of mutations in all 25 genes ever associated with resistance. This may within a short while prevent the need for many phenotypic resistance tests. Although more robust algorithms need to be developed, the recognition of the (sub) species in the M. tuberculosis complex seems possible. The first detailed studies on the population structure of M. tuberculosis strains in The Netherlands provide more resolution in typing but also an interesting observation that a part of the strains are genetically so conserved that they are separated by less than 100 mutations. This demands a more extended and accurate validation and understanding of the utility of WGS in the epidemiology of TB.

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Objective/Background: Globally, approximately 50% of patients with multidrug-resistant tuberculosis (MDR-TB) experience treatment failure. MDR-TB treatment is hindered by adverse events, toxicity of the second-line anti-TB drugs, logistics and costs, especially in low-income countries, and problems with medication adherence. Pharmacokinetic variability is also attributed as one of the reasons contributing to treatment failure. In our reference Tuberculosis Center Beatrixoord (University Medical Center Groningen, Groningen, The Netherlands), we strive to individualize treatment of all MDR-TB patients based on drug-susceptibility testing using minimal inhibitory concentrations and pharmacokinetic parameters. The aim of this work is to give an overview of our efforts to individualize treatment of MDR-TB patients and to provide insights into practical tools that might be implemented in other clinical settings worldwide. Methods: We critically looked at clinical practice guidelines implemented in our center to give an overview of practically applied tools to individualize treatment of MDR-TB patients. Furthermore, we selected studies carried out in our clinic on treatment individualization of MDR-TB patients and combined their results with recent studies in this area to suggest practical tools for implementation in other clinical settings. Results: We regularly perform therapeutic drug monitoring (TDM) of several second-line anti-TB drugs, such as amikacin, kanamycin, linezolid, and moxifloxacin. New analyses of Group D and experimental drugs, such as co-trimoxazole (sulfamethoxazole/trimethoprim), bedaquiline, delamanid, and clarithromycin, have been or are being developed. By implementing TDM methods, variability in pharmacokinetics is often detected and treatment is adjusted, possibly preventing toxicity in patients with very high drug exposure or treatment failure, or resistance in patients with very low drug exposure. Over the past 10 years in the Netherlands, 86% of 104 patients had a successful outcome using a median of six active drugs. Many studies were performed using dried blood spot (DBS) analysis of second-line TB drugs. These studies may be used to implement TDM worldwide, even in low-income countries. Furthermore, several studies are performed to determine limited sampling strategies (LSSs). By limiting the number samples required for adequate sampling, TDM will become easier to implement. Other examples of LSSs included development of oral fluid sampling methods or development of semiquantitative thin-layer chromatography methods. Conclusion: TDM is highly valuable to individualize and optimize treatment of complex MDR-TB patients. TDM is routinely applied in Tuberculosis Center Beatrixoord, and high success rates for treatment of MDR-TB patients have been achieved. DBS and LSS make implementation of TDM feasible, even in low- and middle-income countries.

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Objective/Background: Pyrazinamide (PZA) is an antibacterial used in the first-line regimen against tuberculosis (TB) for its action against dormant bacilli. PZA is also included in the new short regimen to treat multidrug-resistant TB (MDR-TB). However, the prevalence and significance of PZA resistance is not known in Central and West Africa. Methods: Between 2013 and 2016, we collected samples from MDR-TB patients recruited in an observational study implementing the new short MDR-TB regimen in six countries: Burundi (n = 35), Cameroon (n = 135), Niger (n = 57), Central African Republic (n = 35), Democratic Republic of the Congo (n = 99), and Rwanda (n = 16). Resistance to rifamipicine, isoniazide, injectables, and fluoroquinolones was tested by phenotypic (live strains) or genotypic methods (inactivated strains). Resistance to PZA was analyzed through sequencing of the pncA gene. Relevance of mutations was established based on recent literature. Results: From 377 patients with MDR-TB, 354 (94%) samples were successfully sequenced. Among those, 53% (189) presented a mutation in pncA that confers a reported (121), potential (56), or unclear (12) resistance. Furthermore, six isolates presented a mutation associated with PZA sensitivity. The frequency of resistance per country was 26% in Central African Republic, 39% in Niger, 49% in Cameroon, 66% in Burundi, 68% in Democratic Republic of the Congo, and 87% in Rwanda. Isolates presented 109 different profiles of mutations, including 73 occurring only once. Codon 12 was most frequently affected (15 isolates), including 10 isolates with Asp12Ala. These 10 isolates came from three different countries, and presented different profiles of resistance to other drugs. The two next most frequent mutations, Met175Ile and Gln10Pro (8 isolates and 7 isolates, respectively), each suggest clusters of transmission, with similar geographical and resistance characteristics. Moreover, four isolates presented two simultaneous genetic variations, and 11 patients had a mix of sensitive and resistant bacilli. Preliminary data tend to indicate that patients carrying a PZA-resistant isolate had a higher failure rate on the new short MDR-TB treatment regimen (7% vs. 3%). All isolates resistant to injectables (4) and most (19/21) of those resistant to fluoroquinolones, including two extremely-resistant TB isolates, were also resistant to PZA. Conclusion: Similar to other regions in the world, the majority of MDR-TB strains from Sub-Saharan Africa countries are resistant to PZA, albeit with diverse rates between countries. We identified a diverse range of mutations in pncA, with 30% of them not previously reported. The impact of such resistance on the success of the short MDR-TB regimen will require more investigation.

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Expanding tuberculosis (TB)-diagnostic services, including access to rapid tests, is a World Health Organization (WHO) strategy to accelerate progress toward ending TB. Faster and more sensitive molecular tests capable of diagnosing TB and drug-resistant TB have the technical capacity to address limitations associated with smears and cultures by increasing accuracy and shortening turnaround times as compared with those of these conventional laboratory methods. Nucleic acid amplification assays used to detect and analyze Mycobacterium tuberculosis (MTB)-complex nucleic acids can be used directly on specimens from patients suspected of having TB. Recently, several commercial molecular tests were developed to detect MTB and determine the drug resistance (DR) based on detection of specific genetic mutations conferring resistance. The first to be endorsed by the WHO was molecular line-probe assay technology. This test uses polymerase chain reaction (PCR) and reverse-hybridization methods to rapidly identify MTB and DR-related mutations simultaneously. More recently, the WHO endorsed Xpert MTB/RIF, Cepheid Inc, CA, USA, a fully automated assay used for TB diagnosis that relies upon PCR techniques for detection of TB and rifampicin resistance-related mutations. Other promising molecular TB assays for simplifying PCR-based testing protocols and increasing their accuracy are under development and evaluation. Although we lack a practical gold standard for the diagnosis of childhood TB, its bacteriological confirmation is always recommended to be sought whenever possible prior to a diagnostic decision being made. Conventional diagnostic laboratory TB tests are less efficient for children as compared with adults, because sufficient sputum samples are more difficult to collect from infants and young children, and their disease is often paucibacillary, resulting in smear-negative disease. These inherent challenges associated with childhood TB are due to immunological- and pathophysiological-response differences relative to those observed in adults. Several recent meta-analyses showed low sensitivity estimates of PCR-based TB assays for paucibacillary forms of TB (extrapulmonary TB and smear-negative pulmonary disease), which represent the vast majority of childhood TB cases. Despite the lack of evidence regarding use of the rapid molecular assays to identify TB and detect DR in children, and due to the clinical nature of childhood TB, TB-expert groups recommend including rapid methods for TB identification and DR detection in diagnostic algorithms for children suspected of both smear-positive and -negative pulmonary or extrapulmonary TB, both with or without human immunodeficiency virus (HIV)-coinfection, when combined with standard methods (including clinical, microbiological, and radiological assessment) for diagnosing active TB and conventional DR. Since 2011, the WHO has specifically recommended use of the Xpert MTB/RIF test as an initial diagnostic tool for children with suspected HIV-associated TB or multidrug-resistant TB based on successful treatment data related to adults. Implementation of the rapid molecular assays for rapid detection of TB and DR should occur in laboratories with proven capability to run molecular tests and where quality control systems are implemented. Molecular approaches should be more largely tested in children, given their status as the group in whom the diagnostic dilemma is most pronounced. These tests should also be included in specific childhood TB diagnostic algorithms adapted to the local/national context in combination with other strategies for improving diagnostics, including more effective specimen collection.

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Objective/background: The recent call for universal drug susceptibility testing (DST) for all tuberculosis (TB) patients will be difficult to meet in settings where Xpert rollout is limited, such as low prevalence of HIV and Multi-drug Resistant Tuberculosis (MDR) settings. As recommended by World Health Organization (WHO) guidelines, the success of TB treatment is measured by Ziehl–Neelsen (ZN) microscopy or auramine–rhodamine fluorescent microscopy (FM) on sputum, in which conversion to negative smear at 2 months (M) is an important predictor of treatment success, defined as a negative smear at 5M. The sputum smear that fails to convert to negative at 5M are screened for rifampicin resistance. We tested in a prospective study whether an early screen for rifampicin resistance, based on FM results at 2M, could detect MDR patients early, rather than screening all patients with GeneXpert MTB/Rif at baseline. Methods: Between February 2015 and August 2016, we enrolled new TB patients in an IRB-approved prospective cohort study at four health centers in Bamako district. Fresh sputum samples were collected at 2M and 5M to measure FM smear conversion. Patients who failed to show a decline in FM positivity at 2M (moderate or many Acid Fast Bacilli (AFB)) had their sputum tested in GeneXpert to detect rifampicin resistance. Patients who had any AFB seen at 5M were also tested using GeneXpert. Results: Of the 570 patients who were enrolled in the study, 22 (3.8%) died and 27 (4.7%) were lost to follow-up. The prevalence of HIV and TB coinfection was 12.4%, and 65.6% of the patients were male. At 2M, 32 out of 429 patients still had moderate or many AFBs in FM, and were screened by Xpert, of whom 5 (15.6%) tested rifampicin-resistant and were referred for MDR treatment. Of the 310 patients who completed 5M of treatment, 35 (11.3%) met the definition of failure (few or moderate AFB in FM) and had their sputum tested in Xpert; moreover, four (11.4%) demonstrated rifampicin resistance. In total, 67 (21.6% of 310) patients were screened by Xpert, of whom nine were detected to have MDR (or 13.4% of those screened). Conclusion: Although we cannot exclude additional MDR patients having been missed by our screening strategy, our screening algorithm at 2M detected five out of nine MDR patients. Detecting patients at 2M allowed for earlier referral, and potentially less acquired drug resistance and lower mortality. This strategy may be advantageous while awaiting further rollout of Xpert machines that will permit universal DST.

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Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a serious public health challenge towards which new hits are urgently needed. Medicinal plants remains a major source of new ligands against global infectious illnesses. In our laboratories, we are currently investigating locally used ethnobotanicals for novel compounds against zoonotic tuberculosis. The microplate alamar blue assay (MABA) was used to study the anti-TB activity while the CellTiter 96® AQ_ueous Assay, which is composed of solutions of a novel tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine methosulfate) PMS, was used for cytotoxic studies. Correlation coefficients (R²) were used to compare the relationship between antimycobacterial activity of the eight crude extracts against nonpathogenic strains and the pathogenic Mycobacterium bovis. Minimum inhibitory concentration (MICs) values indicated that all the eight tested medicinal plant species had activity against all the three tested mycobacterial strains. Minimum inhibitory concentration value as low as 19.5 μg/mL was observed against non-pathogenic strains M. bovis. Activity of the crude extracts against M. aurum was the best predictor of natural product activity against the pathogenic Mycobacterium bovis strain, with a correlation coefficient value (R²) of 0.1371. Results obtained from the current study validate, in part, the traditional utilization of the tested medicinal plants against tuberculosis. The unripe fruits from Solanum torvum are a potential source of safe and efficacious anti-TB crude drugs as well as a source for natural compounds that act as new anti-infection agents, and thus deserve further investigation towards development of a new class of molecules with activity against sensitive and drug resistant strains of M. bovis.

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Objective/background: The Eastern Mediterranean Region (EMR) has witnessed the largest refugee crisis in history. Overall, 70% of the global refugee populations are from Palestine, Syria, Afghanistan, or Somalia. We reviewed the possible impact of such crisis on the tuberculosis situation in EMR. Methods: We used the available data and information from the World Health Organization and other international and national institutions. Results: Overall, 15 out of 22 countries in the EMR are either engulfed in complex emergencies (10 countries) or suffering from their neighbors' complex emergencies (7 countries), whereas two countries suffer from both. Eighty-five percent of the total population (636 million) in the region lives in these 15 countries. For tuberculosis, these 15 countries account for a significant burden in EMR: 94% of the estimated total incidence of 740,000 cases a year and 95% of the estimated total mortality of 91,000 a year. These countries have yet to show the significant negative impact on tuberculosis epidemiology as such changes take considerable time to manifest. Still, there are reports on health systems impact: access to health facilities, destruction of health facilities, health staff casualties, and shortage of medicines. Conclusion: Complex emergencies pose a significant negative impact on tuberculosis in the EMR. This issue should be raised in the global health and political arena. This is a time bomb for tuberculosis.

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Objective/background: Childhood tuberculosis (TB) is largely a paucibacillary disease and difficult to diagnose. It is difficult to obtain a sputum or gastric aspirate (GA) sample, and patients are often undiagnosed and treated empirically. Stool is a noninvasive specimen not usually used for TB testing in Pakistan. We investigated the value of Xpert MTB/RIF to diagnose Mycobacterium tuberculosis (MTB) in children with pulmonary TB cases, by performing comparative testing of GA and stool samples. Method: We recruited 60 children aged 1–15 years, suspected of TB, from the Department of Pediatrics, Civil Hospital, Karachi, Pakistan and The Aga Khan University Hospital, Karachi, Pakistan. All were immunocompetent. Patients had a Kenneth Jones TB score of ≥5. Paired GA/sputum and stool samples were collected for testing. All GA samples were tested by Xpert MTB/RIF assay and MTB culture, while stool was tested by Xpert MTB/RIF. Results: The study participants included 27 males and 23 females with a mean age of 6 years and a mean TB (Kenneth Jones) score of 7. Stool was received in the laboratory within 1–2 days of the GA sample for all but one participant, who expired. The rates of MTB detection were as follows: 22% (11 cases) based on Xpert MTB testing of GA, 21% (10 cases) based on MTB culture of GA, and 21% (10 cases) based on Xpert MTB testing of stool. No rifampicin resistance was detected. Overall, there was concordance between testing of GA and stool. One case had GA with low positive Xpert and positive MTB culture, but negative stool Xpert result. In another case, there was low positive GA Xpert, positive GA MTB culture, and positive stool Xpert. A positive Xpert MTB stool test was associated with a higher TB score (>5) and a greater bacillary load. All 11 cases of TB diagnosed were put on antituberculous therapy and responded well to treatment. Conclusion: Use of Xpert MTB/RIF assay for stool-based diagnosis of pulmonary TB in immunocompetent children is useful in a resource poor setting. This is a valuable and noninvasive diagnostic alternative for the diagnosis of childhood TB and can be adapted by pediatric arms of national TB programs.

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Background: There is growing evidence that emotional distress expressed in terms of anxiety and depression it is very high among tuberculosis (TB) patients. Objectives: This study aims to determine levels of anxiety, depression and emotional distress in patients with several types of TB and to determine the association between social-demographic and economical factors, clinical variables and anxiety, depression and emotional distress. Methods: A cross-sectional study was performed in a sample of 81 TB patients. A social-demographic and economical questionnaire was used, followed by the Hospital Anxiety and Depression Scale. Results: 38.3% and 49.4% of our sample presented significant levels of anxiety and depression. 44.4% of patients had significant levels of emotional distress. Married subjects, a diagnosis of extra-pulmonary TB and multidrug resistant TB were related to higher risk for anxiety. Gender, extra-pulmonary and multidrug resistant TB were associated to depression. Female gender and cases of extra-pulmonary TB presented a 1,5 times risk for emotional distress. Conclusions: Our study found high rates of anxiety, depression and emotional distress among TB patients. Marital status, gender, type and treatment of TB were related to higher levels of emotional disorder. Mental health services should be an integral part of programs against tuberculosis.

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When the tuberculosis epidemic reached its peak in central Europe in the 1900s (not until the 2000s in sub-Saharan Africa), both contained and disseminated tuberculosis was mainly regarded as a childhood disease. From 1920, before the use of the Bacillus Calmette–Guérin (BCG) vaccine and the possibility of drug treatment, there was a drastic decline in the rate of tuberculosis incidence in the Western world. In 1970, the case rate had declined in Sweden from 500/100,000 individuals to 1/100,000 individuals. We recently studied childhood tuberculosis in the Stockholm area from 1971 to 2015. During this period the case rate had increased from 1/100,000 individuals to 8/100,000 individuals. A major contributing factor has been increased immigration and, more recently, from high incidence countries. While there are very few cases in Swedish children whose parents are also born in Sweden, the case rate is still high in foreign-born children, for example, 450/100,000 foreign-born Somali children. At the beginning of the past 45 years, sentinel (infected in Sweden with no known source person) cases were a reality but they are rarely seen today. This may be attributed to better organization, contact tracing, screening, and early diagnosis in the adult population.

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Objective/Background: Resistances to herbal medicines are still not defined and finding natural remedies against drug resistant Mycobacterium tuberculosis (MTB) has research priority. The antimycobacterial susceptibility method for herbal extracts is unclearly defined and there is no standard method for assessment of the materials against bacteria. In the present study, time kill of three medicinal plants was determined against MTB. Methods: The clinical isolate of MTB from a patient who harbored confirmed tuberculosis was used in the study. Aqueous extracts of Aloe vera leaves, mint, and Hypericum perforatum were prepared using reflux distillation. Disk diffusion methods were conducted in Petri dishes and McCartney bottles containing Löwenstein–Jensen medium to measure the sensitivity of plant extracts in serial concentrations of 0.25–8 mg/mL. A pour plate method was performed by mixing 0.7 mL of each concentration of extract in 5 mL Löwenstein–Jensen medium followed by surface culturing of MTB fresh cells. The time kill method was conducted by bacterial suspension in equal amounts of the extract and viable evaluation in fresh culture at the beginning, and at 24-h, 48-h, 72-h, and 1-week intervals. All cultures were incubated at 37°C for 4 weeks. Inoculum concentrations were considered as a variable. Results: The zones of inhibition of A. vera, H. perforatum, and mint extracts in the disk diffusion method in McCartney bottles were 60 mm, 41 mm, and zero, respectively, but Petri dishes did not have repeatable results. In the pour plate method, an extract concentration up to 1 mg/mL could inhibit cell growth. In mint extract, colony forming was four times more than the others at 0.5 mg/mL. Time kill of 95% of cells occurred when exposed to extracts of A. vera and H. perforatum separately, but was 50% in 24 h and 20% in 10 min. The time kill for mint was 95% in 1week. Conclusion: The results give some scientific basis to the use of plant extracts for growth control of MTB cells. Clinical trials are recommended for assessment of the extract as complementary medicine, as well as for antisepsis.

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A major change of therapy in respiratory medicine has been the transition from oral or parenteral to inhalation therapy, for example, in asthma. Inhalation of anti-infectious drugs has however not a key-role in the treatment of pulmonary infections such as tuberculosis (TB). The inhalation therapy provides several benefits; the target is reached directly with evasion of first-pass metabolism, thereby resulting in reduced systemic side effects. Furthermore, the drug is delivered to an extensive surface area that is rich in lymphoid tissue. The inhalation therapy is also easier to monitor since a more direct response is expected than orally administered drugs. Local side effects are, however, common and may depend on inadequate inhalation technique or devices. However, there are problems to consider regarding the delivery of drugs by inhalation: the anatomic structure of the tracheobronchial tree and the impact of the disease on the normal bronchial lining and the mucus. The latter may have an impact on the absorption of the inhaled drug because the mechanical and immunological defense mechanisms play a crucial role for the balance between clearance and absorption. The inhaled drug is expected to be rather effective in the overt presence of bacteria as in smear-positive cases of TB in which the bronchial tree may be directly connected with the cavitary lesions. Such compartments have more rapidly multiplying TB bacteria than other TB-infected compartments. The hypothesis is that the period of contagiousness is expected to be shorter and the recovery faster if there is an intervention directly against the major burden of TB bacteria. The size of the drug particles is essential to overcome the anatomical barriers. To improve the delivery of drugs, they should be in the form of fine particles, that it <5 μm in size. Particles sized <2 μm can be deposed in the alveoli. To encapsulate drugs for pulmonary delivery in liposomes has several advantages. There will be a prolonged release of the drug in the large surface area of the lungs and a high permeability of the epithelium through the liposome morphology. In general, liposomes are designed as closed spherical vesicles with a wall of a lipid bilayer that encapsulates an aqueous phase in which drugs can be stored. TB treatment with drugs administered by inhalation and liposomes is one future alternative. There are other possibilities for evaluation as well, such as high-dose rifampicin therapy and novel drugs. All new possibilities have to be considered with scientific scrutiny, proper management, and adherence. The clinical community and the patients cannot lose any more opportunities in the management of TB.

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Objective/background: The aims of this talk are to: (a) provide an overview of our method of collecting, quantifying, and sizing infectious aerosols of Mycobacterium tuberculosis; and (b) to review data indicating that cough aerosol cultures of M. tuberculosis are the best predictors of infection and incident disease among household contacts of persons with active tuberculosis (TB). New infection was defined as tuberculin skin test conversion. Methods: We developed a cough aerosol sampling system by placing two Andersen cascade impactor viability samplers inside a cylinder into which patients cough via connector tubing. We recruited sputum acid fast bacilli (AFB) smear-positive patients from the tuberculosis clinic and wards at Mulago Hospital in Kampala, Uganda. Patients were asked to cough as strongly and frequently as they comfortably could for two 5-min sessions of coughing. Results: In a cohort of 96 sputum culture positive index TB cases, 43 (45%) produced culture-positive cough aerosols. Household contacts of TB patients who produced high aerosols (≥10colony forming units (CFU)) were more likely to have a new infection compared with contacts with low aerosol CFU (1–9CFU) and aerosol-negative cases (69%, 25%, and 30%, respectively, p = 0.009). In adjusted multivariate analyses, high cough aerosols were the only predictor of new TB infection (odds ratio [OR] 4.81; 1.20–19.23). In a follow-up (median 3.9 years) of this cohort, 369 (84%) of the contacts could be traced; eight (2%) had developed TB disease. Incident TB disease was associated with larger bacillary load in sputum measured by days to positive in liquid culture (OR 7.9; 0.7–70.5), exposure to a high-aerosol TB case patient (OR 6.0, 1.4–25.2) and marginally to HIV infection in the contact (OR 7.2; 0.7–70.5). Cough aerosol studies of TB patients in Brazil and South Africa are ongoing and appear to be finding similar proportions of cough aerosol cultures among TB patients (personal communication). Conclusion: Cough aerosol cultures of M. tuberculosis are the best predictors of infectiousness and predict incident TB disease among sputum smear-positive patients in Uganda. We propose that cough aerosol cultures are a better surrogate of inhaled dose than sputum smear.

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Objective/background: Over-the-counter availability of antibiotics together with poor access to diagnostics is recognised to promote antimicrobial resistance (AMR) including generation of drug-resistant tuberculosis (DR-TB). In accordance with the End TB Strategy target of ending TB epidemic by 2030, efforts to control DR-TB are ongoing in Eastern Mediterranean Region (EMR) countries, a number of which have well-established facilities for diagnostics, disease care and prevention as well as surveillance. These could serve as models for AMR control. The United Nation's historic 2016 declaration recognises AMR as a threat to health, food production and development, and emphasises the need for global action. In view of this declaration, establishment of collaboration between the DR-TB and AMR activities would be mutually beneficial and lead to strengthening of both programs. Methods: Available information on TB control and AMR programs in EMR was reviewed. To assist with policy and planning strategies for promoting collaboration between AMR and TB, control activities were explored. Results: Review of available information suggests gaps in TB care in many countries in EMR, most of which are linked to limited access to resources. At the same time, the fledgling AMR programs have a lot to learn from the experiences, successes and challenges faced by TB control efforts. A logic model is presented to enhance interprogram collaboration. Conclusions: Given the global commitment and potential availability of resources towards controlling AMR, collaboration between the two programs is discussed towards a more efficient use of resources in the region.

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Objective/background: Molecular-based, rapid drug-susceptibility tests are needed to guide the appropriate use of new drugs and new therapeutic regimens at the programmatic level, and to prevent a further increase in the incidence of drug-resistant tuberculosis (TB). Experts have recognized the need for a global, curated, and standardized analysis and data-sharing platform that provides a one-stop data source for clinically relevant genotypic and phenotypic information on Mycobacterium tuberculosis. Methods: To this purpose, the Relational Sequencing TB Data Platform (ReSeqTB) consortium has critically reviewed the most inclusive set of published data on mutations associated with drug resistance in M. tuberculosis to date, and has graded a comprehensive list of globally prevalent mutations based on the strength of their association with drug resistance. Results and Conclusions: ReSeqTB serves as a single repository for the compilation, curation, and validation of existing and newly created sequences and metadata on M. tuberculosis strains and will use the currently reviewed data set, validated by international experts, as a starting point until sufficient new sequence data are accumulated. This initiative is supported by a global partnership of academic institutions, public health agencies, and nongovernmental organizations including the Critical Path Institute, FIND, the World Health Organization, the New Diagnostics Working Group, the U.S. Centers for Disease Control and Prevention, and the National Institute of Allergy and Infectious Diseases and it is financially supported by the Bill & Melinda Gates Foundation. Key strengths of the ReSeqTB Database include the following:

• A user-friendly interface designed for nonexpert or expert operability.
• A standardized and validated analysis pipeline for variant analyses of M. tuberculosis next-generation sequencing (NGS) data.
• Access to data beyond the published literature with dynamic and iterative updates of new data generated by global surveillance and clinical trials.
• A well-developed legal structure to ensure intellectual property rights and data ownership remain with contributors.
• A structured data-sharing architecture to restrict access to sensitive or unpublished data sets.
• Metadata standardization using CDISC: supports global, platform-independent data standards that enable information system interoperability.
• An emphasis on data quality and rigorous, expert curation with multiple quality control checks for whole-genome sequencing and other metadata.
• Validation of NGS analysis output by an expert committee with grading of resistance conferring mutations based on rigorous statistical standards.
• Regulatory-compliant analysis pipeline and database architecture.

Successful execution of such an extensive database platform requires substantial collaboration from scientists investigating the genetic basis for drug resistance worldwide, and from developers with expertise in database design and implementation.

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Background: Despite the strong association between drug resistance and genetic mutations, the value of molecular diagnosis of drug resistance to guide the treatment of multidrug-resistant tuberculosis (MDR-TB) remains unclear. This is particularly relevant in resource-limited areas, in which it is difficult to implement the drug susceptibility test. Here, we focused on the association of drug susceptibility phenotype and genotype with treatment outcomes in patients with MDR-TB. Methods: In a prospective cohort study, we enrolled 252 consecutive patients with confirmed MDR-TB between 2010 and 2013, and outcomes were followed-up over the 24-month treatment course in terms of clinical manifestation and sputum conversion. All the isolates were tested for phenotypic susceptibility to second-line drugs in the Mycobacteria Growth Indicator Tube based system, and genotypic mutations were assessed by DNA sequencing. Results: Among the 252 MDR-TB isolates, 88 (34.9%) were resistant to fluoroquinolones and/or second-line injectable drugs, of which 65 (73.9%) harbored a mutation in drug resistance-related genes (gyrA, rrs and eis). In addition, 85 individuals (33.7%) were also resistant to pyrazinamide, with 87.1% containing the pncA mutation. Of 252 MDR-TB patients, 207 (82.1%) had known outcomes and 45 (17.9%) were lost to follow-up. Among those with known outcomes, treatment succeeded in 85.8% with plain MDR-TB, 69.7% with initial resistance to either a fluoroquinolone or second-line injective drugs, 37.5% with initial resistance to pyrazinamide, 29.3% with initial extensively drug resistance. In contrast, among those with known outcomes, treatment success and culture conversion depends on the susceptibility to drug especially for pyrazinamide and fluoroquinolones. In multivariate analysis, pyrazinamide resistance and its related pncA gene mutation were independently associated with a lower risk of culture conversion on at 8 weeks and treatment success, while fluoroquinolone resistance was negatively correlated with treatment success. Besides, specific treatment, patient and program variables were also associated with treatment outcome. Conclusion: Drug susceptibility testing for pyrazinamide and fluoroquinolones together with genetic information appears to provide a clinically useful indicator of the treatment outcome of MDR-TB in China.

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Since 2002, there has been a gradual worldwide 1.3% annual decrease in the incidence of tuberculosis (TB). This is an encouraging statistic; however, it will not achieve the World Health Organization's goal of eliminating TB by 2050, and it is being compounded by the persistent global incidence of drug-resistant tuberculosis (DR-TB) acquired by transmission and by treatment pressure. One key to effectively control tuberculosis and the spread of multiresistant strains is accurate information pertaining to drug resistance and susceptibility. Next-generation sequencing (NGS) has the potential to effectively change global health and the management of TB. Industry has focused primarily on using NGS for oncology diagnostics and human genomics, but the area in which NGS can rapidly impact health care is in the area of infectious disease diagnostics in low- and middle-income countries. To date, there has been a failure as a community to capitalize on the potential of NGS, especially at the reference laboratory level where it can provide actionable information pertaining to treatment options for patients. The rapid evolution of knowledge about the genetic foundations of tuberculosis drug resistance makes sequencing a versatile technology platform for providing rapid, accurate, and actionable results for treating this disease. No “plug-and-play” and “end-to-end” NGS solutions exist that provide clinically relevant sequence data from the Mycobacterium tuberculosis complex genome from primary clinical samples (e.g., sputum) in high-burden country reference laboratories, which is where they are most needed. However, such a system-based solution is underdeveloped by Foundation for Innovative Diagnostics (FIND), in collaboration with partners from academia, nongovernmental organizations, and industry. The solution is modular and is designed and developed to perform targeted amplicon sequencing directly from a patient's primary sputum sample. This solution will initially allow reference laboratories to perform reflex NGS that provides a rapid and comprehensive analysis of a patient's M. tuberculosis complex drug resistance profile, thereby facilitating optimization of a patient's treatment, improving treatment outcomes, and reducing the spread of DR-TB. Such a system could also enable countries to implement culture-free drug resistance surveillance programs, which could bypass the need for expensive culture facilities, decrease a country's dependence on external laboratories, and significantly expand the map of global surveillance capabilities. In addition, the introduction of such a system will provide a foundation for NGS to be used for genotypic testing for human immunodeficiency virus-infected patients, surveillance of other diseases, in-country capability for outbreak discovery and management, and a host of other diagnostic benefits that are currently limited to high-income countries.

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Objective/Background: Paratuberculosis (Johne's disease) is a chronic infectious granulomatous enteritis, primarily affecting ruminants, and caused by Mycobacterium avium ssp. paratuberculosis (MAP). The disease is widely prevalent throughout the world with significant economic losses. MAP has also been implicated with human Crohn's disease. There exists a strong correlation between the immune response and development of various types of pathologies in ruminants. The polarization of the immune response, which is critical to clinical outcome of the paratuberculosis infection, is controlled by the differential expression of certain cytokines and inducible nitric oxide synthase (iNOS) in Johne's disease. In previous studies, the role of different cytokines (Th1 and Th2) has been occasionally studied in sheep paratuberculosis. In the present study, we studied differential expression of interferon (IFN)-γ, interleukin (IL)-1α, IL-10, transforming growth factor (TGF)-β, iNOS, and TRAF1 genes in MAP-infected sheep and established relationship with distinct pathologies. Methods: Tissue sections (small intestine, ileocecal junction, and mesenteric lymph nodes) were collected from sheep suspected for Johne's disease and appropriately preserved for RNA extraction, polymerase chain reaction (PCR) analysis, and histopathology. Pathologic grading was done on the basis of nature and extent of cellular infiltration, granuloma formation and abundance of acid-fast bacilli. Six sheep each with pauci (PB)- and multibacillary (MB) lesions and six healthy control sheep were selected for cytokine studies. MAP in tissue extracted genomic DNA of sheep was quantified by a quantitative PCR assay. Tissue extracted RNA was reversed transcribed to prepare c-DNA from which quantitative reverse transcription PCR (qRT-PCR) was performed to amplify IFN-γ, IL-1β, IL-10, TGF-β, β-actin, TRAF1, and iNOS with Quantitect SYBR Green Master Mix. qRT-PCR data were analyzed using 2^−ΔΔCT method using β-actin gene as a control. All qRT-PCR results were compared by using one-way analysis of variance (least significance difference and Duncan tests) for p-value using SPSS (version 7.5) for expression of each gene in tissues from infected and control sheep. Results: In the small intestine, PB sheep showed significant enhancements in the expression of IL-10, TGF-β, iNOS, and IFN-γ in comparison to similar tissues from uninfected control sheep. IL-1α expression was significantly reduced (p <0.01). The expression of IL-10 in the mesenteric lymph node (MLN) tissue of PB sheep was significantly increased (p <0.01) as compared with the control sheep. MB sheep revealed significantly enhanced expression of TGF-β mRNA and reduction in the expression of IL-1α in comparison to control sheep. In the MLN of MB sheep, the expressions of IL-10 and TGF-β were significantly (p <0.01) increased, and IFN-γ was significantly downregulated in comparison to uninfected control sheep. When the cytokine expression was compared between two distinctly infected groups, the MB sheep showed highly significant decrease (p <0.01) in the expression of iNOS and IFN-γ in the small intestine and IFN-γ in the MLN tissues. Conclusion: The present study indicated that IFN-γ and iNOS were found to play important role in the induction of Th1 type immune response in PB sheep. MB sheep had significant reduction in expression of IFN-γ and iNOS and elevation of IL-10 and TGF-β, which was typical of Th2 cytokine pattern. Elevated expression of IL-10 and TGF-β in PB cases possibly suggests the role of T-regulatory cells and may follow an independent mechanism not typical of Th1 pattern. In view of significantly reduced expression in both forms of the disease, IL-1α may not be an important cytokine in ovine paratuberculosis.

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Several studies have been done in relation to recurrence of tuberculosis (TB) following completion of treatment. However, recurrence of TB is still a major problem from a public health perspective in high-burden countries, where no special attention is being given to this issue. Disease recurrence is an important indicator of the efficacy of antituberculosis treatment. The rate of recurrence is highly variable and has been estimated to range from 4.9% to 25%. This variability is not only a reflection of regional epidemiology of recurrence but differences in the definitions used by the TB control programs. In addition to treatment failure related to medication adherence, there are several key host factors that are associated with high rates of recurrence. The widely recognized host factors independent of treatment program that predispose to TB recurrence include: malnutrition; human immunodeficiency virus; substance abuse including tobacco use; comorbidity such as diabetes, renal failure and systemic diseases, especially immunosuppressive states; and environmental exposure such as silicosis. With improved understanding of the human genome, proteome, and metabolome, additional host-specific factors that predispose to recurrence are being discovered. Information on temporal and geographical trends of TB cases as well as genotyping might provide further information to enable us to fully understand TB recurrence and discriminate between reactivation and new infection. The recently launched World Health Organization End TB Strategy emphasizes the importance of integrated, patient-centered TB care. Continued improvement in diagnosis, treatment approaches, and defining host-specific factors are needed to fully understand the clinical epidemiological and social determinants of TB recurrence.

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Aims and objective: Despite effective treatment of pulmonary tuberculosis (TB) patients, destruction of lung parenchyma may lead to complications including repeated infections. These infections are often misdiagnosed or wrongly identified as TB recurrence, and hence are not treated effectively. The frequency and severity of these infections vary with the extent of damage, and are much more prominent in patients with post-TB bronchiectasis and fibrocavitary diseases. This presentation will focus on the epidemiology, treatment, and management of post-TB infections and challenges, and the impact of these infections on public health in high-TB-burden countries. Methods: Published literature and review articles were evaluated to address this objective. Results: Apart from conventional agents of pneumonia, patients with post-TB bronchiectasis and post-TB fibrocavitary diseases are prone to develop chronic pulmonary aspergillosis and nontuberculous mycobacterial infections. A high burden of chronic pulmonary aspergillosis has been reported in TB-endemic countries. Similarly, prior TB increases the risk of acquiring nontuberculous mycobacterial infections. Diagnosis and management of chronic pulmonary aspergillosis and nontuberculous mycobacterial infections require expertise and high-level care. Conclusion: Limited diagnostic and therapeutic capacities compounded by nonavailability of essential antimicrobials in most high-TB-burden countries pose great challenges to physicians involved in the management of these infections. These infections affect the overall outcome and lead to high cost for public health systems.

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Objective/background: Molecular methods for the detection of Mycobacterium tuberculosis (MTB) are widely used for the early diagnosis of tuberculosis (TB). The objectives of this study were to evaluate the performance of two molecular techniques for the detection of MTB for both pulmonary and extrapulmonary specimens in comparison with conventional culture techniques in Kuwait and to correlate the results with clinical and histopathological diagnosis. Methods: A total of 9,594 consecutive pulmonary (n = 5,597) and extrapulmonary (n = 3,997) specimens received by the Kuwait National TB Reference Laboratory from November 2011 to December 2015 were processed for direct smear microscopy for acid-fast bacilli using a Zhiel-Neelsen stain, Xpert MTB/RIF assay, ProbTec polymerase chain reaction (PCR), and growth in fully automated MGIT 960 system tubes. The clinical and histopathological diagnoses were correlated with the results. Results: For pulmonary specimens, the overall sensitivity and specificity of both molecular methods together were 90.7% and 99.7%, respectively. The sensitivity and specificity of the Xpert MTB/RIF assay were 90.1% and 99.4%, respectively. The sensitivity and specificity of ProbTec PCR were 86% and 99.6%, respectively. For extrapulmonary specimens, the overall sensitivity and specificity of both molecular methods together were 87% and 97.8%, respectively. The sensitivity and specificity of Xpert MTB/RIF assay were 89.5% and 97.2%, respectively. The sensitivity and specificity of ProbTec PCR were 73% and 97.8%, respectively. When clinical and histopathological correlations were made with the assumed false-positive results from both molecular methods, the corrected sensitivity and specificity for pulmonary specimens were 91% and 99.9%, respectively, and 89.6% and 99.8% for extrapulmonary specimens, respectively. Conclusion: The performance of the Xpert MTB/RIF assay was superior to ProbTec PCR for the early detection of MTB from both pulmonary and extrapulmonary specimens.

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Objective/background: Despite the important role that the African region plays in a global tuberculosis (TB) epidemiological context, many countries in the region still lack data on the prevalence of specific Mycobacterium tuberculosis strains and drug resistance. This is the case for Angola, which presently lacks any data concerning drug-resistance rates and prevalence of specific M. tuberculosis genotypes and respective population structure. In this study, we made the first characterization of the genetic diversity and drug resistance of M. tuberculosis complex strains circulating in Luanda, Angola's most important setting concerning TB epidemiology. Methods: We have analyzed 89 M. tuberculosis isolates recovered from the same number of patients. All clinical isolates were genotyped by spoligotyping and 24-loci mycobacterial interspersed repetitive unit–variable number of tandem repeats (MIRU–VNTRs). First-line drug-susceptibility testing was performed by the standard BACTEC 960 Mycobacteria Growth Indicator Tube (MGIT) procedure. Results: We have detected 33 different spoligotype profiles corresponding to 24 different shared international types (SITs) and nine orphan profiles. SIT 20 (LAM1) was the most prevalent (n = 16, 18.2%) followed by SIT 42 (LAM9; n = 15, 17.1%). Overall, the M. tuberculosis population structure in this sample was dominated by LAM (64.8%) and T (33.0%) strains. Twenty-four-loci MIRU–VNTR analysis revealed that a total of 13 isolates were grouped into five distinct clusters. Drug-susceptibility testing revealed a worrying situation concerning resistance rates. Drug-susceptibility data showed that 22 (24.7%) of the 89 clinical isolates were resistant to one or more antibacillary drugs of which four (4.5%) were multidrug resistant (MDR). Drug-resistant isolates were found across distinct clades and MIRU–VNTR clusters. Conclusion: This first cross-sectional study conducted in Luanda, Angola, provides a framework for future studies and programmatic management of TB in Angola. We provide sufficient evidence for cluster-based transmission with a high predominance of LAM strains, with differential geographic dispersion. The moderate rate of MDR-TB found in this sample has major public health implications and highlights the need for further studies specifically focused on MDR-TB transmission.

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Aims and objectives: The WHO estimate that 8.8 millions new cases of tuberculosis occurred in 2010, (between 8.5 and 9.2 millions) linked to 1.45 millions death cases. The load of children tuberculosis is estimated at 10–15 per cent of the total load. In 2014 more than one million children have developed the disease. The children just as adults are exposed to contract and develop the multi resistant forms of the tuberculosis, constituting a major issue for the disease control. The children less than 5 years of age are the most exposed to present the most serious and more often deadly forms of the illness. Further, in many developing countries, the lack of pediatric forms of the tuberculosis drugs makes it difficult to control the problem. The tuberculosis diagnosis among the children is based on a set of arguments: the presence of a tuberculous person excreting bacillus, exposition and receptivity conditions of the child (the level of his immunity, the level of under nutrition, associated pathologies etc…). The diagnosis is also based on the research of the symptoms and other signs suggestive of tuberculosis: tuberculin skin test, thoracic radiography, interferon-gamma test. The aim of this study, is to describe and analyze the features and difficulties of the biological diagnosis of tuberculosis among the children and to find a strategy for the improvement of the results. Methods: It's a retrospective study from 2002 to 2015, dealing with pediatric patients' records from whom a bacteriological diagnosis was requested. We took advantage of the methods used on the laboratory to establish a diagnosis: microscopy, culture, study of the susceptibility to the tuberculosis drugs in solid medium and molecular biology. Results: From 2002 to 2015, only 207 strains were isolated from the children samples, aged from 0 to 15 years predominantly female sex (sex ratio is 0.53) with an average age of 9 years. The detailed results of the diagnosis methods of tuberculosis and the drugs resistance will be presented. Conclusions: Tuberculosis in children is often undiagnosed or difficult to diagnose, most developing countries still using ancient methods which can recognize only the developed tuberculosis. It's necessary to evaluate the issue's importance in order to improve the diagnosis conditions (systematic culture and susceptibility test in children), and to ensure the availability of the effective treatment (the pediatric formulation of the essential drugs).

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In 2014, the World Health Organization (WHO) recommendation to include the endorsed rapid molecular technologies (Xpert MTB/RIF, line probe assays) into surveillance systems and surveys allowed the testing of more tuberculosis (TB) patients for drug resistance at country level than ever before. The whole genome sequencing (WGS) approach is emerging as a more powerful tool for epidemiological and drug-resistant routine surveillances, promising a rapid and simultaneous screening of all the clinically-relevant mutations for the determination of resistance to the first-, second-line, and new anti-TB drugs. In addition, WGS can support the conventional contact tracing for epidemiological studies with high discriminatory power by tracking the circulating strains and their relatedness. These features make WGS, moreso than the conventional molecular tools, an ideal tool to monitor transmission and drug resistance trends in countries, providing deep and wide information in a standardized way. WGS technologies have already been adopted in many supranational and reference laboratories at the centralized level, and several research groups are working to reduce the complexity and costs of these platforms, from sample preparation to the downstream analysis and interpretation of sequencing reads, with the final aim to expand the use of WGS to all laboratory levels. The landscape of the platforms available for next-generation sequencing (NGS) is rapidly enriching. It includes high-throughput instruments that can be used for centralized surveillance studies on a large scale, and “benchtop” sequencers that conversely can reach more peripheral settings for rapid and non-extensive surveys. Traditionally, WGS is performed on genomic DNA samples extracted from clinical isolates to ensure the required high DNA quality and quantity for the following library preparation and sequencing reaction steps. Nevertheless, the researchers are trying to apply the WGS to early primary cultures and in particular directly to sputum samples, including specific procedures to remove non-mycobacterial genetic material and to enrich the Mycobacterium tuberculosis (MTB) genome. The targeted NGS approach that takes advantage of the amplification of selected regions of the MTB genome for genotyping and drug resistance determination could represent the most effective method to avoid the need of culturing MTB prior to sequencing, also enabling the implementation of NGS for surveillance purposes in resource-limited settings without infrastructures and equipment for growing TB cultures. Classical sequencing and NGS approaches have been successfully used in a recent study conducted in five countries with high burden of TB and multidrug resistant tuberculosis (MDR-TB) and aimed at investigating levels of resistance to pyrazinamide among patients with TB by pncA sequencing [doi: 10.1016/S1473-3099(16)30190-6]. This work innovatively demonstrated that the establishment of strong links between national (peripheral and reference laboratories) and supranational laboratories, with the former possibly processing indirect or direct samples and generating sequencing data, and the latter supporting them for bioinformatics analysis and data interpretation, will soon make WGS and targeted NGS the preferred tools to conduct public health surveillances in TB field, thus helping the strategies adopted by TB control programs at local and national levels.

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Background/objective: We urgently need novel treatments for multidrug-resistant tuberculosis (MDR-TB). Autologous mesenchymal stromal cell (MSC) infusion is one such possibility due to its potential to repair damaged lung tissue and boost immune responses. We aimed to assess the safety and effectiveness of MSC to improve treatment outcomes among MDR-TB patients. Methods: We analyzed treatment outcomes for 108 Belarusian MDR-TB patients receiving chemotherapy. Thirty-six patients (cases) also had MSCs collected, extracted, cultured, and reinfused (average time from chemotherapy start to infusion was 49 days) in optimal dose; another 36 patients (without MSC treatment) were “study controls”. We identified another control group: 36 patients from the Belarusian national surveillance database (surveillance controls) 1:1 matched to cases. Results: Successful outcomes were observed in 81% of cases, 42% of surveillance controls, and 39% of study controls. After adjusting for age, odds of a successful outcome were 6.5 (95% confidence interval, 1.2–36.2, p = 0.032) times greater for cases than surveillance controls. Adjusting for other potential confounders increased the effect estimate while maintaining statistical significance. Cases were less likely (p = 0.01) to be culture negative at 2 months than surveillance controls, indicating a poorer initial prognosis in cases before (or shortly after) infusion. Radiological improvement was more likely in cases than in study controls. Conclusion: MSC treatment could vastly improve treatment outcomes for MDR-TB patients. Our findings could revolutionize therapy options and have strong implications for future directions of MDR-TB therapy research

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Even in the 21st century, we are losing the battle against eradication of tuberculosis (TB). In 2015, 9.6 million people were estimated to have fallen ill with TB, of which 1.5 million people died. This is the real situation despite the well-structured treatment programs and availability of effective treatment options since the 1950s. The high mortality rate has been associated with other risk factors, such as the HIV epidemic, underlying diseases, and decline of socioeconomic standards. Furthermore, the problem of drug resistance that was recognized in the early days of the chemotherapeutic era raises serious concerns. Although resistance to a single agent is the most common type, resistance to multiple agents is less frequent but of greater concern. The World Health Organization estimated approximately 5% of all new TB cases involved multidrug-resistant (MDR)-TB. The estimation for MDR-TB is 3.3% for new cases, and 20.5% for previously treated cases. Failure to identify and appropriately treat MDR-TB patients has led to more dangerous forms of resistant TB. Based on World Health Organization reports, 5% of global TB cases are now considered to be extensively drug resistant (XDR), defined as MDR with additional resistance to both fluoroquinolones and at least one second-line injectable drug. XDR-TB had been reported by 105 countries by 2015. An estimated 9.7% of people with MDR-TB have XDR-TB. More recently, another dangerous form of TB bacillus was identified, which was named totally drug resistant (TDR-TB) or extremely drug resistant TB. These strains were resistant to all first- and second-line anti-TB drugs. Collectively, it is accepted that 2% of MDR-TB strains turn to be TDR-TB. This number, however, may not reflect the real situation, as many laboratories in endemic TB countries do not have proper facilities and updated protocols to detect the XDR or TDR-TB strains. Nevertheless, existing data emphasize the need for additional control measures, such as new diagnostic methods, better drugs, and more effective vaccines to prevent the spread of these strains around the world.

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Objective/Background: The objectives of this study were to highlight the key challenges and the strategic directions to scale-up tuberculosis (TB) care in the Eastern Mediterranean Region (EMR); to review TB burden in EMR, and evaluate the progress toward Millennium Development Goals (MDGs) targets by 2015; to identify the main obstacles for optimal TB care toward TB elimination; and to provide strategic directions to address the key challenges. The Eastern Mediterranean Region is composed of 22 countries with 648 million populations. The region has estimated TB incidence of 116 per 100,000. The estimated number of incident TB cases in 2015 was 749000 TB case. Methods: The paper is based on the Annual World Health Organization Global TB report on implementation of TB control strategy. Results: The EMR is a middle TB- and MDR-TB-burden region with 22 countries. As for MDGs, the region was able to meet the mortality target and revert TB incidence but could not achieve the prevalence target. The main challenges are limited infrastructure, human capacity, funds, use of new diagnostics tools and new medicines, and involvement of all stakeholders and community in TB care and control in addition to complex emergencies. Conclusion: The year 2015 marks a transition from the MDGs to the Sustainable Development Goals and from the Stop TB Strategy to the End TB Strategy. The region will move into three main directions: complete pending tasks, mitigate the impact of the complex emergencies on TB control, and move toward TB elimination.

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Multidrug-resistant tuberculosis (MDR-TB), and even more severe forms of drug resistance, cause significant problems and costs for national TB control programs and constitutes an increasing public health concern globally. In parts of the former Soviet Union, the prevalence of MDR-TB is as high as 50% and one third of all newly detected TB patients are infected with MDR strains. Such strains transmit and certain MDR-TB clones constitute an important part of the problem, especially in high MDR-TB burden areas. There are several actions that should be given priority to control this situation. A first important step is timely detection of all patients infected with resistant strains, which makes possible prompt change of standard TB chemotherapy to more effective combinations of drugs. This is important both from the public health and clinical perspectives, since it renders the individual patient noninfectious and subsequently cured. Early detection of MDR-TB also allows infection control to be focused where it is most needed. Strengthened infection control measures are crucial for limiting the ongoing spread of resistant TB in hospitals and elsewhere. In addition, a sustainable drug supply must be ensured to guarantee that all patients are initiated on effective treatment and can avoid interruptions due to drug shortages. An extra focus should be put on vulnerable cases, such as immunosuppressed individuals, prisoners, drug addicts, and migrants, in whom TB is generally more frequent and difficult to control than in the normal population. Finally, political support is needed to ensure necessary infrastructures, human and financial resources to effectively control drug resistant TB.

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Pyrazinamide (PZA) is included in the 2016 World Health Organization multidrug-resistant tuberculosis treatment guidelines and is a key component of most ongoing clinical trials investigating novel antibiotic combinations. PZA resistance is associated with worse tuberculosis treatment outcomes. Unfortunately, for such an important drug, phenotypic susceptibility testing is extremely challenging. The exacting bacterial growth conditions required to induce susceptibility to the drug reduce the accuracy of the susceptibility assay, even in experienced laboratories, and widespread testing is not performed. This situation is unacceptable for such a valuable and important drug. A more complete understanding of the mechanism of action of PZA would be expected to lead to improvements in this situation. Although the exact mechanism of action of PZA is not known yet, it is widely accepted that PZA is a prodrug requiring transformation to pyrazinoic acid, the active form, by the mycobacterial enzyme encoded by the pncA gene. Most clinical resistance indeed appears to be a result of a diverse range of mutations in this gene and sequencing of the pncA gene has been shown to have excellent predictive power for PZA resistance. The wider availably of pncA sequencing in combination with databases of the phenotypic implications of these mutations has helped make genetic testing for PZA resistance a practical proposition. For the past decades, it has been generally accepted that an extracellular low pH is required for PZA activity but work in our laboratory ^[1] and others ^[2] has recently challenged this assumption. Alternative bacterial stresses, apart from a reduced pH of the growth media (such as reduced temperature), can also induce a PZA-susceptible phenotype. The characterization of spontaneous in vitro-resistant pyrazinoic acid mutants selected under neutral pH conditions suggests a key role for the pantothenate/coenzyme A biosynthetic pathway. This has profound implications for the mechanism of action of PZA as well as potentially the bacterial population against which PZA is active in the host. These findings will be discussed as well as their implications for further research and the future of PZA susceptibility testing.

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Aims & objectives: Socially and economically disadvantaged or “vulnerable” people are at high risk of tuberculosis (TB) and also contribute to active chains of TB transmission. Included in such vulnerable populations are children, women, prisoners, people living with human immunodeficiency virus, the homeless, and displaced people. The ongoing active transmission of TB among such populations is made more difficult to assess and control by difficult access, health inequities, poverty, and other chronic and debilitating health conditions at individual, domestic, and community levels. Methods: The 22 Eastern Mediterranean Region member states encompass diverse sociopolitical and socioeconomic situations with far-reaching effects on vulnerable populations in each country, thereby threatening the control of TB. Here, we examined the impact of these populations on the incidence and transmission of TB in light of these risks. Results: Approximately 60% of the regional population comprises children and adolescents ≤19 years of age, increasing the population at risk. Additionally, up to 11% of the population suffers from mental- or substance-abuse disorders, while >50% of the world refugee populations live in the Eastern Mediterranean Region. Conclusion: TB control requires a strategic approach at the country level to access these vulnerable populations.

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Objective/background: Mycobacterium tuberculosis is an obligate pathogenic bacterial species in the family Mycobacteriaceae and the causative agent of most tuberculosis (TB) cases. Until today, the only approved TB vaccine is Bacille Calmette Guerin (BCG), which has been used since 1921. While BCG provides fairly effective protection for infants and young children, its efficacy in adults is variable around the world. This could be due to several parameters including strains of the vaccine and exposure of individuals to different environmental bacterial infections. The situation is complicated by the emergence of multidrug resistant strains of M. tuberculosis. This urged the demand to develop new improved vaccines and immunotherapies against TB. Development of nonpathogenic recombinant constructs delivering M. tuberculosis-specific antigenic proteins provides the chance to evaluate candidates to be included in diagnostic tools and preventive vaccines. In our study, we are introducing some of the major M. tuberculosis genes in Escherichia coli and Mycobacterium smegmatis. Methods: DNA corresponding to the genes Rv3891, Rv3020, Rv0287, Rv3875, Rv3874, Rv3872, Rv2346c, and Rv3619 were PCR-amplified from M. tuberculosis genomic DNA and visualized on gel electrophoresis at the expected DNA size. Products were subsequently ligated to the plasmid pGEMTeasy and used to transform TOP10 E. coli. Transformed colonies were selected on appropriate media. At the second stage, genes-DNA were subcultured in expression vectors pDE22 and pGESTH1; the recombinant plasmids were finally used to transform. M. smegmatis and E. coli, respectively. Expression of proteins in E. coli was confirmed by Western blotting and in M. smegmatis by reverse transcriptase polymerase chain reaction (RT-PCR). Results: Amplified genes were successfully cloned and transformed in E. coli and M. smegmatis. Colonies of recombinant bacteria were detected on appropriate media. Western blotting and RT-PCR confirmed the expression of our corresponding proteins in both the bacterial vehicles. Conclusion: Positive results of cloning and expression suggest that the constructed clones are ready tools for further assessment of their immunogenicity and can be included in improved diagnostic tools and vaccines against TB.

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Objective/Background: Interferon (IFN)-γ release assays (IGRA) are designed for diagnosing tuberculosis (TB) infection. The new IGRA, QuantiFERON-TB Plus (QFT-Plus), is based on the enzyme-linked immunosorbent assay detection of IFN-γ after stimulation with Mycobacterium tuberculosis TB1 and TB2 antigens. TB1 elicits a cellular-mediated immune (CMI) response by CD4 T cells, and TB2 contains peptides recognized by both CD4 and CD8 T cells. The aim of the study is to characterize the CMI to QFT-Plus peptides in active TB and latent TB infection (LTBI) at baseline and during or after specific treatment (follow-up). Methods: We enrolled 7 individuals with active TB and 11 individuals with LTBI at baseline and followed them during the treatment, either for active diseases or preventive therapy. Peripheral blood mononuclear cells were stimulated with QFT-Plus antigens (TB1, TB2, and mitogen). Cytokine profile (IFN-γ, tumor necrosis factor-α, interleukin-2) and phenotype (CD45RA, CD27) of CD4 and CD8 T cells were characterized by flow cytometry. Results: All the individuals responded to mitogen. CD4 T-cell responses to TB1 and TB2 were similar in both individuals with active TB and those with LTBI evaluated over time. Differently, we found a higher number of TB2-associated CD8 T-cell responders in individuals with active TB than in those with LTBI. For individuals with active TB, there was no change in the specific response overtime. Differently, in individuals with LTBI, the number of CD8 responders to QFT-Plus antigens increased during preventive treatment (TB1 = 5/11 [45%], TB2 = 5/11 [45%]) compared with that at the time of enrolment (TB1 = 1/11 [9%], TB2 = 1/11 [9%]). Moreover, we analyzed the effector memory profile of T cells responding to QFT-Plus antigens. The largest component of antigen-specific CD4 T cells (65%) had a central memory (CD45RA−CD27+) phenotype at enrolment and during follow-up. In contrast, specific CD8 T cells, which were analyzed only at follow-up because they were almost absent at baseline, were characterized by a large component with naïve (CD45RA+CD27+) phenotype (40%) and a minor component with central memory (25%) features. Conclusion: To our knowledge, this is the first report characterizing CD4 and CD8 T-cell responses of individuals with active TB and with LTBI, followed overtime, to QFT-Plus antigens by flow cytometry. The results, although preliminary, may help in identifying better tools for monitoring therapy, especially in those with LTBI undergoing preventive treatment.

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Objectives: Tuberculosis (TB) is an infection disease caused by an organism called Mycobacterium tuberculosis (tubercle bacilli). Tuberculosis remains the most common infection worldwide (Organization & World Health Organization, 2013). Yemen situated in WHO Eastern Mediterranean Region and ranked as intermediate TB-burdened countries. TB considers as one of the main health problems; it ranks as the fourth in the priority of the public health issue (NTCP, 2010). The study was conducted to evaluate the Socio-Demographical variables on tuberculosis treatment outcome. Methods: A Prospective cohort multicenter study was carried out among Tuberculosis (TB) patients, to find out the socio-demographic characteristics and factors affecting the treatment outcome. The study was conducted in two major prevalence TB cities in Yemen i.e. Alhodiah city and Taiz city. Questionnaires were given to the TB patient during their registration in TB health center after the confirmation of their TB diagnosis. All Patients were followed up until the end of their treatment. Result: A total of 413 smear positive, smear negative and extrapulmonary tuberculosis were involved in the study. The responses rate among overall TB patients at the beginning of treatment was 67%. Patients were followed up and were interviewed again at the end of intensive phase and end of treatment. Survey shows that majority of TB patient were smeared positive pulmonary (66.1) followed by Extra Pulmonary (27.6) and lowest were from Smear Negative Patient (6.3). Prevalence of TB diagnosis with respective to male and female were same. The majority of Tb cases were found in patient age 16–25 (41.9), urban residence (68.8), illiterate (59.8) unemployment (59.1), chewing khat (61.7), monthly income were less than 50dollars/month (67.3). No stigma (59.6) and good knowledge of TB patient (86.4) was found good in the-the majority of TB patient. Conclusion: By evaluating the socio-demographic risk factors, pharmacist and health care providers can optimize and indicate the factors which play a role in successful and unsuccessful treatment outcome.

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Objective/background: Accurate and rapid detection of drug-resistant strains of tuberculosis (TB) is critical for early initiation of treatment and for limiting the transmission of drug-resistant TB. Here, we investigated the accuracy of Xpert MTB/RIF for detection of rifampicin (RIF) resistance, and whether this detection predicts the presence of multidrug resistant (MDR) TB in Southwest Ethiopia. Methods: Smear- or culture-positive sputa obtained from TB patients with increased suspicion of drug resistance were included in this study. GenoType MTBDRplus line-probe assays (LPAs) and Xpert MTB/RIF tests were performed on smear-positive sputum specimens and on cultured isolates for smear-negative specimens. We performed routine drug-susceptibility testing using LPA as the reference standard for confirmation of RIF and isoniazid (INH) resistance. Results: First-line drug-susceptibility results were available for 67 Mycobacterium tuberculosis complex-positive sputum specimens using the LPA test, with our preliminary results indicating that 30% (20/67) were MDR-TB, 3% (2/67) were RIF monoresistant, 6% (4/67) were INH monoresistant, and 61% (41/67) were susceptible to both RIF and INH. Relative to routine RIF-susceptibility testing (LPA), Xpert MTB/RIF detected all RIF resistance correctly, with 100% sensitivity and 97.8% specificity and a positive-predictive value of 95.7%. Of the 23 RIF-resistant strains according to Xpert MTB/RIF, 87% (20/23) were resistant to both RIF and INH (MDR), 8.7% (2/23) were RIF monoresistant, and 4.3% (1/23) were sensitive to RIF according to the LPA test. A high proportion of RIF resistance was documented among patients previously categorized as failure cases (50%, 10/20), followed by relapse cases (31.6%, 6/19) and defaulters (28.6%, 2/7). Conclusion: Xpert MTB/RIF was highly effective at identifying RIF-resistant strains in smear- or culture-positive samples. RIF resistance based on Xpert MTB/RIF results could be used to estimate MDR and allow rapid initiation of MDR-TB treatment in regions with high levels of drug-resistant TB.

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Aims and objective: Paratuberculosis or Johne's disease is a chronic infectious granulomatous enteritis, mainly of cattle, sheep, goats, and other domestic and wild animals caused by Mycobacterium avium subsp. paratuberculosis (MAP). Currently, MAP has been recognized as an important animal pathogen with significant zoonotic and public health concerns. The early detection of infected animals using suitable diagnostic methods helps in developing control and preventive strategies for the herd. Therefore, the present study was aimed to determine the comparative efficacy of certain diagnostic methods used in the identification and confirmation of MAP in the ovine tissues with distinct pathology of paratuberculosis. Methods: The ileum and mesenteric lymph node (MLN) tissues were collected from 38 sheep infected with paratuberculosis from organized farms of Rajasthan. These animals were further classified as paucibacillary (PB; n = 15) or multibacillary (MB; n = 23) on the basis of histopathological findings and mycobacterial loads. The ileum and MLN tissues of these animals were subjected to IS900 and 251 gene polymerase chain reaction (PCR) and bacterial culture. The tissue sections from MB, PB, and uninfected control sheep groups were stained using indirect immunoperoxidase technique (IPT) and Ziehl–Neelsen (ZN) method. Results: On bacterial culture examination of the ileum and MLN tissues using Herrold's egg yolk medium, MAP was isolated in 14 (60.9%) MB and 5 (33.3%) PB sheep. Of 38 sheep, IS900 PCR detected 21 (55.2%) positive for MAP, of which 19 (82.6%) were MB and 2 (13.3%) were PB sheep. Similarly, 251 gene PCR detected 25 (65.7%) sheep positive for MAP infection, of which 21 (91.3%) were MB and 4 (27%) were PB sheep. Thus, 251 gene PCR was found superior to IS900 PCR in the detection of MAP from the tissues. In PB sheep, IPT and ZN tests were positive in 87.5% and 50% of ileum sections and 70% and 37.5% in MLN sections, respectively. In MB sheep, IPT and ZN tests detected all animals as positive for MAP organisms or antigen and had equal sensitivity in the detection of MAP. The overall sensitivity of IPT was found superior (95%) to ZN staining (80%) in the demonstration of acid-fast bacteria or its antigen in the tissues. Conclusion: The sensitivity of all the tests in the detection of MAP was lower in PB sheep than in MB sheep. Bacterial culture detected MAP in only 50% of sheep and was found less sensitive than other tests used in the present study. Comparing the overall sensitivity of both the PCR assays, 251 gene PCR was found superior to IS900 gene PCR. The sensitivity of IPT was found superior (95%) to the ZN staining (80%) in the demonstration of acid-fast bacteria in the tissues. In the present study, IPT was found superior in the detection of MAP in PB and MB form of ovine paratuberculosis. This test can be used in the confirmation of post mortem diagnosis and research of paratuberculosis along with histopathology. However, 251 gene PCR assay was found easier to perform than IPT and could be used as paratuberculosis screening test in endemic sheep farms for blood and fecal samples.

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Contrary to classical dogma, molecular epidemiological studies and mathematical modeling clearly show that the global drug-resistant tuberculosis (TB) epidemic is driven by transmission. Furthermore, there is mounting evidence demonstrating that amplification of resistance occurs during treatment, possibly as a result of insufficient active drugs being administered. To control the drug-resistant TB epidemic, it will be essential to implement new, rapid, and highly sensitive and specific diagnostic methods to decrease the diagnostic delay associated with culture-based testing. To date, three molecular-based diagnostic tests have been endorsed by the World Health Organization: MTBDRplus (Hain Lifescience, Nehren, Germany) Xpert MTB/RIF (Cepheid, Sunnyvale, United States) MTBDRsl (Hain Lifescience, Nehren, Germany). Implementation of the MTBDRplus assay reduced the laboratory turnaround time from 55 days to 27 days. This was further reduced to 1day with the implementation of the Xpert MTB/RIF assay. However, time to initiation of multidrug-resistant TB (MDR-TB) treatment was not significantly reduced, remaining at approximately 17 days from receipt of drug-susceptibility testing (DST) results. In an attempt to reduce the time to initiation of MDR-TB treatment, some guidelines have recommended initiating MDR-TB treatment based on the diagnosis of rifampicin resistance alone (within 5 days). However, this implies treating MDR-TB blindly until routine culture-based DST results are available (mean, 54 days). This strategy may be highly effective in countries where second-line treatment has only recently been introduced, but may have significant consequences for patients with resistance beyond MDR-TB sensu stricto. Implementation of the MTBDRsl assay promises to reduce the time for DST for fluoroquinolones and second-line injectable drugs. These tests will form the foundation for DST for the implementation of the recently recommended shortened MDR-TB regimen. The impact of the implementation of these tests on treatment outcome using either the standard or shortened MDR-TB remains to be determined.

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Objective/background: Pakistan ranks sixth among high-tuberculosis (TB)-burden countries worldwide and fifth worldwide for diabetes incidence. Although both these factors are independently known, the rate of diabetes among TB patients is not well known. We aimed to determine the prevalence of diabetes among patients with TB presenting at tertiary care health centers in Karachi, Pakistan. Methods: A total of 216 patients with TB were recruited and their blood samples were taken for testing of glycosylated hemoglobin (HbA1c) and random blood sugar. Diabetes was defined as HbA1c>6.5% and random blood sugar>180 mg/dL, pre-diabetes as HbA1c 5.7–6.4%, and normoglycemia as HbA1c<5.7%. Results: Data for 211 patients were available and showed that 24 (11.4%) patients had diabetes. Of these, 17 were newly diagnosed, while seven were known diabetics. Prediabetes was identified in 45 (21.3%) cases. Of the TB patients, 165 were newly diagnosed, while 46 were retreatment cases. The majority of patients (60%) were underweight with a body mass index of <18.5. Conclusion: This study identified 11.4% diabetics among TB patients presenting to a tertiary care facility. Despite the high diabetes incidence in Pakistan, 71% of the diabetics in the group studied did not know their status. Given the negative impact of diabetes on treatment outcomes of TB, it is important that screening for diabetes be included as initial workup for TB patients. Identification and management of diabetes would result in improved outcomes for TB treatment.

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Objective/background: Pulmonary hypertension (PH) can be a complication of patients with severe pulmonary tuberculosis (TB). We aimed to study the correlation between pulmonary artery (PA) diameter (PAD) as measured by computed tomography (CT) and mean PA pressure (mPAP) as measured by echocardiography. We also aimed to determine the accuracy of PAD in diagnosing PH in patients with pulmonary TB. Methods: We retrospectively investigated the correlation between PAD measured using CT and mPAP measured using echocardiography in 132 patients with TB and PH, and 68 patients with TB but without PH, admitted to the TB intensive care unit at Masih Daneshvari Hospital in Tehran, Iran. We used logistic regression analysis to determine the relationships between PAD, PA diameter to ascending aorta (AA) ratio, and area of PA to area of AA ratio with mPAP. Using receiver operating characteristic analysis, we examined the utility of the PAD in predicting PH (mPAP ≥25 mmHg). Results: PAD had a significant correlation with mPAP (p <0.005 and r = 0.47). Also, PA:AA ratio and area of PA to area of AA ratio had significant correlation with mPAP (r = 0.48 and r = 0.47, respectively; p <0.001). The threshold of 29 mm for PAD was determined using ROC. This index had a sensitivity of 0.55, specificity of 70.2 and area under curve of 0.66. Conclusion: Although PAD and PA:AA ratio are useful in assessing of presence of PH, we conclude that these CT parameters are not sufficient for ruling in or ruling out PH in this group of patients.

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New scientific approaches are necessary: The current strategies for controlling tuberculosis (TB) are not sufficient. Improved prophylactic and diagnostic tools are imperative, being crucial for decreasing TB incidence and mortality and for preventing outbreaks. Furthermore, new and better drugs are badly needed, particularly considering the increase in cases with multidrug-resistant strains. The current TB vaccine—the Bacillus Calmette–Guérin vaccine—has a preventive impact on disseminated TB in children, but little effect on the most common form of TB, that is, lung TB in adults and young adults. For many years extensive scientific efforts have been made in order to develop new vaccines against TB that are better and more effective than Bacillus Calmette–Guérin. No such vaccine exists, however, to date. During the last few years it has become increasingly clear that TB patients can be infected with more than one strain and that a previous TB infection increases rather than decreases the risk for getting a new one. Mycobacterium tuberculosis organisms are thus not capable of inducing protective immunity to such an extent that a new TB infection is prevented. This phenomenon highlights the problems of developing effective vaccines against TB. A new TB vaccine based on general immunological protection models would in all probability only have a limited capacity to hamper TB incidence and mortality. The question whether or not it is feasible to make a vaccine of sufficient efficacy must therefore be discussed. Prophylaxis is practically always far better than therapy and we all wish we had an effective TB vaccine. However, considering the problems with vaccines, scientific efforts could well focus on developing new therapies rather than new vaccines. New scientific approaches are highly necessary and we need ideas and visions. Some examples of recent projects will hereby be presented. One study concerns the mycobacterial cell envelope and its unique macromolecules as targets for new drugs. Another study concerns new ways of administrating the drugs which could enhance the effects of new as well as of already available drugs. In addition, what can be learnt from cancer therapy—is supporting the patient's own defense by immune modularly methods a possible approach? We also need to look back since ample knowledge on TB has been assembled during many years. Unfortunately some of this valuable knowledge is about to be forgotten, particularly, the experience from the time when TB was an incurable disease.

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Background: GenoType MTBDRplus is a molecular assay for detection of Mycobacterium tuberculosis resistance to isoniazid (INH) and rifampicin (RMP), the two major anti-tuberculosis (TB) drugs. Identification of INH resistance is largely based on the occurrence of mutations in the katG gene, coding for the catalase-peroxidase, or in the promoter region of the inhA gene, coding for the NADH-dependent enoyl-ACP reductase. For testing the RMP resistance, mutations in the rpoB gene, coding for the RNA polymerase β subunit, particularly in the RMP resistance determining region (RRDR) of the gene are investigated. The GenoType MTBDRplus assay has been validated in several countries. The aim of this study was to evaluate the ability of the assay to detect INH and RMP resistance among strains of M. tuberculosis, isolated from Pakistani TB patients, and phenotypically identified as multidrug-resistant (MDR), that is resistant to both INH and RMP. Material/methods: The study included a collection of 100 MDR M. tuberculosis strains isolated from as many Pakistani TB patients over a 9-month period (i.e. between January and September 2014). Drug susceptibility testing was performed using the standard 1% proportion method on Löwenstein-Jensen medium, with INH and RMP critical concentrations of 0.2 mg/L and 40 mg/L, respectively. Genomic DNA was extracted by the cetyl-trimethyl ammonium bromide (CTAB) method. The GenoType MTBDRplus assay (Hain Lifescience, Germany) was done following the manufacturer's instructions. Results: In the katG gene, with MTBDRplus assay, a specific mutation associated with INH resistance (i.e. G944C transition, conferring Ser315Thr amino acid change) was detected in 66 (66%) of the strains. Thirty-four (34%) strains did not carry the katG mutation detected by the assay. Mutations in the mabA-inhA promoter region were detected in 10 (10%) strains (C-15T – in 10 strains, and T-8C – in 2 strains). Seventy-seven (77%) strains tested harboured a mutation in rpoB gene. Mutations in the rpoB gene were of four types: C1349T, A1304T, C1333G, and TC1324CA found in 63 (63%), 11 (11%), 8 (8%), and one (1%) strain, respectively. Of the 100 strains designated as MDR by the proportion method, GenoType MTBDRplus confirmed this phenotype in only 62 strains. The results of GenoType MTBDRplus and the conventional drug susceptibility method were consistent in 70% (70/100) for INH, and 77% (77/100) for RMP. Conclusions: As evidenced in this study, the major concern with the GenoType MTBDRplus assay were false negative results. In comparison to conventional drug susceptibility testing, the assay was unable to detect 30 (30/100; 30%) strains resistant to INH and 23 (23/100; 23%) strains resistant to RMP. The GenoType MTBDRplus failed to identify 38 MDR (38/100; 38%) strains. Resistance in those strains probably originate from mutations in other codons and/or genes than those covered by the test. For detecting INH and RMP resistance in TB cases, especially in high TB incidence countries, such as Pakistan, molecular approaches should still be a complement rather than areplacement to conventional drug susceptibility testing.

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Nyaditum resae (NR) is a galenic preparation of heat-killed Mycobacterium manresensis (hkMn). This is a new species that belongs to the Mycobacterium fortuitum complex, and it is present in drinking water—thus, regulatorily speaking, it is considered a food supplement. Preclinical studies in the murine model of active tuberculosis (TB) in the C3HeB/FeJ strain have demonstrated that daily administration of NR containing 10³–10⁶ hkMn for 14 days was able to stop the progression toward active TB ^[1]. The mechanism of action was linked to the induction of low dose tolerance and was related to the increase of Tuberculin Purified Protein Derivative (PPD) memory-specific Tregs (CD4⁺CD25⁺CD39⁺ cells) after ex vivo incubation of splenocytes for 7 days. This increase of Tregs was related to the increase of interleukin (IL)-10 in the spleen and in the reduction of IL-17 in the lungs, where there was also a reduction in bacillary load and the pathology caused by a reduction of neutrophiles' infiltration ^[2]. Two randomized, double-blind placebo-controlled clinical trials (CTs) have been conducted in humans. The NYADATREG study (Clinicaltrials.gov identifier NCT02076139; 2013–2014) was aimed to evaluate the safety and the immunogenicity of two concentrations of NR (containing 10⁴ hkMn and 10⁵ hkMn) versus placebo (all administered orally everyday for 14 days) in tuberculin-positive and tuberculin-negative volunteers (total n = 51). The results demonstrated an excellent safety record, with no differences between groups in terms of adverse effects. A significant increase in PPD-specific memory regulatory T cells was also detected in both NR groups ^[3]. The NYADAPETRICS study (Clinicaltrials.gov identifier NCT02581579) is evaluating the safety and immunogenicity of NR 10⁵ hkMn (capsule format, orally) in the pediatric population. Currently, an efficacy study (randomized, double-blinded, placebo-controlled CT) is being conducted in Georgia. This NYADAGEORG trial includes close contacts of active TB cases with positive sputum not tributaries of chemoprophylaxis (<5-year-old children and HIV-positive individuals), which will receive NR (containing 10⁵ hkMn) or placebo (orally, every day for 14 days). A total of 3300 participants will be recruited in four medical centers around Tbilissi. The participants are monitored by telephone for up to 2 years to evaluate the incidence of active TB. The hypothesis is that the NR group will exhibit a 40% reduction in expected TB incidence. Thus, the anticipated TB incidence will be 3% in the NR group versus 5% in the placebo group. The CT is projected to end by 2021 (Clinicaltrials.gov identifier NCT02897180). The administration of the food supplement NR appears to be a new, easy, safe, and reliable method for reducing the risk of developing active TB, and new CTs must be encouraged to discern the particular efficacy power according to different population characteristics.

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Objective/background: The risk of mortality and morbidity among tuberculosis (TB) and human immunodeficiency virus (HIV) coinfected patients is significantly higher than that of patients infected with TB alone. The aim of this study was to evaluate the survival of TB-HIV patients in a TB-referral center during a 10-year follow-up. Methods: All TB-HIV patients in our referral center were enrolled in the study from 2003 to 2014, and patients were divided into two groups: HIV-TB patients without a history of TB treatment (new cases of TB) and HIV-TB patients with a history of TB treatment. Both groups were treated based on World Health Organization TB-treatment guidelines, and multivariate analysis was performed to evaluate risk factors of all-cause mortality. Results: During the study, 22 HIV-TB patients with a history of TB treatment and 263 HIV-TB patients with newly diagnosed TB were included. Baseline demographic and clinical characteristics were similar, except that miliary TB (98% vs. 2%) and mortality (97% vs. 3%; p = 0.06) were more likely in HIV patients with newly diagnosed TB. During TB treatment and subsequent follow-up, two patients did not respond to treatment and 92 (32.3%) patients died, whereas the cure rate was 60%. Pneumothorax [hazard ratio (HR): 3.17], coinfection (herpes zoster, toxoplasmosis, cytomegalovirus infection, Pneumocystis jiroveci, candidiasis, and other opportunistic infection; HR: 1.75), CD4<100cells/mL (HR: 1.96), thrombocytopenia (HR: 2.29), and lack of treatment with antiretroviral agents (ART; HR: 2.82) were significantly associated with all-cause mortality according to multivariate analysis. Conclusion: Our retrospective review of coinfected TB-HIV patients hospitalized in Tehran showed that the management and monitoring of coinfection, pneumothorax and other adverse effects, as well as early initiation of ART, improved patient survival.

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Objectives/Background: Mycobacterium africanum that causes 40% of tuberculosis (TB) in West Africa grows more slowly in culture and has similar transmission capacity compared with Mycobacterium tuberculosis, but M. africanum-exposed contacts progress more slowly to active disease. The presence of lipid body (LB) containing M. tuberculosis complex (MTBC) cells in sputum samples has been associated with mycobacterial transcriptomes indicating slow or no growth and persister-like antibiotic tolerance. Slow-growing bacilli have been found to display a persister-like phenotype with the accumulation of LBs and drug tolerance. Our previous study showed that the body mass index and lung damage resolution on chest X-ray were significantly improved slower in M. africanum-infected patients posttreatment than in M. tuberculosis-infected patients; however, the reason for this remains unclear. Therefore, we hypothesized that these differences could be either due to significant differences in drug resistance between the MTBC lineages or a difference in their content of persisters, as indicated by the percentage of LP-positive bacilli in sputum. Methods: Sputum isolates collected before treatment from patients with TB were subjected to drug susceptibility testing using the BD BACTEC MGIT 960 SIRE kit. The percentage of acid-fast bacilli (AFB) and LB-positive bacilli in pretreatment sputum was determined by a dual staining procedure using Auramine O and LipidTOX Red neutral lipid stain, respectively, and fluorescence microscopy imaging. Results: Out of the 77 isolates tested, 9 showed resistance to at least one drug and only 2 showed multidrug (rifampicin and isoniazid) resistance among M. tuberculosis-infected patients. The percentage of AFB-positive smears was similar between the two groups (p = 0.821), whereas that of LP-positive bacilli was significantly higher (p = 0.0059) in M. africanum-infected patients' sputa (n = 24) than in M. tuberculosis-infected patients' sputa (n = 36). In addition, the bacillary lengths were significantly higher in M. africanum-infected patients' sputa than in M. tuberculosis-infected patients' sputa (p = 0.0007). A high frequency of LP-positive bacilli in pretreatment sputum was associated with a poor body mass index and lung damage on chest X-ray improvement following anti-TB treatment in both the groups (r² = 0.022; p = 0.017). Conclusion: The slow clinical recovery of M. africanum-infected patients compared with M. tuberculosis-infected patients posttreatment may be at least partially associated with the persistence of drug-tolerant “fat and lazy” bacilli.

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Tuberculosis (TB) is among the top infectious causes of morbidity and mortality worldwide and is associated with frequent pulmonary damage despite microbiological cure. Patients with treated TB may remain lifelong sufferers of disabling structural and functional sequelae of the disease, which subsequently impair quality of life. Long-term follow-up studies have revealed that many patients with treated pulmonary TB show signs of permanent impairment of their lung function. Impairment is variable in pattern and severity, ranging from none to severe, and shows restrictive, obstructive, or mixed patterns. Patients who presented with recurrent tuberculosis had a 2.8–3.0-fold higher likelihood of developing abnormal lung function at the end of treatment than those with a first episode of TB. A variety of noninfectious pulmonary disorders are also common in post-TB patients. (1) Parenchymal disorders that include thin-walled cavities (open negative syndrome), and lung fibrosis with structural destruction and scar carcinoma. (2) Airway disorders that include subglottic stenosis, chronic obstructive air flow obstruction, bronchiectasis, tracheobronchial stenosis, anthracofibrosis, and broncholithiasis. (3) Vascular lesions such as Rasmussen aneurysm. (4) Pleural lesions that range from pleural thickening to severe fibrothorax. (5) General complications that include cor pulmonale, secondary amyloidosis, and chronic respiratory failure. The prevalence of these abnormalities among patients completing anti-TB treatment is alarmingly high. In fact, some studies have suggested greater morbidity from the sequelae rather than from the disease itself. It is important to be aware of the full spectrum of these disorders to facilitate early diagnosis and management.

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Aim & objective: In India, tuberculosis (TB) is a foremost health problem, and the emergence of multidrug-resistant (MDR) and extensively drug resistant (XDR) strains of Mycobacterium tuberculosis (M. tuberculosis) has further complicated the situation. Although various mechanisms have been proposed to elucidate the emergence of resistance, our knowledge remains insufficient. The formation of a very complex network and drugs of proteins are countered by their efflux/modification or target over-expression/modification. The analysis of the over-expressed proteins and their qualitative and phenotypic evaluation before and after the development of drug-resistance may be the most appropriate tool to understand the mechanisms of the mechanism of development of drug-resistance. Most studies are performed on distinct strains. Therefore, the objective of this study was to compare the proteomic information of sequential isolates of M. tuberculosis Beijing type from a single patient who developed MDR-TB during the course of anti-tuberculosis therapy. Methods: In this study, a clinical isolate of M. tuberculosis was grown in Middlebrook 7H9 broth medium for 2 weeks, and the cell lysate of isolates was prepared by sonication and centrifugation. We compared and analyzed the whole cell lysate proteins of M. tuberculosis sequential clinical isolate from a patient with pulmonary TB before and after the development of drug resistance using two-dimensional gel electrophoresis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and bioinformatics tools. Results: The genotypes of both isolates remained homologous, showing no re-infection. The first isolate (before treatment) was sensitive to all the first-line drugs, sequential isolate was found resistant to rifampicin (RIF) and isoniazid (INH) and developed mutations in rpoB, katG and inhA. The concentrations of 17 protein spots were found to be consistently over-expressed in RIF- and INH-resistant isolates. The most prominent and over-expressed proteins found during the development of drug resistance were wag31, Rv2714, GarA, SSB, FabG4, Probable lipase, Rv3924c, Rv3204A, Rv2031c, Rv3418c and GroES. The InterProScan and homology searches generated insights into the possible functions and essential domains of the proteins. Rv1827, Rv2626c, Rv2714, Rv2970c, Rv3208A, and Rv3881c showed significant in silico interaction with RIF and INH; thus, the over-expression in the drug-resistant isolates could be compensating the inhibited/modulated molecules. Other proteins, which are over-expressed but do not unveil good binding with drug, might be indirectly associated with RIF and INH. Conclusions: This proteomic study provides an understanding about the proteins that are over-expressed during the development of drug resistance. These over-expressed proteins, identified here, could prove useful as vaccine candidate, immunodiagnostic and possibly drug-resistant or chemotherapeutic markers in future.

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Passive case finding remains the mainstay of tuberculosis (TB) control in several resource-limited countries. Although TB prevalence and mortality rates have declined in recent years, an estimated 3 million cases a year still go undetected and/or unreported, and delays in the diagnosis and treatment of TB continue to be widespread. It has been shown that identifying and treating individuals with latent TB infection (LTBI) prevents progression to active TB disease and is one of the key components of TB control strategies. However, neither systematic screening of LTBI nor a policy of isoniazid preventive therapy exists in several resource-limited countries for TB close contacts, leading to the vicious cycle of infection/reinfection in household. Moreover, no reliable data exist for TB contacts for efficient control measures to be set up. In recent years, many countries, especially developing countries, have witnessed an upsurge in the use of mobile phones. Should we want to achieve the post-2015TB elimination goal? For this purpose, it is necessary to formulate new strategies utilizing the widespread availability of new communication technologies (e.g., smartphone apps). This can provide an enhanced means to curb the disease, especially in resource-limited countries still lacking preventive measures and effective strategies to combat this old foe.

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Objective/Background: Mycobacterial infections including tuberculosis, leprosy, and buruli ulcer are among the most prevalent, debilitating, and deadly tropical diseases, especially in Sub-Saharan Africa. The development of drug resistance to the currently available drugs and the poor compliance emphasize the need for new chemotherapeutic agents. This study was designed to evaluate the in vitro activity of Cleistopholis patens, Annona reticulata, and Greenwayodendron suaveolens against Mycobacterium smegmatis. The safety on normal liver cells was also assessed. Methods: The crude extracts, fractions, and subfractions were tested against M. smegmatis and for cell cytotoxicity on WRL-68, normal human hepatocyte using microdilution resazurin-based assays. The phytochemical screening was performed using standard methods. Results: Most of the extracts, fractions, and subfractions inhibited the growth of M. smegmatis with minimum inhibitory concentration (MIC) values ranging from 6.25 μg/mL to 125 μg/mL. The subfractions P12 and P29 from G. suaveolens twig were more potent with MIC values of 6.25 μg/mL and 25 μg/mL, respectively. Fruit crude extract and root CH₂Cl₂ fraction from A. reticulata also showed activity with MIC values of 50 μg/mL and 25 μg/mL, respectively. Crude extracts from the twig and stem bark of C. patens displayed inhibition at MIC values of 125 μg/mL and 100 μg/mL, respectively. Majority of active extracts showed no cell cytotoxicity, except the extract from C. patens with IC₅₀ ranging from 41.40 μg/mL to 93.78 μg/mL. The chemical investigation of the promising extracts revealed the presence of phenols, alkaloids, glycosides, triterpenes, and acetogenins. Conclusion: The results achieved from this preliminary antimycobacterial drug discovery study supported the traditional claims of C. patens, A. reticulata, and G. suaveolens in the treatment of mycobacterial infections. Meanwhile, further fractionation is required to characterize the active ingredients.

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Objective/Background: The aim of this work was to study the efficacy and safety of quercetin and polyvinylpyrrolidone (QP) in the treatment of patients with newly diagnosed destructive pulmonary tuberculosis (TB) in comparison with standard antimycobacterial therapy. Materials and methods: The study involved 124 patients aged between 20 years and 70 years with newly diagnosed destructive pulmonary TB. Patients were allocated to two groups. The first (control) group received standard antimycobacterial and pathogenetic therapy and included 31 (25.0 ± 3.89%) patients. The second (main) group of patients received QP therapy in addition to chemotherapy and included 93 (75.0 ± 3.89%) patients. All patients received standard chemotherapy, consisting of oral isoniazid (0.3g), rifampicin (0.6g), pyrazinamide (2g), ethambutol (1.2g), and/or an intramuscular injection of streptomycin (1g) with a dose reduction after the intensive phase of therapy. The anti-TB drugs were procured through the centralized national supply system in Ukraine. QP was used at a dose of 0.5g in 100 mL 0.9% sodium chloride solution intravenously once per day for 10 days, starting on admission to hospital. Results: Intoxication symptoms in the second group were reduced after 1.33 ± 0.15 months, whereas in the first group, intoxication symptoms were reduced following 2.64 ± 0.20 months (p <0.001). Moreover, respiratory symptom regression in the second group was observed after 1.43 ± 0.30 months, whereas in the first group, it was after 2.33 ± 0.30 months (p <0.05). Bacillary excretion period evaluated within 3 months was reduced, as it was shown by 97.67 ± 1.63% in the main group compared to 72.41 ± 8.45% (p <0.05) in the control group. In addition, the period of cavity healing was reduced to 2.86 ± 0.15 months in the main group compared to 3.43 ± 0.20 months (p <0.05) in the control group. Residual radiological lung damage findings (mild or slight or even no signs) were observed in 84 (90.32 ± 3.07%) patients in the main group versus 22 (70.97 ± 8.15%) patients in the control group. Significant residual radiological lung damage findings were observed in nine (9.68 ± 3.07%) patients in the main group and in nine (29.03 ± 8.15%) patients in the control group (p <0.05). In addition, QP increased anti-TB drug tolerance by 20.42% and had an immunomodulatory effect. Conclusion: Administration of QP combined with chemotherapy in patients with newly diagnosed destructive pulmonary TB resulted in a rapid reduction in disease manifestation.

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Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis. Despite the availability of novel therapeutic approaches, TB is considered as one of the leading causes of death due to infectious diseases worldwide. Alveolar macrophages are the first line of defense against M. tuberculosis; they ingest and sequester the bacilli within granulomatous structures. Control and resolution of the infection requires activated T lymphocytes as well as Th1 cytokines. There are two forms of TB: active TB and latent TB. Latent TB is a state in which M. tuberculosis survives in the body without causing overt signs and symptoms. People with latent TB are noncontagious. However, M. tuberculosis can become active in the body, multiply, and cause overt TB. Sarcoidosis, on the other hand, is an autoimmune disease of unknown etiology which can affect multiple systems of the body. Nonspecific constitutional symptoms, such as fever, fatigue, malaise, and weight loss, are present in approximately one-third of patients. Chest X-ray usually shows hilar and mediastinal lymphadenopathy. Although the lungs are the most common sites of inflammation, sarcoidosis can also involve other organs, such as the eyes (intraocular and adnexal), skin, lymph nodes, salivary glands, heart, spleen, liver, and the nervous system. Recent investigations have provided further insights into the genetic basis of sarcoidosis and the way genotype determines the clinical presentation and phenotype of patients. Histopathologic features are usually insufficient for diagnosis of sarcoidosis. Diagnosis of sarcoidosis in endemic areas for TB can become a great challenge. Both TB and sarcoidosis are granulomatous diseases; TB is characterized by caseating granulomas, whereas sarcoidosis is characterized by noncaseating granulomas. New cases of sarcoidosis are increasingly being diagnosed in areas endemic for TB due to increased orientation of physicians and availability of diagnostic modalities. However, it is often difficult to differentiate sarcoidosis from TB, especially when caseous necrosis is not seen and acid-fast staining is negative in the biopsy specimen of patient with TB. Granulomatous inflammation in sarcoidosis is believed to be caused by the presence of a persistent poorly degradable unknown antigen in combination with a nonresolving host response. M. tuberculosis has been extensively studied as a possible cause of sarcoidosis. Results suggest that granulomas form in the lungs as a result of the immune response to inhaled M. tuberculosis and serve as the central site of host–pathogen interaction during M. tuberculosis infection. M. tuberculosis DNA detection in sarcoidosis samples by traditional polymerase chain reaction (PCR) has been used for the pathological study of sarcoidosis; however, it is likely that real time quantitative PCR analysis of specific mRNAs and microRNAs will be necessary as a sensitive, precise, and rapid diagnostic test for detecting trace of TB in Sarcoidosis. In conclusion, diagnosis of sarcoidosis in areas with a high burden of TB poses a significant challenge. Improved diagnostic tests including genetic tests can improve our knowledge and help in distinguishing these two diseases.

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Aim and objective: To highlight the need for post-treatment rehabilitation of TB patient; including social psychological and economic aspects. Methods: Patient interaction, informal interviews, field observations and secondary research. The minimum duration of treatment (for drug-susceptible TB) is 6 months. Treatment for drug-resistant TB can be from anywhere between 18 and 24 months. In some cases treatment can last for more than 5 years. Despite this, little attention is given to the social, psychological and economic impact extended treatment can have on the lives of individuals. Due, to a large extent, to the high levels of stigma that continue to exist in many high-burden countries, people affected must face obstacles even before diagnosis. These challenges persist at every stage of the treatment process (diagnosis, accessing appropriate care, treatment and post-treatment), but impact the individual's lives in various ways. Research has shown that despite diagnosis and treatment being provided for free, most individuals have to face high out-of-pocket expenditure (on transport, additional medication, nutritional supplementation etc.). In many countries, these expenses often have a significant negative impact on family finances. In most national programs, pre-treatment counselling is not included. This means that those affected are not prepared for the implications (in terms of time, money and physiological effects) that their treatment can have. Results: The side effects of TB medication (especially DR-TB) are sometimes extreme, and require extensive therapy and/or rehabilitation. Some side effects such as hearing-loss are often permanent. Nutritional support is another key factor that receives little attention in national programs. Adding to all these physiological challenges is the constant stigma and discrimination that individuals face from friends, family and even healthcare workers. Returning to jobs or to their education is a challenge as many workplaces and educational institutions do not provide for extended periods of leave or absence. This is costly both in terms of time and money. In families where the primary “bread winner” needs to undergo intensive treatment, the reduced income can have a devastating effect. Conclusion: Therefore despite having completed treatment, many people continue to suffer. Hence it is necessary that robust systems for post-treatment rehabilitation are put in place, and are designed based on the needs of those affected. Peer to peer counselling can be a low-cost high-impact strategy to address some of these issues. Institutional provisions, under the national response in each country, must be put in place if individuals are to successfully complete treatment, and return to their normal lives.

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Objective/background: Tuberculosis is a major public health problem and the emergence of drug resistance complicates the situation even more. It is therefore crucial to implement all conclusions from the studies that aim at a better understanding of the molecular mechanisms which govern the emergence and the evolution of drug resistance. The aim of this study is to assess the degree of involvement of the inhA and katG genes in the acquisition of isoniazid resistance in clinical strains of Mycobacterium tuberculosis. Methods: The inhA and katG genes were sequenced in 21 strains of M. tuberculosis with different resistance profiles and from different regions. Results: Analysis of the sequences obtained by comparison to those of the reference strain H37Rv showed that 95.2% had mutations. KatG S315T was the most common mutation (85.7%). The mutation katG T275A was revealed in two strains (9.5%). Two different point mutations in the inhA gene and its promoter region were identified as C-15T and G56A at a frequency equal to 14% and 10%, respectively. The G56A mutation is a new silent mutation. Our study showed no correlation between found mutations and multidrug resistance. Among the 21 strains studied, only one strain showed no mutations. Conclusion: In terms of this study, we characterized the mutations involved in resistance to isoniazid. katG S315T was by far the most frequent mutation, followed by C-15T. The frequency of these mutations was concordant with those reported in literature including those in intermediate tuberculosis endemic countries.

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Introduction: Extensively drug-resistant tuberculosis (XDR-TB) is defined as tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) strains that are multidrug resistant (MDR) and also resistant to a fluoroquinolone and to one injectable aminoglycoside or capreomycin. Whilst resistance in MTB has been associated with single nucleotide polymorphisms (SNPs), efflux pumps are thought to play a role in conferring resistance to MTB but little is known about them. Methods: We studied XDR MTB (n = 10) strains characterized by whole genome sequencing (WGS; http://www.ebi.ac.uk/ena/data/view/PRJEB7798). Phenotypic susceptibility testing was performed by the MGIT 960 (Becton, Dickinson and Co., NJ, USA) method. All XDR MTB strains were resistant to at least seven drugs whilst one XDR MTB strain, X54 was resistant to isoniazid, rifampicin, pyrazinamide, streptomycin, ethambutol, fluoroquinolones, capreomycin, kanamycin, amikacin, and ethionamide. The mRNA expression of efflux candidate genes Rv0194, Rv2688c, Rv1634, drrA, and drrB was determined in XDR MTB strains as compared with the ATCC reference strain, H37Rv, and drug-susceptible (DS) MTB (n = 9) strains using the relative quantification method normalized to 16S rRNA. Results: The mRNA expression levels of efflux genes Rv2688c (p = 0.0037), Rv1634 (p = 0.0042), drrA (p = 0.0078) and drrB (p = 0.0003) were upregulated in XDR-TB strains as compared with DS MTB strains. Conclusion: The differences between XDR-TB and drug-susceptible isolates suggest that the increased expression levels of MTB efflux pump genes may contribute to drug resistance in extensively drug-resistant tuberculosis. Future studies are needed to determine whether combining efflux pump inhibitors to antitubercular drugs would be effective to treat resistant tuberculosis.

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Objective/background: To determine the occurrence of active pulmonary tuberculosis (TB) due to Mycobacterium bovis in abattoir workers, butchers, livestock farmers, and veterinarians and to document their knowledge and practices regarding bovine TB (bTB). Methods: A cross-sectional study was conducted among abattoir workers, butchers, livestock farmers, veterinary doctors, and veterinary assistants. Sputum samples were collected from the respondents with a chronic cough and data on sociodemographic conditions, knowledge, and practices regarding TB were obtained. The chi-square test was used for statistical analysis. Results: A total of 141 abattoir workers, 317 butchers, 50 livestock farmers, five veterinary doctors, and three veterinary assistants took part. Four of 16 coughing abattoir workers and one of 50 coughing livestock farmers were positive for M. bovis by polymerase chain reaction analysis. Duration of work as an abattoir worker was significantly associated (p <0.05) with prevalence of zoonotic TB. Age, education, and type of work carried out by workers were not significantly associated with zoonotic TB. None of the abattoir workers, butchers, and livestock farmers had undergone any sort of formal training related to their work. The knowledge of abattoir workers, butchers, livestock farmers, and veterinary assistants regarding transmission of bTB from animals to humans and the symptoms of TB in humans was very poor. Most of these workers did not use protective equipment and appropriate safe working techniques and were considered at high risk of acquiring zoonotic TB. Conclusion: Zoonotic TB is a significant public health issue among professionally exposed groups in Peshawar, Pakistan, and suggests a need for further detailed investigations of the disease in this and similar areas.

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Objective/background: Buruli ulcer, known as necrotizing skin disease caused by Mycobacterium ulcerans, has emerged as the most prevalent mycobacteriosis after leprosy and tuberculosis. Accordingly, it has been classified by the World Health Organization as a neglected disease with high significance in tropical areas, including Cameroon. So far, the control of the disease relies mainly on the rifampin-streptomycin combination. Despite its efficiency, it has shown considerable issues, including availability and side effects. Therefore, more effective and safer drugs are urgently warranted. For this fact, natural plant-based products have always been of great importance in drug discovery. Therefore, the present study was initiated to assess the antimycobacterial properties of four medicinal plants against M. ulcerans. Methods: The methanolic and aqueous crude extracts prepared from Ficus binjamina, Ficus elastica, Ficus saussureana, and Terminalia superba were screened against M. ulcerans using the resazurin microtiter assay method. The phytochemical screening of promising extracts was performed to reveal bioactive components that might explain the exhibited activity. Results: Out of the 24 tested extracts, 11 extracts showed promising activity with minimal inhibitory concentration ranging from 62.5 μg/mL to 250 μg/mL. The phytochemical screening revealed the presence of phenols, flavonoids, tanins, triterpenes, glucosides, and saponins. Conclusion: The obtained results further strengthened the exploitation of these extracts as potent hits in the treatment of Buruli ulcer. Meanwhile, further studies are required to fully characterize the bioactive compounds.

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Introduction: Extensively drug-resistant tuberculosis (XDR-TB) has emerged as one of the biggest threats to public health and TB control programs worldwide. XDR-TB is caused by Mycobacterium tuberculosis (MTB) strains resistant to rifampin and isoniazid, as well as to a fluoroquinolone and to at least one injectable aminoglycoside. Drug resistance in MTB has primarily been associated with single nucleotide polymorphisms (SNPs) in particular genes. However, it has also been shown that efflux pumps may play a role in resistance of MTB. Upregulation of drug efflux pumps can decrease the intracellular concentration of drugs and reduce their efficacy. Methods: Whole genome sequencing was performed on 32 XDR-TB clinical isolates. Sequence data were used to investigate SNPs in efflux pump genes as compared with the H37Rv reference genome. Results: Of the XDR MTB strains, eight (21.62%) were wild type for rpsL, rrs (500 region), and gidB genes, but had non-synonymous (ns) SNPs (aspartic acid to histidine) in the drrA efflux pump gene at position 3273138. Three of eight (37.5%) XDR MTB strains, wild type for rpsL, rrs (500 region), gidB, and gyrB genes were phenotypically streptomycin sensitive and five (62.5%) XDR MTB strains were streptomycin resistant, while all XDR MTB strains, wild type for rpsL, rrs, gidB, and gyrB genes were resistant to fluoroquinolone (ofloxacin) and ethambutol. In addition, three XDR MTB strains wild type for rpsL, rrs, gidB, and drrA genes showed nsSNPs (isoleucine to valine) in the major facilitator superfamily, Rv1634 efflux pump gene at position 1839306. Conclusion: Our data show an nsSNP in the drrA efflux pump gene that may result in upregulation of drug efflux mechanisms in MTB strains. It is therefore imperative to understand the mechanism of efflux and its role in drug resistance, which will enable the identification of new drug targets and development of new drug regimens to counteract the drug efflux mechanism of MTB.

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Objective/Background: Bovine tuberculosis (BTb) is mainly a disease of cattle, although it continues to infect human populations across the world. Operation of a test and slaughter plan in Iran since 1981 has lowered the frequency of BTb from >5% to <0.14% at the national scale. In 2015, unusual uncontrollable epidemics of BTb were detected in two cattle farms in municipal suburbs of Qazvin and Isfahan. These farms had a tuberculin-test-certified record of BTb-free status for the past 5 consecutive years, with no new cattle registered with either of the two herds during this time period. Routine tuberculination of the bovids in 2015 resulted in the detection of tuberculin-positive animals that were subsequently removed from the herds. Serial tuberculin tests improved the situation, as new reactors were found each time. The aim of this research is based on isolation and identification of Mycobacterium from infected animals in both farms. Methods: To investigate the situation, major mesenteric/mediastinal lymph nodes from the culled reactor animals along with specimens from bulk milk tanks, trapped rats living on the farms, and environmental specimens were collected and subjected to bacterial culture. Tuberculin-positive cattle were also subjected to paratuberculosis enzyme-linked immunosorbent assay (ELISA), ESAT-6 ELISA, and gamma-interferon tests. Results: In bacterial culture, Mycobacterium bovis, Mycobacterium microti, and Mycobacterium avium subsp. paratuberculosis were isolated from collected specimens at both farms. Conclusion: There is circumstantial evidence supported by previous studies to expect a high frequency of M. avium subsp. paratuberculosis infection in Iranian cattle/sheep farms. This observation might explain the large skin reaction size seen at the avian tuberculin injection site in tested animals in these farms. Introduction of a third infection with M. microti, possibly by rodents visiting the farms, might have triggered immunological reactions that have ended the surge of BTb. If correct, we assume that a technical review of the Iranian test and slaughter scheme against BTb is required to address persisting cases of BTb in disease-free farms, as described here.

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Aim and objectives: Buruli ulcer (BU) is a neglected tropical disease caused by a mycobacteria, Mycobacterium ulcerans. The WHO recommended Rifampicin-Streptomycin combination side effects and poor compliance, leaves rural populations with no choice than to patronise indigenous remedies. This study is aimed at validating medicinal plants used in traditional medicine to treat BU by investigating the in vitro efficacy and safety as well as their composition in active molecules. Methods: A short report-based survey was used to identify medicinal plants used traditionally for BU treatment. Maceration of collected plant samples in methanol, hydroethanolic, ethanol, dichloromethane, and hexane, resulted in a total of 67 extracts assessed for antimycobacteria activity against Mycobacterium smegmatis and Mycobacterium ulcerans using the Resazurin Microtiter Assay. The cytotoxicity effect of promising extracts was assessed on normal human liver cells using the MTT assay. The bio-guided fractionation of the promising extracts led to the isolation of active compounds. Results: Majority of plants prepared as infusion, decoction, poultice, and macerate were administered topically. Significant antimycobacterial activity with MIC values ranging from 16 to 250 μg/mL was recorded against M. smegmatis (25 extracts) and M. ulcerans (17 extracts).¹ Most of antimycobacterial extracts showed no significant cytotoxicity against normal human hepatocytes. ¹The isolation guided by the biological activity revealed nine compounds with significant in vitro anti-M. ulcerans activity (MIC = 16–128 μg/mL). Conclusions: The results completed support the use these plants in the indigenous knowledge against BU. Further analyses of active principles might lead to new drug toe fight against BU.

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Background: Observations on Tuberculosis/HIV co-infection in addition to epidemiologic molecular studies have recently provided strong evidence for the state of immune system as the major determinant of the TB imaging spectrum. However, the presence of any correlation between radiographic findings and the degree of immunosuppression in HIV+ patients still remains controversial. The present study aimed to investigate the TB radiographic manifestation in HIV+ patients and its relationship to the CD4 cell count. Method and material: Chest radiography of 15 HIV+ patients with a definite diagnosis of pulmonary Tuberculosis in Masih Daneshvari Hospital, between 2013 and 2014, were retrospectively reviewed. Radiographic findings and severity were categorized as typical (upper lobe infiltration/cavity) and atypical (middle/lower lobe opacity, adenopathy, pleural effusion and normal X-ray). Demographics and CD4+ cell count were also recorded. Data analysis was performed using SPSS version 23 (frequency and mean for descriptive quantitative variables and Logistic regression analysis for correlation, p <0.05). Results: Of a total 15 patients (86.7% men and 13.3% women), 78.6% had CD4+ counts <350 (mean ± SD; 229.15 ± 199.45). The most common radiographic findings in descending order of frequency were adenopathy (53.3%), pleural effusion (26.7%) and cavitation (6.7%) with an overall atypical presentation of 93.3%. This study failed to reveal any statistically significant correlation between CD4+ cell count and radiographic manifestation as well as severity. Conclusion: In CD4+ cell count <500, the dominant radiographic pattern of Tuberculosis is atypical presentation. At this level of immunity, CD4+ T cell dysfunction may play a deterministic role in TB radiographic manifestation.

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Objective/background: The histological diagnosis of Mycobacterium tuberculosis (MTB) has long been a diagnostic challenge in the anatomical pathology field despite availability of different laboratory methods. Immunohistochemistry (IHC) could not only confirm granulomatous tissue involvement but also demonstrate MTB antigen immunolocalization. This study tries to clarify the details of IHC staining for MTB with pAbBCG. Methods: A total of 50 patients undergoing simultaneous biopsy and tissue culture with positive tissue culture for MTB during 2005–2009 were selected from the MRC Department at Masih Daneshvari Hospital, Tehran, Iran. Using the archives of the Pathology Department of this hospital, which is a referral center for pathological lung lesions, hematoxylin and eosin slides of the selected patients were evaluated. Twenty-three confirmed TB granulomatous tissue samples with adequate tissue and number of granulomas were chosen and studied by Ziehl–Neelsen and IHC staining with pAbBCG. Results: A total of 23 cases were evaluated, of which 17 (73.9%) were males. The types of tissue obtained from study cases were as follows: pleura (9 cases, 39.1%), lymph node (cervical, axillary, and thoracic [9 cases, 39.1%]), and lung tissues (5 cases, 21.7%). IHC staining was positive in all samples, whereas Ziehl–Neelsen staining was positive in nine cases of 23 (39.1%). IHC showed positive coarse granular cytoplasmic and round, fragmented bacillary staining. In this study, epithelioid cells clearly showed more positive staining at the periphery rather than at the center of granuloma. There is also positive staining in endothelial cells, fibroblasts, plasma cells, macrophages, and lymphocytes outside the granuloma. Conclusion: Detection of TB in tissue slides is still based on the histological pattern of the granuloma, which has several differential diagnoses with different treatments. Presence of mycobacterial antigens and tissue morphology can be evaluated using the IHC technique. Considering the criteria of positive IHC staining of TB granulomatous reactions, this stain not only highlights the presence of mycobacterial antigens for tissue diagnosis, but also could morphologically localize their distribution in different cells. Pathologists must be familiar with adequate staining pattern, elimination of background staining, and type of selected antibody. This method is especially important for application in countries with high prevalence of TB as a technique with early diagnostic value in tissue specimens. Early diagnosis using this technique can reduce related morbidity and mortality and decrease the rate of complications due to misdiagnosis and mistreatment of TB.

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Objective/background: Tuberculosis (TB) has been declared as a global emergency by the World Health Organization in 1993 and still remains one of the world's biggest threats. Worldwide, 9.6 million people have been estimated to have fallen ill with TB in 2014: 5.4 million men, 3.2 million women, and 1.0 million children. To reduce this burden, detection and treatment gaps must be addressed and new tools developed (Global TB report 2015). Methods: Seroreactivity of the purified excretory secretory (ES) antigens ES-31, ES-43, ES-41, and ES-6 have been assessed in pulmonary TB (fresh, relapse, chronic, and latent), extrapulmonary TB, and in human immunodeficiency virus-TB coinfection. Results: Analysis of immune response to these purified antigens by indirect and sandwich enzyme-linked immunosorbent assay (ELISA) using sensitive penicillinase enzyme-immuno assay, showed ES-31 antigen as having good diagnostic potential in pulmonary TB and in certain groups of extrapulmonary TB, in particular tuberculous lymphadenopathy, tuberculous meningitis, whereas ES-41 was found to be more seroreactive in abdominal and bone and joint TB. ES-43 antigen was primarily recognized by serum antibodies in relapse cases, while ES-6 was useful in contacts. Antigen assay was found to be more sensitive than antibody-based assay for detecting TB with human immunodeficiency virus coinfection. Immunomonitoring for the presence of antigens in TB patients under antitubercular treatment showed that ES-31 antigen assay was useful in determining the effectiveness of therapy and the patient's compliance. User-friendly peroxidase ELISA has been standardized for the detection of circulating mycobacterial ES-31 serine protease (free antigen and immune-complexed antigen) with 70–75% sensitivity and 90% specificity and with a limit of detection of antigen at 1 ng/2 μL (0.5 μg/mL serum). In-house developed SEVA TB ELISA assay using a cocktail of antigens (ES-31+EST-6) and a cocktail of specific antibodies is being routinely done for screening of patients suspected of TB attending Kasturba Hospital—a tertiary health care center. The characterization of ES-31 antigen protein showed that ES-31 is a 31-kDa protein antigen with zinc containing serine protease as well as lipase activities and shown to be a chymotrypsin-like protein which has the catalytic triad responsible for both the activities. Addition of serine protease inhibitors: (1) pefabloc; (2) 3,4 dichloroisocoumarin; (3) phenyl methyl sulfonyl fluoride (53–76%); and metallo-protease inhibitors: (1) EDTA; (2) 1,10 phenanthroline (46–61%), lipase inhibitor, orlistat (61%), or anti-ES-31 serine protease antibody (89%) inhibited the Mycobacterium tuberculosis (MTB) H₃₇Ra growth in axenic culture which is further confirmed by a decreased amount of ES-31 protein secreted in the culture filtrate. The importance of excretory secretory ES-31 protein for the survival of MTB H37Ra and H₃₇Rv bacilli has been shown by 77% and 78% growth inhibition in macrophage culture by protease inhibitor pefabloc and was further confirmed by the enhancement of growth of TB bacilli in the presence of ES-31. Conclusion: Inhibition of ES-31 leads to the growth inhibition of MTB bacilli, suggesting that ES-31 is important for entry and multiplication of bacilli and an important drug target for exploring new drugs for TB based on protease and lipase activities of ES-31 protein.

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Objective/background: We investigated the relationship between high-resolution computed tomography (HRCT) imaging manifestation of tuberculosis (TB) in childhood and the results of sputum smear. The aim of this study was to identify an alternative indicator of infectivity to prevent disease transmission through special isolation methods in children who have a clinical condition that suggests TB. Methods: This retrospective comparative study was performed on 95 children under the age of 15 years with a diagnosis of TB based on World Health Organization criteria and laboratory data (smear and culture positive for Mycobacterium tuberculosis). Investigations were performed on children admitted to the National Research Institute of Tuberculosis and Lung Disease for detection of TB between 2008 and 2012. Samples were collected from direct smear or gastric lavage method. We also performed HRCT on all of the children. The children were divided into two groups based on the results of their smear test. A multivariate analytical model was used for comparison of HRCT abnormalities in these two groups. Results: Consolidation, tree-in-bud pattern, upper lobe nodular infiltration, and cavitation were the most prevalent findings in the positive smear group. Lymphadenopathy, consolidation, collapse, and nodular infiltration in the upper lobe were dominant in the negative smear group. Conclusion: We found an association between cavity, tree-in-bud, and upper lobe nodular infiltration, and smear positivity in children with TB. Furthermore, we also found an association between lymphadenopathy and collapse in the negative smear group. Moreover, the positive smear group had radiologic manifestation of postprimary TB, whereas the negative smear group had primary TB manifestation.

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Objective/Background: To determine the culture yield and time to detection of mycobacterial growth between samples decontaminated using 0.7% chlorhexidine and sodium hydroxide–N-acetyl-l-cysteine (NaOH–NALC) and cultured on the Löwenstein–Jensen (LJ) medium. We also aimed to determine the contamination rate between the 0.7% chlorhexidine and NaOH–NALC decontamination methods. Methods: The study was carried out on 68 sputa samples (42 smear positives and 26 smear negatives). Of these 68 samples, 46 were collected from men and 26 from women with an approximate average age of 27 years. All the sputum samples were decontaminated using the standard NaOH–NALC and 0.7% chlorhexidine methods. The concentrates were cultured in parallel on LJ media in which reading of the slope for mycobacterial growth was obtained daily for the first 2 weeks and then weekly until week 8. The mycobacterial recovery rate, time to detection, and contamination rate were then compared. Results: The overall recovery rate of mycobacterial growth on samples treated with both decontamination methods inoculated on LJ media is 51.5% (35/68). Specifically, mycobacterial growth rates on samples treated with 0.7% chlorhexidine and standard NaOH–NALC on LJ media were 61.8% (42/68) and 54.4% (37/68), respectively. However, the growth of Mycobacterium tuberculosis complex was faster on samples treated with 0.7% chlorhexidine than those treated with NaOH–NALC (average, 32 ± 5 days vs. 33 ± 5.2 days, respectively). The contamination rate on samples treated with 0.7% chlorhexidine was 1.5% (1/68), whereas on those treated with NaOH–NALC, the rate was 4.4% (3/68). Conclusion: The 0.7% chlorhexidine decontamination method is rapid and has less contamination rate in terms of mycobacterial recovery compared with the standard NaOH–NALC method. Therefore, the 0.7% chlorhexidine decontamination method would be an ideal alternative option for decontamination of sputum samples and recovery/isolation of M. tuberculosis in resource-poor countries.

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Objective/background: Mycobacterium avium subspecies paratuberculosis (MAP) has been reported in Iranian cattle since 1965. Little is known on the population genetics of MAP in this Middle-Eastern State. A principle scope of this study was therefore genetic characterization of MAP isolates collected from farm animals in Iran. Methods: Sixteen field isolates of MAP collected from bovine (n = 8), ovine (n = 3), and caprine (n = 2) hosts in Alborz, Fars, Isfahan, Qazvin, Tehran, and Zanjan provinces were subcultured on mycobactin J-supplemented Herrold's egg slopes in order to provide the required genomic material. The laboratory strain MAP III & V was included as the reference strain. IS900-RFLP (Restriction Fragment length Polymorphism) was conducted using BsteII restriction enzyme. Application of IS900-RFLP genotyping on 16 Iranian MAP isolates in the present study classified them into six observable but similar types represented by two clustered and four orphan types. The laboratory strain MAP III & V displayed a totally different pattern easily distinguishable from that of Iranians. Results: Detection of six genotypes among 16 wild isolates is an indication of a population with a potentially high level of diversity. We assume that, with inclusion of more field isolates, it is very likely that even higher diversity may be observed within the studied isolates. The different patterns displayed by the Iranians and the laboratory strain in this work might explain the independent evolutionary pathways these have gone through to evolve from their ancestral clones. Conclusion: Further description on population genetic of MAP in Iran urges for more epidemiological work using similar and alternative standard genotyping system.

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Objective/background: Antituberculosis drugs (ATDs) efficiently combat Mycobacterium tuberculosis either through direct molecular interactions or its metabolites. However, a variety of adverse effects have been reported, leading to frequent interruptions of treatment. In order to investigate possible metabolic disturbances resulting from antituberculosis treatment, the uric acid level of patients on ATDs was measured in the Southwest region of Cameroon. Methods: This hospital-based cross-sectional study involved 96 tuberculosis patients on ATDs and 32 controls who were neither on ATDs nor any other treatment that could increase uric acid levels. The hospital records of consenting participants were reviewed for medical history and questionnaires were issued. About 2-mL venous blood was collected and analyzed using spectrophotometer to determine uric acid levels. Results: Hyperuricemia was observed in 56/96 (58.3%) of the studied group as compared with four of 32 (12.5%) in the control group (p <0.001). Our results indicated that treatment duration was significantly associated with hyperuricemia (p = 0.0016), while gender (p = 0.1275) was not. Conclusion: Hyperuricemia is associated with ATDs, with treatment duration being a significant factor. The disorder should be closely monitored, especially during the intensive phase of treatment.

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Childhood tuberculosis (TB) has been long neglected but has gained attention in recent years. In 2012, the World Health Organization annual report included an estimate for childhood TB for the first time, and in the following year, the TB Alliance received a grant from UNITAID (International Drug Purchase Facility) to develop pediatric TB formulations. Qatar is a low-incidence country. In this observational study, laboratory-confirmed cases of TB were analyzed from 2013 to 2015 and included patients aged ≤14 years. Microscopy and GeneXpert MTB/RIF (Cepheid, USA) and MGIT 960 (Becton, Dickinson and Company, USA) automated culture systems were used to confirm cases at the National TB Reference Laboratory, Doha, Qatar. A total of 24 positive cases were identified in this pediatric population, 21 with Mycobacterium tuberculosis complex (MTBC) and three with Mycobacterium other than tuberculosis (MOTT). Out of 21 MTBC cases, 19 were direct polymerase chain reaction (PCR) positive and two were smear and PCR negative but culture positive were later confirmed by PCR. Most of the positive specimens were extrapulmonary from pus and tissues biopsies. While six were from pulmonary, out of that, five were sputum and one was from gastric aspirates. Niacin Strip Test (Becton, Dickinson and Company, USA) was used to identify the Bacillus Calmette–Guérin (BCG) vaccine strains from MTBC infections, and seven patients were infected with BCG. Keeping in mind that there were 500–600 laboratory-confirmed cases of TB in adults, childhood tuberculosis is not a major problem in Qatar. Lack of sensitivity of niacin test due to identification various niacin accumulating BCG strains is documented worldwide, further testing with more stringent molecular methods will certainly increase the number of BCG isolates in this study population.

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Background/objectives: Pediatric tuberculosis is usually a primary infection presenting mainly as mediastinal or hilar adenopathy in computed tomography (CT) scan. In this study, we study the distribution and other CT scan characteristics of mediastinal lymphadenopathy in childhood tuberculosis. Methods: Chest CT scans of 75 cases of pediatric tuberculosis at Masih Daneshvari Hospital in Tehran, Iran, from 2009 to 2013 were studied regarding characteristics of mediastinal lymphadenopathy. Results: Mean ± standard deviation age of cases was 11.2 ± 4.6 years. Lymphadenopathy (mediastinal/hilar) was detected in 94.7% of cases. Most of the lymphadenopathies were located in the lower paratracheal (81.7%), upper paratracheal (69.1%), hilar (53.5%), and subcarinal (47.9%) stations. Perilymph node fatty stranding, lymphadenopathy conglomeration, bronchial pressure by the lymph nodes, and lymph node calcification were noted in 74.6%, 52.11%, 4.23%, and 5.6% of cases, respectively. Bilateral, right, and left lung parenchymal involvement were reported in 45%, 25%, and 8% of cases, respectively. Lung parenchymal involvement was significantly correlated with lymphadenopathies in subcarinal (p <0.001), hilar (p <0.001), subaortic (p = 0.03), lower paratracheal (p = 0.037), and axillary (p = 0.006) stations. Right- and left-sided pleural effusions were observed in 12.7% and 7% of cases, respectively. Conclusion: Attention to distribution and characteristics of mediastinal lymphadenopathy can help differentiate tuberculosis from other causes of pediatric mediastinal lymphadenopathy.

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Aims and objectives: Age increasing is caused physiological changes in the human body, such as reducing the power of the immune system. Weakened immune systems are more susceptible to bacterial infections like tuberculosis. So, this present study was performed for evaluating the prevalence of tuberculosis among Iranian elderly patients admitted to the infectious ward of a hospital. Methods: This systematic review and meta-analysis study has been done based on PRISMA guidelines. A comprehensive search was conducted in Iranian and International databases included: Magiran, Iranmedex, IranDoc, SID, Medlib, Scopus, PubMed, Science Direct, Cochrane, Web of Science, Springer, Wiley Online Library as well as the Google Scholar search engine in the period 1990–2016 by two independent researchers using the Mesh keywords. All of the reviewed studies that had inclusion criterion were been evaluated. The diagnosis of tuberculosis were considered results of physical examination, PPD (Purified Protein Derivative) test, Blood tests, Imaging tests and sputum test. The data were analyzed by using random effects model with the software Stata-Ver.11.1. Results: Five studies with a total number of 2,956 elderly patients were included. The prevalence of tuberculosis among Iranian elderly patients admitted to the infectious ward of the hospital was estimated to be 15% (95%CI: 1–30). The relationship between prevalence of tuberculosis with a year of study was not statistically significant (p = 0.371). Conclusion: This will be the first systematic review of tuberculosis prevalence among elderly patients admitted to the infectious ward in Iran. This study showed a high prevalence of Tuberculosis and it is recommended considering tuberculosis as a differential diagnosis in elderly patients with infectious symptoms.

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Objective/background: Heterogeneous mixtures of cellular and caseous granulomas coexist in the lungs of tuberculosis (TB) patients, with Mycobacterium tuberculosis (Mtb) existing from actively replicating (AR) to dormant, nonreplicating (NR) stages. Within cellular granulomas, the pH is estimated to be less than 6, whereas in the necrotic centres of hypoxic, cholesterol/triacylglycerol-rich, caseous granulomas, the pH varies between 7.2 and 7.4. To combat TB, we should kill both AR and NR stages of Mtb. Dormant Mtb remodels lipids of its cell wall, and so lipophilic drugs may be active against NR Mtb living in caseous, lipid-rich, granulomas. Lipophilicity is expressed as logP, that is, the logarithm of the partition coefficient (P) ratio P _octanol/P _water. In this study, the activity of lipophilic drugs (logP>0) and hydrophilic drugs (logP ≤0) against AR and NR Mtb was measured in hypoxic conditions under acidic and slightly alkaline pHs. Methods: The activity of drugs was determined against AR Mtb (5-day-old aerobic cells: A5) and NR Mtb (12- and 19-day-old hypoxic cells: H12 and H19) in a Wayne dormancy model of Mtb H37Rv at pH 5.8, to mimic the environment of cellular granulomas. Furthermore, AR and NR bacilli were grown for 40 days in Wayne models at pH 6.6, 7.0, 7.4, and 7.6, to set up conditions mimicking the caseous granulomas (hypoxia+slightly alkaline pH), to measure drug activity against NR cells. Mtb viability was determined by colony-forming unit (CFU) counts. Results: At pH 5.8, lipophilic drugs (rifampin, rifapentine, bedaquiline, PA-824, clofazimine, nitazoxanide: logP ≥2.14) reduced CFU of all cells (H12, H19, and A5) by ≥2log₁₀. Among hydrophilic drugs (isoniazid, pyrazinamide, ethambutol, amikacin, moxifloxacin, metronidazole: logP ≤0.01), none reduced H12 and H19 CFUs by ≥2log₁₀, with the exception of metronidazole. When Mtb was grown at different pHs the following Mtb growth was noted: at pH 6.6, AR cells grew fluently while NR cells grew less, with a CFU increase up to Day 15, followed by a drop to Day 40. AR and NR Mtb grown at pH 7.0, 7.4, and 7.6 showed up to 1 log₁₀ CFU lower than their growth at pH 6.6. The pHs of all AR cultures tended to reach pH 7.2–7.4 on Day 40. The pHs of all NR cultures remained stable at their initial values (6.6, 7.0, 7.4, and 7.6) up to Day 40. The activity of drugs against H12 and H19 cells was tested in hypoxic conditions at a slightly alkaline pH. Under these conditions, some lipophilic drugs were more active (>5 log CFU decrease after 21 days of exposure) against H12 and H19 cells than clofazimine, nitazoxanide, isoniazid, pyrazinamide, amikacin (<1 log CFU decrease after 21 days of exposure). Testing of other drugs is in progress. Conclusion: Lipophilic drugs were more active than hydrophilic agents against dormant Mtb in hypoxic conditions at pH 5.8. The Wayne model under slightly alkaline conditions was set up, and in hypoxic conditions at a slightly alkaline pH some lipophilic drugs were more active than other drugs against NR Mtb. Overall, these models can be useful for testing drug activity against dormant Mtb under conditions mimicking the environments of cellular and caseous granulomas.

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Objective/Background: Bacterial persistence is the hallmark of tuberculosis (TB) and poses the biggest threat to the success of any antitubercular drug regimen. The DevR/DosR dormancy regulator of Mycobacterium tuberculosis belongs to the NarL subfamily of response regulators and is essential for M. tuberculosis persistence in macaque models of TB. The DevR/DosR crystal structure revealed a unique (αβ)₄ topology instead of the classical (αβ)₅ structure found in the receiver domain of other regulators in this subfamily. It was proposed that phosphorylation may culminate in the formation of a DNA-binding-competent dimeric species via α10–α10 helix interactions. Here, we deciphered the role of the α10 helix in activation of the DevR/DosR response regulator in M. tuberculosis. Methods: Wild-type (WT) and mutant DevR [α10-helix-deleted DevR (DevR_Δα10)] proteins were cloned in suitable plasmids and expressed in Escherichia coli and M. tuberculosis strains. An in vitro phosphorylation assay was performed using acetyl phosphate, and the dimeric/oligomeric status of WT DevR and mutant proteins in the presence or absence of phosphorylation was assessed by glutaraldehyde-based in vitro cross-linking, followed by western blot analysis. Additionally, recombinant M. tuberculosis strains expressing WT and mutant DevR proteins were assessed for dormancy regulon gene expression under aerobic and hypoxic conditions by western blot analysis. An electrophoretic mobility shift assay was performed to assess the in vitro DNA-binding activity of DevR proteins to the target DNA, and biophysical characterization was performed using circular dichroism spectroscopy, fluorescence spectroscopy, and thermal shift assays. Results: Our results revealed that DevR structure and activity are modulated by phosphorylation-dependent α10 helix dimerization. In its hyperphosphorylated state, DevR_Δα10 is defective in DNA binding and exhibits an open and less stable conformation. The combined results of in vitro cross-linking and genetic analysis established an essential role for the α10 helix in postphosphorylation dimerization of DevR and gene activation. The importance of the α10 helix for dormancy regulon induction in M. tuberculosis established the α10–α10 helix interaction as a novel target in the DevR-signaling pathway for developing inhibitors against DevR, a key regulator of hypoxia-triggered dormancy. Conclusion: This study established the importance of the α10 helix for DevR activation in M. tuberculosis and proposed a novel molecular tool to screen small-molecule inhibitors targeting dimerization of DevR in the absence (inactive state) or presence of phosphorylation (active state) to combat latent TB infection. This concept can be extended to screen inhibitors against response regulators where dimerization is crucial for their activation.

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Objective/background: Nontuberculosis mycobacteria (NTM), defined as any mycobacterial strain other than Mycobacterium tuberculosis complex, are a diverse group of pathogens that cause a substantive, but often unappreciated worldwide burden of illness. NTM cause illness similar to M. tuberculosis, but generally do not respond to classic tuberculosis (TB) drug regimens. Here, we evaluated GenoType Mycobacterium CM/AS (Hain Lifesciences) for rapid identification of NTM and compared its results with those of other biochemical tests. Methods: During the study interval from February 2015 to August 2015, samples were tested by GenoType Mycobacterium tuberculosis complex (MTBC) for differentiation of MTB complex, and NTM isolates obtained from patients were analyzed with the GenoType Mycobacterium CM assays for common mycobacteria. Results: All samples tested were M. tuberculosis (typical), except samples from sputum that was negative according to Geno Type MTBC results. All isolates were analyzed with the Geno Type Mycobacterium CM (for common mycobacteria) assays, which correctly identified the species as Mycobacterium chelonae, Mycobacterium intracellulare, Mycobacterium kansasii, and Mycobacterium simiae. Conclusion: GenoType testing of Mycobacteria species using GenoType MTBC and GenoType Mycobacterium CM constitutes a reliable, rapid, simple, and easy-to-interpret assay. Moreover, it appears suitable for use in our region, since it identified all mycobacterial species.

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Objective/Background: The susceptibility to tuberculosis (TB) depends upon different factors, and the risk of developing diseases after infection with Mycobacterium tuberculosis ranges from 5% to 10%. This suggests that besides the mycobacterial itself, the host genetic factors may determine the differences in host susceptibility to TB. Among the important risk factors, cytokines and especially tumor necrosis factor alpha (TNF-α) genes, are thought to be responsible for regulating the protective immune responses. The TNF-α gene that encodes the cytokines TNF-α is located within the class III region of the major histocompatibility complex (MHC). The TNF-α gene and its receptors have significant suppressive effects on bacterial growth into macrophages. Tumor necrosis factor receptor 1 (TNFR1) is more responsible when apoptosis is needed but tumor necrosis factor receptor 2 (TNFR2) is involved in cell survival. TNF-α conducts its replicative effects on immune cells via the latter. Up to now, several polymorphisms within the promoter region of the TNF-α gene have been shown to be associated with susceptibility or resistance to TB in different ethnic groups. By contrast, the correlation of TNF-α gene with their receptors such as TNFR2 in susceptibility to TB has not been resolved yet. In this study, we aimed to analyze the single nucleotide polymorphisms (SNPs) in the TNF-α gene at the –238, –308, –857, and –863 positions, and compare the susceptibility to TB with polymorphisms at TNFR2 (T587G). Methods: One hundred fifty-one tuberculosis patients (n = 151) and 83 control subjects (n = 83) were included in this study. Polymorphisms in the TNF promoter region, namely TNF (SNP), –238, –308, –857, and –863 were studied using polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP). For TNFR2 polymorphisms at 587 positions, the following primers were used to amplify a 242 base pair (bp) product: 5'-TTCTGGAGTTGGCTGCGTGT-3' and ACTCTCCTATCCTGCCTGCT-3'. PCR products of TNFR2 digested with 2U enzymes of NLA III. Results: The current study found a strong correlation between two polymorphisms in different loci of TNF-α gene including 857 C/C (85; 56.2%) and TNF 238 A/A 127 (84.1%). However, there were no associations between polymorphisms of the TNF-α gene and its receptor, that is, TNFR2. Conclusion: Concerning our current study, screening assessments for TNF-α-857 and A238GSNPs in Iran would be important in order to make future decisions for preventive treatments before getting the disease among individuals who are at high risk considering their genotyping.

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Objective: Multidrug-resistant tuberculosis (MDR-TB) is caused by Mycobacterium tuberculosis strains that do not respond to isoniazid and rifampicin, the two most effective first-line anti-TB drugs. Here, we designed and produced antibodies based on biomarkers that exist only in MDR-TB. Methods: Bacilli were cultured for 4 weeks at 37°C, and protein extraction was performed by sequential extraction. Bacterial cells were sonicated, centrifuged at 5000rpm for 45min, and the supernatant was collected and subjected to multiple rounds of treatment to prior to protein isolation. Protein concentration was determined using the Bradford method, and extracted proteins (50 μg) from each strain (drug-sensitive- and MDR-TB isolates) were visualized on polyacrylamide gels (5–15%) with Coomassie Brilliant Blue R-250 staining. Three extracts were mixed and dialyzed against 0.1M ammonium bicarbonate (pH 8.0), followed by mass spectrometry. Specific polyclonal antibodies against purified MDR-TB proteins were purified by affinity chromatography and prepared in rabbits using three booster injections. The ELIZA test was performed for evaluation the antibody production. The antibody was treated with normal oral flora to remove any non specificity and cross reactivity. Analyses of different protein patterns (drug-sensitive- and MDR-TB) were performed by western blot. Results: Our revealed that the MDR-TB strains contained specific antigens, and that the protein profiles of drug-sensitive TB strains differed from those of the MDR-TB isolates. Five bands from the MDR-TB fractions were detected as diagnostic antigens and were not observed in drug-sensitive-TB fractions. Western blot results showed that the MDR-TB antigenic fractions showed immunogenic bands at 50.0 kDa and 70.0 kDa, with the five antigenic MDR-TB-specific bands were identified as Rv3248c, Rv0350, Rv0440, Rv0475, and Rv3588c. Conclusion: Western blot data revealed dynamic properties of antibody responses that led to actionable findings for further research. Moreover, specific anti-mycobacterial antibodies, such as MDR-TB antibodies, can be essential tools in the identification of species-restricted antigens, such as drug-resistant TB antigens. The MDR-TB antibodies described here might promote identification of mycobacterial antigens during the course of infection, which could be helpful for the development of newer TB-vaccine candidates or therapeutic agents for improved TB treatment or diagnosis.

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Aims and objective: To develop novel immunodiagnostic test for tuberculosis. Methods: The selected novel proteins named as Rv2145c (SS1), Rv1437 (SS2), Rv1827 (SS3), and Rv2970c (SS4) were cloned, expressed and purified under specific conditions. Additionally, monoclonal antibodies (mAbs) were developed using these recombinant antigens via hybridoma technology. Hybridoma clones were screened and positive clones were selected for further experiment. The mAbs were purified from cell culture supernatant using protein A/G column chromatography. The diagnostic potential of these recombinant antigens and mAbs were investigated using a well characterized cohort of tuberculosis patients (Pulmonary-TB, Extra-pulmonary, MDR-TB) and healthy subjects sera using ELISA. Monoclonal antibodies (mAbs) raised against these antigens were used to detect the mycobacterial antigens. Result The selected recombinant antigens showed superior activity in the developed TB detection test. The observed sensitivity was 98.6% (SS4), 97.9% (SS1), 97.1% (SS3) and 92.7% (SS2), whereas specificity was 100% (SS1), 98.2% (SS4), 93.6% (SS3) and 87.5% (SS2). The purified mAbs also demonstrated good activity in the diagnosis of TB. Conclusion: The novel recombinant antigens and mAbs generated against these antigen showed very good activity for the immunodiagnosis of TB. The combination of these recombinant antigens and mAbs could be used as novel biomarker for detection of active TB directly from patient sera.

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Objective/background: “The captain of all these men of death”, is the apt sobriquet for the age-old disease tuberculosis (TB). Despite the availability of many drugs, cases of increasing resistance in the forms of multi-drug and extensively drug-resistant TB and persistence [characteristic of Mycobacterium tuberculosis (MTB)] make the eradication of TB a nightmare. Approval of bedaquiline by the Food and Drug Administration focused attention on quinoline scaffolds for development of new anti-TB agents. Lysine ɛ-aminotransferase (LAT) in MTB plays a pivotal role in regulating amino acid synthesis, which in turn affects mycobacterial persistence. Here, developed quinoline inhibitors that targeted LAT with an objective to eliminate dormant forms of mycobacterium. Methods: Using e-pharmacophore approaches, quinolone (PBD: 2CJD) leads were found to inhibit lysine binding to LAT. To investigate structural activity relationships, 21 analogues were synthesized and characterized based on the identified lead molecules. Results: Among the derivatives, N-(pyridin-2-yl methyl)-2-(4-(quinolin-4-yl) piperazin-1-yl) acetamide was identified as a potent molecule, with an IC50 for LAT of 1.04 μM. In nutrient-starved and zebra fish models, this molecule exhibited logarithmic reductions of 2.1- and 2.2-fold, respectively, at a concentration of 10 μg/mL. The compound also exhibited good activity against persistent forms of mycobacteria (biofilm model), showing logarithmic reduction of 2.8-fold. Additionally, the hit molecule showed concentration-dependent kill kinetics against dormant forms of mycobacteria, and were devoid of cytotoxicity against RAW cell lines 264.7 at concentrations of 50 μM. Conclusion: Our results indicated that the hit molecule showed activity against both active and persistent forms of infection, which is ideal for new anti-TB agents. This molecule requires further pharmacokinetic and dynamic screening for development as new drug candidate.

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Background: Duration of digestion/decontamination has a considerable impact on yield of mycobacteria especially from sterile body fluids and pus specimens. Additionally, duration of digestion/decontamination affects the contamination rates. This study evaluates the effect of digestion/decontamination protocol for 15 and 20min versus inoculation of media directly from the sample on contamination rates as well as the yield of mycobacteria from pus and sterile fluids other than cerebrospinal fluids. Methods: Pleural fluid (n = 60), pus (n = 48) and ascitic fluid (n = 12) specimens were cultured for mycobacteria and evaluated for contamination and mycobacterial yield using three different processing methodologies: without digestion/decontamination with 5% NaOH-NALC (D/D), D/D for 15min and D/D for 20min. All samples >3 mL in volume were spun at 3000 RCF for 15min, whereas those less than 3 mL were used as is. They were simultaneously processed using the three different methods as mentioned above, and inoculated on LJ media and MGIT. Smear was made from samples treated for 20min and stained with fluorescent stain. Kinyoun staining was done on smears with dubious findings. Mycobacterial culture yield and contamination rates were recorded at 6 weeks as recommended by the Global Laboratory Initiative (GLI) laboratory manual 2014. Results: Pleural fluid and pus contamination rates were substantially lowered by increasing decontamination time from 15 to 20min, but it did not have any effect for ascitic fluid (Table 1). The 5-min difference in the decontamination procedure improved mycobacterial culture yield for pus samples by 10%, but there was no substantial effect on pleural and ascitic fluids. Prolonged decontamination did not compromise the culture yield in any of the mentioned specimens. Conclusion: In areas where specimen delay is common and sterility of collection procedure cannot be ensured, digestion/decontamination with NaOH-NALC for up to 20min can reduce contamination rates without considerably compromising mycobacterial culture yield. However, one should be alert to the possibility of decreased viability, and culture should be supplemented with molecular methods.

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Objective/Background: Bovine tuberculosis is one of the zoonotic diseases and important in terms of both public health and its impact on the decrease in animal products and heavy economic losses. Consequently, its control is warranted. The key in controlling bovine tuberculosis is to test and slaughter the infected cows. In this method, animals >3 months of age undergo a comparative tuberculin test, and the positive cases are sent to a slaughterhouse. This study reviews the trend of coverage process of testing and slaughtering of infected cows in Iran during 2000–2014 to evaluate the bovine tuberculosis control in this period. Methods: In the current study, the data related to the control program of bovine tuberculosis and domestic animal population in Iran were taken from the Agriculture Ministry. Next, the yearly percentage of the tested cows and the proportion of positive cows to tested cows were calculated, and the process was drawn. Results: The results show that the coverage of a tuberculin testing process has been constant. The lowest percentage of test coverage was in 2005 with 11.8%, and the highest percentage of test coverage was in 2008 with 19.2%. On average, 15.7% of cows underwent the program of tuberculin test over this period. The results showed that the proportion of positive cases to tested cows increased in 2008 and 2009, and subsequently decreased in the following years. In addition, 0.13% of tested cases in this period were positive. Conclusion: The results of this study show that the coverage of a tuberculin testing process has been constant during this period. If more animals were covered by the program, more positive cases would have been detected and removed; this will have better effect in controlling the tuberculosis. The proportion of positive tests showed that it had been increasing in 2008 and 2009, and subsequently decreasing, which indicates decreased prevalence and efficacy of the bovine tuberculosis control program in the cattle herd that underwent the tuberculin test in these years.

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Objective/background: Multidrug-resistant tuberculosis (MDR-TB) is a major public health problem. The diagnosis of MDR-TB is of paramount importance in establishing appropriate clinical management and infection control measures. Rapid detection of MDR-TB allows the establishment of an effective treatment regimen, minimizes the risk of further resistance, and limits the spread of drug-resistant strains. The aim of this study is to determine the genotypic characterization of MDR-TB isolates from extra pulmonary tuberculosis (EPTB) cases in tertiary care centers in Northern India. Methods: This study was a prospective study. In total, 756 extra pulmonary specimens were collected from patients with suspected tuberculosis in two tertiary care hospitals in Northern India. Specimens were processed for Ziehl–Neelsen staining, culture, and first-line drug susceptibility test using BacT/ALERT 3D system and GenoType MTBDRplus assay for genotypic analysis of MDR-TB. MDR-TB strains were further processed by novel multiplex polymerase chain reaction for rapid identification of Beijing and non-Beijing strains associated with MDR-TB. Results: Of these 164 Mycobacterium tuberculosis complex isolates, 100 (60.9%) strains were fully susceptible and 64 (39.1%) strains were resistant. We noted that the prevalence of MDR-TB among EPTB was 22 (13.4%). The prevalence of MDR-TB was 11.4% in new cases and 19.1% in previously treated cases (p <0.05). Ser531Leu mutation was the predominant mutation noted, and Ser315Thr mutation was more prevalent among the MDR-TB isolates (p <0.05). The proportion of Beijing strains was significantly higher among MDR-TB strains (72.7%, p <0.05). Conclusion: The prevalence (13.4%) of MDR-TB among EPTB was high, and the most prominent mutations in rpoB, katG, and inhA genes were S531L (67.3%), S315T1 (94.5%), and C15T (20%), respectively. Beijing stains are significantly associated with MDR-TB among EPTB in this region. We found that the transmission of prominent mutations contributes to an unexpected increase in primary resistance, including MDR-TB cases in Northern India.

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Background: World Health Organization (WHO) declared tuberculosis (TB) as a global public health emergency and recommended DOTS as a standard strategy for controlling the disease. TB is one of the major causes of infectious diseases in the world, and 25% of all avoidable deaths in developing countries. About a third of the world's population is estimated to be infected with tubercle bacilli, and hence at risk of developing active disease. The objective of the study was, therefore, to evaluate the impact of DOTS strategy on smear-positive pulmonary tuberculosis case finding and their treatment outcomes in Gambella Regional State, Ethiopia. Methods: A retrospective health facility-based descriptive study was employed. Quarterly data were collected by using WHO structured reporting format for TB case finding and treatment outcome from all DOTS implementing health facilities in the region. Results: A total of 10,024 TB cases (all forms) were registered and reported between the periods from 2003 up to 2012. Out of these, 4100 (40.9%) were smear-positive pulmonary TB, 3164 (31.6%) were smear-negative pulmonary TB and 2760 (27.5%) had extra-pulmonary TB. An average case detection rate (CDR)¹ CDR: Percentage of smear-positive TB cases detected among the total number of TB cases estimated to occur. 1 of 40.9% (SD=0.1) and treatment success rate (TSR)² TSR: A sum of TB cases who completed treatment and who declared cured 2 of 55.7% (SD=0.28) for smear-positive pulmonary TB including other forms of TB were reported for the specified years period. Additionally, the average mean values of treatment defaulter and treatment failure rates were 4.2% and 0.3%, respectively. Conclusions: The recommended TSR set by WHO was achieved as it was already been fulfilled more than 85% from 2009 up to 2011 in the region and the reported CDR was far below (40.9%) for smear-positive pulmonary TB including other forms of TB from the target. Therefore, extensive efforts should be established to maintain the achieved TSR and to increase the low level of CDR for all forms of TB cases through implementing alternative case finding strategies.

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Background: Pleural tuberculosis (TB) is common among HIV-infected patients. In the absence of HIV infection, the yield of mycobacteriological study is low and usually invasive procedures, including pleural fluid analysis and pleural biopsy, are necessary. The present study aimed to determine the yield of mycobacteriological study of sputum and pleural fluid among HIV-infected patients. Methods: This retrospective case–control study involved HIV-positive and HIV-negative patients with new pleural TB admitted to the National Research Institute of Tuberculosis and Lung Disease, Tehran, Iran, for 5 years. The results of sputum and pleural fluid smear for acid-fast bacilli (AFB) and mycobacterium culture were extracted and compared between the two groups. Results: In the study period, 343 patients were admitted due to pleural TB, of which 42 were HIV-positive patients. We randomly selected 132 HIV-negative patients as controls. In total, 57.1% of HIV-infected patients had positive sputum smear for AFB compared with 38.6% of controls (p = 0.04). Positive culture of pleural fluid was more frequent among the HIV-positive patients than among the controls (63.6% vs. 29.5%, p = 0.001). There was no significant correlation between CD4 cell count and sputum or pleural fluid results. Mycobacteriological assay was enough for diagnosis in 66.6% of HIV-positive patients compared with 49.2% in controls. After adjusting for other factors and multivariate analysis, HIV remained independently and significantly associated with positive culture of pleural fluid. Conclusion: The diagnostic yield of mycobacteriological studies is higher among HIV-infected patients with pleural TB than among HIV-negative patients. This may decrease the need for pleural biopsy among them. Therefore, a diagnostic approach to pleural TB may be different among HIV-infected patients. In this group of patients, it is prudent to perform sputum and pleural analysis for the detection of AFB before pleural biopsy.

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Introduction: Pakistan ranks fifth in high tuberculosis (TB)-burden countries and seventh among countries with high prevalence rates of diabetes mellitus (DM). DM is a risk factor for TB and worsens disease outcomes. Furthermore, Mycobacterium tuberculosis (MTB) infection can induce glucose intolerance and worsen glycemic control in diabetes. Suppressor of cytokine signaling (SOCS)-1 and -3 molecules regulate cytokine signaling and are important in maintaining an immune balance. In TB, interleukin (IL)-6 upregulation induces SOCS3, which is also a negative regulator of insulin signaling. This research focuses on the mechanism by which SOCS1 and SOCS3 affect insulin resistance and increased susceptibility to TB. Methods: We studied gene expression in peripheral blood cells of patients with diabetes (n = 10) and healthy endemic controls (EC, n = 11) both with and without MTB infection. Mycobacterial antigen (PPD) and mitogen-stimulated SOCS1, SOCS3, interferon-gamma (IFN-γ), IL-6, and tumor necrosis factor alpha (TNFα) mRNA expression levels were determined using real-time polymerase chain reaction. Results: MTB antigen-stimulated mRNA levels of IFN-γ was 10-fold higher, SOCS1 was 4 times greater, TNFα was 10-fold higher, and IL-6 was 2-fold greater in patients with DM than in ECs. Overall levels of PPD-stimulated IL-6 was higher in patients with DM than in ECs (p = .036). Mitogen-induced mRNA levels of IFN-γ were 30-fold higher, SOCS3 was 20 fold higher, and SOCS1 was 4-fold higher in patients with DM than in ECs. Conclusion: Increased proinflammatory cytokine production in response to MTB antigens in diabetes would lead to exacerbated pathology and reduced inflammatory control at the site of MTB infection. This would in turn hamper the resolution of inflammation, resulting in unfavorable disease outcomes.

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Background: Central nervous system (CNS) infections caused by Mycobacterium tuberculosis (MTB) are the most severe forms of extrapulmonary TB (EPTB) due to high levels of mortality and neurological morbidity. Limited studies are available on CNS-TB animal-model development, despite the steady rise in cerebral-TB cases in India over the past decade. This study describes the development of a murine model of CNS-TB using a clinical strain (C3) isolated from the cerebrospinal fluid (CSF) of CNS-TB patients. Methods: Groups of mice were infected intravenously with an MTB C3 strain isolated from the CSF of CNS-TB patients in order to mimic the dynamics of actual infection. Brain and lung tissue were evaluated for bacterial burden, as well as histopathology and surrogate markers of TB infection at 30- and 50-days post-infection. Results: Mice infected intravenously with MTB C3 strains showed progressive development of CNS disease, with high bacillary burden in the lungs during the initial stage (30 days), which eventually disseminated to the brain at a later stage (50 days). All C3-infected mice showed elevated levels of mycobacterial antigens and antibodies, as well as increased T cell adenosine deaminase activity in brain homogenates, which explicitly correlated with mycobacterial load in the brain and chronic brain pathology. High mortality rates (60%) were associated with mice infected with the C3 strain as compared to those of controls. Conclusion: Our findings demonstrated the design of a novel murine model of CNS-TB using a C3 strain and that replicated events of EPTB dissemination. This model will promote efforts to understand the pathogenesis CNS-TB infection for development of improved therapeutic interventions in the future.

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Objective/background: Molecular epidemiology methods are very useful for differentiating between strains, assessing their diversity, and measuring the prevalence of the most circulating strain in an area. Various molecular typing methods using different molecular markers have been utilized worldwide, such as restriction fragment length polymorphism (RFLP), spoligotyping, Mycobacterial Interspersed Repetitive Unit – Variable Number of Tandem Repeat (MIRU-VNTR), and Double repetitive element-PCR (DRE-PCR) typing, for simultaneous detection and epidemiologic typing of Mycobacterium tuberculosis. The present study is conducted to assess the genetic diversity of M. tuberculosis by IS6110-RFLP and spoligotyping in patients attending a tertiary care hospital in eastern Uttar Pradesh, North India. Methods: A total of 83 representative isolates of M. tuberculosis were included in this study. These isolates were subjected to spoligotyping and IS6110-RFLP DNA fingerprinting techniques as described previously. Results: The spoligotype patterns were compared with SpolDB4.0; patterns of 64 out of 83 M. tuberculosis isolates were matched with the available data, while 19 isolates were found to be orphan, that is, absent in the SpolDB4.0 database. The majority of the M. tuberculosis strains (56.5%) belong to central Asian (32.5%), ill defined T (13.2%), and Beijing (10.8%) families. On IS6110-RFLP analysis, in 19.2% (16/83) of these isolates, IS6110 element was not found (0 copy number strains). Further, 15.6% (13/83) isolates were found to be low-copy-number strains having less than six copies of IS6110 element, and the remaining 65.0% (54/83) were multiple-copy-number strains with six or more copies of the element. On comparing the results of spoligotyping and IS-6110-RFLP, a total of 47 isolates were clustered by spoligotyping; out of these isolates, 40 were found to be unique by IS6110-RFLP. Conclusion: Spoligotype analysis resulted in the grouping of a much larger number of isolates within apparently identical clusters compared with IS6110-RFLP typing, while IS6110-RFLP was not found to effectively distinguish between zero- and low-copy-number isolates. Therefore, we concluded that, in India, the use of both the techniques simultaneously for DNA fingerprinting of M. tuberculosis could be a better approach.

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Background/objective: Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis (PTB), also called as Johne's disease, and is considered as the cause of irreparable economic losses in livestock industry. This bacterium is excreted in large amounts through the milk and feces of infected animal. For the detection of the PTB, indirect ELISA has been highly considered as a simple method with high sensitivity and specificity. Accordingly, this study aims at designing a system of indirect ELISA for the detection of PTB. Methods: In this study, 100 serum samples from 10 herds of dairy farms in Tehran and Alborz provinces, where PTB has been proven by culture, were selected and surveyed using the IDXX commercial kit, and the in-house ELISA system was designed. To design ELISA system, surface antigens and real positive and negative serum samples were used, and checkerboard titration was performed. To determine the cutoff point, the results of the commercial kit were used as gold standard and mean+2SD formula was also used in this regard. Results: According to the commercial ELISA kit results (15 positive samples and 85 negative samples), the best concentration of antigen and antibody dilution were evaluated as 1.2 μg and 1/100, respectively. Furthermore, the cutoff point was determined as 0.44. The sensitivity and specificity were evaluated as 67% and 100%, respectively. Conclusion: Surface antigens in MAP are sensitive to detect the infected animals, and the indirect ELISA system can be used to detect antibody in the early stages of the disease.

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Objective/background: Increasing resistance to various first-line and second-line drugs has become a major concern in India. However, it is not known if some genotypes are more associated with second-line drug resistance. Thus, the main aim of this study was to find out the predominant genotype associated with second-line drug resistance. Methods: During the study, a total of 234 multidrug resistant (MDR) strains of Mycobacterium tuberculosis, isolated between 2008 and 2015, were randomly selected and screened for pre-extensively drug-resistant (XDR) and XDR patterns using second-drug susceptibility testing with BACTEC MGIT 960. All the MDR isolates were tested against ofloxacin (2 μg/mL), kanamycin (2.5 μg/mL), amikacin (1 μg/mL), and capreomycin (2.5 μg/mL). Based on the resistance pattern pre-XDR was defined as M. tuberculosis isolates resistant to fluoroquinolone alone. The identified pre-XDR and XDR isolates were further characterized using spoligotyping. The spoligo patterns obtained were compared and analyzed using SITVIT_WEB Unweighted Pair Group Method with Arithmetic Mean, and Minimum Spanning Tree was derived using MIRU-VNTRplus. Results: Among the 234 MDR strains of M. tuberculosis, 85 (36.3%) were detected as pre-XDRs and 15 (6.4%) as XDRs. All the pre-XDR strains were ofloxacin resistant, whereas among the XDR strains, 10 (66.6%) were resistant to ofloxacin, kanamycin, and capreomycin, four (26.6%) were resistant to ofloxacin, kanamycin, and amikacin, and one (6.6%) isolate was resistant to ofloxacin and kanamycin. Upon spoligotyping analysis, the Beijing lineage was found to be the single most dominant lineage among the pre-XDR strains (38.8%) followed by CAS (30.5%), X (7%), T (5.8%), Haarlem (3.5%), EAI (2.3%), and MANU (2.3%). Among the XDR isolates, seven (46.6%) belonged to Beijing, three (20%) belonged to CAS, and one (6.6%) to each of the EAI, T, URAL, and X lineages. Within the Beijing family, ST1 was the most common in both pre-XDR (94%) and XDR isolates. All the isolates belonged to the ST1 sublineage. Conclusion: The Beijing lineage was found to be the single most dominant genotype among the pre-XDR and XDR isolates.

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Objective/Background: Morphological study of a mice of tissue necrosis stages in experimental organ-preserving tuberculosis (TB) pharmacotherapy using quercetin and polyvinylpyrrolidone (QP). Methods: A total of 32 laboratory mice of C57BL/6JLacSto strain were used in the experiment. The animals were divided into five groups (Group 1–5), with six to seven mice in each group: Group 1, Mycobacterium tuberculosis (MBT)-uninfected mice; Group 2, MBT-infected mice; Group 3, MBT infected and treated with anti-TB preparation (ATP); Group 4, MBT infected and QP treated; and Group 5, MBT infected and treated with ATP and QP. The mice were infected through caudal vein injection with the MTB H37Rv strain. The QP preparation, which belongs to the capillary-stabilizing-remedy group, was used for the research. The ATP included isoniazid and streptomycin. Thus, the drug doses for the mice contained the following drugs: isoniazid (10%, 5 mL), 45 mg/kg; streptomycin (1g), 90 mg/kg; and QP (0.5g), 45 mg/kg of the body weight of a mouse. The medicines used in the experimental studies on the mice were applied as follows: isoniazid and streptomycin, administered intramuscularly once a day; and QP, administered intraperitoneally according to a schedule (on the 5th day after the introduction of the infection every 2 h, and then every 12 h; on the 6th day and 7th day two times a day every 12 h). Results: QP produced a strict delineation of caseous necrosis from the unaffected parts of the connective tissue with fibrosis in the center and a large number of Langerhans cells, which was not observed in the control groups without QP. The combination of QP and ATP had more pronounced effects. In MBT-infected mice, where QP was not used, unlike the group where QP was used, adipose dystrophy of hepatocytes was observed. Thus, the hepatoprotective effect of QP against TB can be suggested. Conclusion: Under the influence of QP, the separation of caseous necrosis of granulomas from unaffected areas begins through connective tissue with fibrotization in the central part and a large number of Langerhans cells and lymphocytes that are not observed in the control groups. The interaction of QP with anti-TB drugs shows more obvious effects: fast tendency of epithelioid cellular tubercles to fibrotization and separation of TB granulomas through connective tissue. In addition, in the control groups of animals infected with TB, in contrast to the experimental groups, fatty degeneration of hepatocytes is observed. Thus, we have shown the hepatoprotective function of QP against TB.

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Objective/Background: Interferon (IFN)-γ-release assays (IGRAs) are designed for the diagnosis of tuberculosis (TB) infection. The new IGRA called QuantiFERON-TB Plus (QFT-Plus) is based on enzyme-linked immunosorbent assay (ELISA) detection of IFN-γ following Myobacterium tuberculosis-antigen stimulation with TB1 and TB2 antigens. TB1 elicits a cell-mediated immune response by CD4 T cells and TB2 elicits a response from both CD4 and CD8 T cells. Here, we characterized variations IFN-γ release detected by ELISA to QFT-IT and QFT-Plus in patients with active TB and latent TB infection (LTBI) at baseline and during or after specific treatment (follow-up). Methods: We studied seven patients with active TB and 10 patients with LTBI at baseline and during treatment either for active disease or preventive therapy. IFN-γ release detected by ELISA to QFT-IT and QFT-Plus was concomitantly evaluated over time. Statistical analysis was performed using a nonparametrical test for a paired dataset (Wilcoxon test). Results: All participants responded to the mitogen, with all active-TB patients responding to QFT-IT or QFT-Plus at baseline. The responses did not change over time either qualitatively (number of responders) or quantitatively (IFN-γ release evaluated as IU/mL). Among the LTBI group, although all participants responded to both QFT-IT and QFT-Plus and the responses did not change over time, the quantitative responses to QFT-Plus showed a different trend. Specifically, response to TB2 was significantly lower at follow-up as compared with that observed at baseline (p = 0.004), whereas the response to TB1 was not significantly different (p = 0.16). Conclusion: To our knowledge, this is the first report characterizing IFN-γ responses to QFT-Plus antigens in participants with active TB and LTBI over time. The data need to be confirmed in larger settings; however, we showed that monitoring IFN-γ release in response to TB2 can be used to evaluate preventive therapy immune changes. This can be useful also as a tool for public health control strategies in settings where preventive treatment is recommended.

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Objective/background: The GenoType MTBDRsl test rapidly detects resistance to ethambutol, fluoroquinolones, second-line aminoglycosides (amikacin [AMK] and kanamycin [KAN]), and cyclic peptides (capreomycin [CAP]) in Mycobacterium tuberculosis. According to data from Global Drug Resistance Surveillance Report (2007), Armenia is counted as a high-burden country for multidrug-resistant tuberculosis (MDR-TB). The estimated burden of MDR-TB in 2012 was 9.4 (7–12) and 43 (38–49) among retreatment TB cases. A total of 92 laboratory confirmed cases were reported to the World Health Organization (57 new and 35 previously treated) out of 511 cases tested for MDR-TB. Methods: A set of 77 drug-resistant TB isolates during 2011 and 2012 period, being either acid-fast bacterium positive or negative but culture-positive resistant to isoniazid, rifampin, or both according to the GenoType MTBDR plus assay, were consecutively tested using GenoType MTBDRsl. rrs gene analysis and the results from GenoType MTBDRsl were compared with phenotypic drug resistance testing. The DNA preparation method was performed as recommended by the manufacturer (Genotype MTBDR plus version 1.0 and Genotype MTBDRsl version 2.0 Hain Lifescience Nehren, Germany). Results: Aminoglycosides are key drugs for the treatment of MDR-TB. A total of 77 drug-resistant TB and four extensively drug-resistant M. tuberculosis isolates from Armenian TB patients were analyzed to characterize mutations within rrs and to compare with phenotypic drug resistance testing. Simultaneously, the following were identified: 65 (84.41%) rrs wild type (WT), 1 (1.3%) rrs WT MUT1 and MUT2 (WT; A1401G and G1484T), 1 (1.3%) rrs WT1, MUT1 (A1401G), 9 (11.7%) rrs WT1, MUT1 (A1401G), and 1 (1.3%) rrs WT1, MUT1. Mutation at position 1401 in rrs leads to resistance to KAN (7/77 = 9%), AMK (9/77 = 11.68%), and CAP (5/77 = 6.49%). Eleven (14.28%) streptomycin-resistant strains had a rrs mutation. Conclusion: Isolates with rrs structural gene mutations were cross-resistant to streptomycin, KAN, CAP, and AMK. Detection of the A1401G mutation appeared to be 100% specific for the detection of resistance to KAN and AMK. Being the first assessment, these data establish the presence of phenotypic drug-resistant and extensively drug-resistant strains using molecular profiling and are helpful in understanding aminoglycoside resistance on a molecular level.

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Objective Tuberculosis: (TB) is a devastating disease that remains a major health threat worldwide. The appearance of Mycobacterium tuberculosis strains resistance to current antibiotics is a growing problem, both in the third world and in developed countries. Completion of genomic sequencing of M. tuberculosis provides a strong foundation for subsequent identification of proteins to aid the understanding of protein function and the discovery of new drug targets or a TB vaccine. This study employed a proteomics approach to identify proteins from antibiotic resistant M. tuberculosis isolates and compare them to drug-sensitive isolates to determine the role of T cells in multidrug-resistant (MDR)-TB patients against M. tuberculosis-purified proteins (Rv0147) as compared with healthy subjects. Methods: Proteins were extracted by Triton X-114 detergent-phase separation and precipitated by adding saturated ammonium sulfate to the supernatant. Following isoelectric focusing, proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Mass spectrometry was performed, and protein sequences were determined. Peripheral bloom mononuclear cells (PBMCs) were cultured, and autologous T cells were isolated from PBMCs by negative selection. Cells were subsequently cultured at 37°C in 5% CO2, followed by stimulation with 10 μg/mL of the protein candidate (Rv0147) for 72 h. Culture supernatants were assayed for interleukin (IL)-10 and interferon (IFN)-γ by enzyme-linked immunosorbent assay. Results: The identified proteins included Rv3057c, Rv0009, Rv3161c, Rv3614c, Rv0685, Rv2986c, Rv0443, Rv2114, Rv3311, Rv0831, Rv3804, and Rv3614c, and our results showed that the majority of upregulated or overexpressed proteins belonged to pathways associated with cellular metabolism, cell wall integrity, respiration, or cell membrane construction. Additionally, Rv1876 from MDR-TB isolates was predicted to be involved in the expression of bacterioferritin exclusively in MDR-TB-related resistance to first-line TB drugs. Furthermore, Rv2031c (HspX) was induced under oxygen-deficient conditions, and hypothetical protein (Rv2744c) and two membrane- and cell-wall-fraction proteins (Rv0379 and Rv1886c) were also identified. Analysis revealed increased percentages of INF-γ and decreased IL-10 levels in MDR-TB patients as compared with those observed in normal subjects. Conclusion: Four identified membrane or membrane-associated proteins, including bacterioferritin, GroEs, HspX, and Ef-Tu, may be potential targets for the development of novel prophylactic diagnostics and therapeutic strategies against TB. Our results suggested that T cells stimulated by the protein candidate Rv0147 may be shifted to T helper 1 status in MDR-TB patients.

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Objective/Background: Mycobacterium species comprise one of the most frequently-reported causative agents in granulomatous lesions of fish. Also, mycobacteria have been documented as fish-born zoonoses. Ornamental aquaria, commercial aquaculture, and wild fisheries therefore produce a potential risk of infection for humans through physical contact and consumption of fishes. A number of fish mycobacteriosis and fish-born zoonotic cases have been reported to date in Iran. Methods: In order to examine the frequency of mycobacteria existence in fish supplied to the public in seafood retail outlets, whole fresh fish from cold water (n = 50) and tropical (n = 50) audible fish were obtained from five stores in Karaj, the central city of Alborz province. The head, tail, and offal of these fish were sampled during the necropsy and used for mycobacterial culture on Lowenstein–Jensen slopes. Results: In total, 15 acid-fast isolates were collected including 10 from tropical fish. The 16S ribosomal RNA and hsp genes of all isolates were polymerase chain reaction amplified and sequenced. Mycobacterium fortuitum was the most frequent mycobacterium identified in the study panel according to the Basic Local Alignment Search Tool search results. Work is still ongoing to characterize the other cultured mycobacterial isolates. Conclusion: The detection of 15 culturable mycobacterial isolates from 100 audible fish is an indication of bacteria highly active in the colonization in fish populations. Assessing the potential zoonotic risk raised from exposure of human to fishes in Iran demands search for evidence of linkages between mycobacteria infecting human cases and fishes.

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Tuberculosis is one of the most important zoonotic diseases in the world. Rapid diagnosis of the disease and identification of species is extremely important for proper treatment of the disease as some species of the complex are resistant to the first-line of tuberculosis drugs. The aim of present study was molecular identification of Mycobacterium tuberculosis (MTB) complex isolates from Kermanshah Province, Iran, which were submitted to the Tuberculosis Reference Laboratory at Razi Vaccine and Serum Research Institute (Tehran, Iran). To identify the genus Mycobacterium, all isolates were subjected to 16S rRNA polymerase chain reaction (PCR), and PCR-IS6110 was subsequently used to confirm that the isolates belonged to MTB complex. Finally, region of difference (RD) typing was used to identify the species in the complex. The results of 16S rRNA and IS6110 PCR analysis showed the presence of 543-bp and 245-bp bands, respectively. Furthermore, 146bp, 172bp, 235bp, and 369bp at RD1, RD4, RD9, and RD12, respectively, were observed during RD typing. Thus, based on the results, all isolates were identified as MTB. It is worth mentioning that most tuberculosis cases are identified on the basis of acid-fast bacilli detection, and antibiotic therapy is immediately initiated subsequently. Moreover, it should be noted that some of these acid-fast positive cases might not be of genus Mycobacterium, and thus, the antibiotics prescribed might threaten the health of the patients. Additionally, if the identified bacilli are not within MTB complex, the drug therapy would differ. However, Mycobacterium bovis, which is a member of MTB complex and is resistant to pyrazinamide, requires exact strain identification. Based on the findings, individual isolates should be identified by RD typing methods, which could clearly discriminate the species from each other.

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Objective/Background: Tuberculosis (TB) is a major global threat to human health, especially in low-income countries. The diagnosis of TB is challenging because of the limitations of specificity and sensitivity with the current diagnostics. Novel, selective biomarkers for TB would be of great practical value. Exosomes are bioactive vesicles with 30–100nm in diameter, which are secreted from almost all cell types and are found in virtually every human body fluid. Exosomes transport micro-RNAs (miRNAs), which are post-transcriptional regulators of gene expression, around the body and allow miRNAs to modulate biological pathways in target cells. Our aim was to investigate the potential of exosomal miRNAs as biomarkers by examining their release from human monocyte-derived macrophages (MDMs) after infection with Mycobacterium using miRNA sequencing. Methods: Human monocytes were obtained from blood and driven to an MDM phenotype using standard protocols. MDMs were infected with Mycobacterium bovis Bacillus Calmette–Guérin (BCG) or left uninfected as control. Exosomes were collected 72 h postinfection from the cell culture medium and subjected to RNA isolation. Small RNA libraries were constructed and RNA sequencing performed. The raw reads were filtered to eliminate adaptor and primer sequences, and the sequences in FASTQ format were run against the mature human miRNA sequences available in miRBase using BLAST software using a Linux operating system. Micro-RNAs were identified using E = 0.01 or 1. Results: Infection of MDMs with BCG lead to the release of a number of exosomal miRNAs. These mainly consisted with Let-7 family members, miR-155, miR-146a, miR-145, and miR-21 all of which were predicted to target important immune-related genes and pathways. Conclusion: This study provides evidence for the release of specific miRNAs from BCG-infected MDMs. These results need to be confirmed and the presence of this panel of miRNAs tested in the blood of patients to determine their selectivity and specificity as a diagnostic in patients with TB.

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Aims and objectives: Extrapulmonary disease accounts for a significant proportion of all cases of tuberculosis (TB) in endemic areas. With increasing prevalence of human immunodeficiency virus (HIV), the case numbers are rising; extrapulmonary involvement can be seen in >50% of patients with concurrent AIDS and TB. In both HIV-positive and HIV-negative cases, due to diagnostic difficulties, extrapulmonary disease is often recognized late and, hence, although completely curable in many cases, as far as bacterial eradication is concerned, is not without consequence. Methods: Sequelae of extrapulmonary TB were explored through literature review. Additionally, case files of patients presenting to the TB clinics in a tertiary care hospital in Karachi, Pakistan were examined for sequelae during variable periods of follow up. Results: The sequelae of TB can be divided into:

• those of ongoing inflammation, for example, vasculitis in central nervous system infection leading to neurologic deficit, or amyloidosis with renal failure in longstanding, inappropriately managed cases, or where the diagnosis is missed;
• healing with fibrosis, for example, intestinal obstruction, pericardial constriction, infertility;
• loss of function secondary to bone and joint deformity, for example, gibbus formation and paraplegia in spinal TB.

Conclusion: Early reliable diagnosis and anti-TB treatment, often with steroids, is essential for control of disease and prevention of complications. Patients need to be monitored clinically and supported psychologically, logistically, and socially to return to lead productive lives after extrapulmonary TB infections.

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Objective/Background: Nontuberculosis mycobacteria (NTM) are a diverse group of microorganisms that cause a variety of diseases in humans including skin, respiratory, and gastrointestinal tract infection. Generally, NTM are classified into two categories: rapid (<7 days) and slow growing (>7 days). In this study, we aimed to investigate NTM frequency and prevalence in environmental samples. Additionally, we tried to identify various subtypes of isolated rapid growing mycobacteria (RGM). Methods: Through a prospective descriptive cross-sectional study, water and soil samples were gathered from four neighboring towns around Tehran, the capital of Iran, at different geographic directions. Every 100m² of the studied areas gave one sample containing 6g of soil in 3–5 cm depth deposited in 50 mL sterile water as sampling media. After digestion and decontamination, DNA from culture-positive specimens (RGM) were extracted using phenol–chloroform methods. Then the molecular identification of species and subspecies were performed using 16s–23s rRNA and hsp65 gene. Results: In total, 341 RGM were found, out of which 322 (94.4%) were identified and 20 (5.8%) could not be identified. The most frequent RGM was, Mycobacterium fortuitum (72; 22%), Mycobacterium senegalense (58; 17.7%), Mycobacterium parafortuitum (44; 13.4%) and Mycobacterium conceptionense type 1 (24; 7.2%), and Mycobacterium cheloni type 1 (20; 6.0%). As shown in [Table 1], M. fortuitum had more subtypes (8), and the frequency of subtypes 1 (27.7%), 4 (16.6%), and 5 (13.8%) were higher. Among subtypes of M. senegalense, subtype 1 had a higher frequency (70.4%) in comparison to subtype 2 (29.5%). M. cheloni had just one subtype.{Table 1} Conclusion: Our results showed M. fortuitum as the most prominent strain isolated from environmental samples. The frequency was similar in different places, irrespective of climatic variations. Availability of various subtypes of M. fortuitum might indicate a large circulation of this RGM in soil and water of Iranian territory. This high prevalence of M. fortuitum might raise the risk infection, especially in children, immunocompromised patients, diabetics, and cancer cases.

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Objective/background: Incidence of tuberculosis (TB) in Golestan Province is consistently higher than other provinces of Iran. This study aimed to determine the rate of drug resistance to first-line antibiotics in Mycobacterium tuberculosis (MTB) isolates recovered from new cases in this province. Methods: The sputum and broncho-alveolar lavage samples from 3828 patients who were suspected for active TB were collected from March 2015 to July 2015. All specimens were subjected to smear microscopy and culture. Drug susceptibility testing to rifampicin, isoniazid, ethambutol, and streptomycin was performed on Löwenstein–Jensen medium using proportion method. Results: Of 3828 clinical specimens, 40 were culture-positive for MTB. The mean age of patients was 48.6 ± 17.5 years and 23 (57%) patients were male. Thirty-eight patients were native Iranians while two (5%) were immigrant patients from Turkmenistan and Afghanistan. A set of 34 (85%) isolates were pan-susceptible, six (15%) were resistant to at least one drug, and one isolate (2.5%) was multidrug resistant. Conclusions: The rate of drug resistance in this study area point to the necessity for further enforcement of TB treatment and disease control management. Future studies are recommended for assessments of drug resistance pattern of MTB isolates in Golestan Province.

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Introdution: Mycobacterium avium ssp paratuberculosis (MAP) causes paratuberculosis (Johne's disease) in ruminants. As a species, M. avium comprises M. avium subsp. hominissuis and a number of clones that are known to have evolved from this subspecies, namely M. avium subsp. avium (MAA), M. avium subsp. silvaticum, and MAP. Despite the very high genomic similarity of MAP and MAA, the insertion sequence IS900, which is 1,451-bp long, is now understood to be exclusively present in 10–20 copies in the genome of MAP. In the present study, a multidiscipline polymerase chain reaction (PCR)-based algorithm targeting16SrRNA, IS6110, IS901, IS1245, and IS900 markers has been employed to differentiate between six laboratory strains of M. avium complex (including MAP 316F, III&V, and 2e plus MAA D4), Mycobacterium tuberculosis DT, and Mycobacterium bovis AN5 strains used at the Razi Institute (Tehran, Iran) for the preparation of paratuberculin, avian, human, and bovine tuberculin, respectively. Materils and methods: Three laboratory strains of III&V, 2e, and 316F were subcultured on Herrold's egg yolk medium, whereas the MAA strain of D4 along with M. bovis AN5 and M. tuberculosis DT were subcultured on Lowenstein–Jensen slopes. All the inoculated culture tubes were incubated for 8 weeks at 37°C. Eventually, their genomic DNA was extracted according to the method of van Soolingen. Five individual PCRs were conducted on these templates to amplify 16SrRNA (genus-specific marker shared by all mycobacteria), IS900 (MAP-specific marker), IS901 (MAA-specific marker), IS1245 (M. avium complex (MAC)-specific marker), and IS6110 (M. tuberculosis complex (MTC)-specific marker) loci. Results: Consequently, a 543-bp amplicon was amplified by all the six strains in PCR against 16SrRNA, an indication of their identity as members of Mycobacterium genus. A 245-bp fragment was detected in only IS6110-PCR with M. bovis AN5 as well as M. tuberculosis DT. In the IS1245 assessment, the MAA strain of D4 produced a 427-bp amplicon, whereas none of the other studied strains produced this amplicon. A 1,108-bp amplicon fragment of the IS901 marker was successfully produced by MAA strain, whereas no PCR product was achieved in amplification of all the three MAP strains. In IS900-nested PCR, the three MAP strains produced the expected 400-bp and 298-bp fragments Conclution: However, no amplification was observed with other strains. Two main achievements of this work are the development of an efficient means of differentiation between the six Razi laboratory mycobacterial strains and characterization of the genomic profile of these strains, a capability that is vital when cross contamination is potentially an important concern.

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Objective/background: Tuberculosis (TB) remains the leading cause of AIDS-related deaths among adults in countries with resource limitations. The emergence of the Xpert MTB/RIF rapid molecular assay and its subsequent World Health Organization endorsement in 2010 transformed the TB-diagnostic landscape. Xpert provided diagnostic accuracy that was far superior to that of the sputum-smear microscopy test previously used. The detection of mycobacterial lipoarabinomannan (LAM) antigen in urine has emerged as a potential point-of-care test for TB. LAM antigen is a lipopolysaccharide present in mycobacterial cell walls which is released from metabolically active or degenerating bacterial cells and appears to be present only in people with active TB. Urine-based testing has advantages over sputum-based testing because urine is easy to collect and store and lacks the infection control risks associated with sputum collection. A previously study reported that urinary-LAM testing is a rapid, low-cost, ante-mortem diagnosis for human immunodeficiency virus (HIV)-associated TB. The objective of this study was to investigate the levels of LAM in HIV patients referred to the Mashih Daneshvari Hospital Tehran, Iran. Methods: Urine from 31 HIV patients without TB, 33 HIV patients with pulmonary TB, and eight HIV patients with extrapulmonary TB was analyzed for LAM using enzyme-linked immunosorbent assay kits (Mybiosource, San Diego, CA, USA). Results: The plasma levels of LAM in pulmonary TB/HIV patients was 7.67 ± 2.3 ng/ml compared with 4.5 ± 1.6 ng/ml in extrapulmonary TB/HIV and 6.7 ± 1.2 ng/ml in HIV patients without TB. There was no significant difference in urine LAM levels between the three groups. Conclusion: Our results highlight the limitations of using urine LAM levels for differentiating HIV-associated TB patients in Iran.

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Objective/Background: The incidence of resistant strains of Mycobacterium tuberculosis (MTB), including multi-drug resistant, extensively drug resistant, or totally drug resistant, is one of the major problems of health policies worldwide. The accumulation of mutations causes multi-drug resistant strains. Mycobacterium abscessus has a plasmid called pMab2401 containing the trfA1 gene in its integron part. The aim of the present study was to investigate the possible existence of the trfA1 gene in clinical strains of MTB for the first time. Methods: Bioinformatics analysis in GenBank revealed an absence of any integrons or internal components in MTB. Several specific primers for different genes and the trfA1 gene of M. abscessus were used in a touch-down (60–52) amplification program and followed by loading on gel electrophoresis. Products were extracted and were sequenced. Sequencing results were analyzed carefully and compared with those in the databank. Results: Bands of 500bp were resulted with pair primers in an amplification reaction by clinical MTB that has 94% identity with a fragment in most plasmids and phages M13. It should be noted that such an independent replication system-like structure has not been reported to date in MTB strains. Conclusion: The trfA1 gene is depends on the replication process. It is necessary to investigate other probable new areas that may be of concern with drug resistance in clinical isolates of MTB.

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Introduction: Mycobacterium haemophilum is a slow-growing nontuberculous mycobacterium (NTM) that can cause ulcerating cutaneous or subcutaneous nodular skin lesions in immunocompromised and immunocompetent patients. Acid-fast staining cannot distinguish NTM from M. tuberculosis; culturing at two temperatures with iron-supplemented media and polymerase chain reaction (PCR) are needed for optimal detection of M. haemophilum. Case presentation: A 32-year-old man with end-stage renal disease, undergoing hemodialysis twice a week, presented with multiple, painless, nonpruritic nodular lesions. A formalin-fixed paraffin-embedded tissue block from his finger lesion was sent to the Department of Pathology, Masih Daneshvari Hospital for consultation. The lesions were primarily diagnosed to be dermatofibroma by another pathologist. On microscopic examination, vague granuloma with areas of necrosis was observed. The diagnosis was established by positive acid-fast staining, negative PCR results for M. tuberculosis complex, and positive nested PCR results for M. haemophilum. Conclusion: Cutaneous lesions in immunocompromised patients with positive results in acid-fast staining and negative results for M. tuberculosis should be further assessed using skin culture and molecular techniques to identify rare, atypical mycobacterial species like M. haemophilum.

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Objective/background: Children living in contact with smear-positive pulmonary tuberculosis (TB) patients are highly exposed to TB infection. Our objective was to estimate the incidence of TB in children living in contact with a Smear Positive (M+) pulmonary tuberculosis (PTM+) index case during 2 years following exposure. Methods: This was a descriptive, cohort, prospective, multicenter study of children aged from 6 months to 15 years in contact with a PTM+ case. The recruitment of children has been based on the diagnosed PTM+ index case and taken in charge by the Services of Control of Tuberculosis and Respiratory Diseases located in Algiers during 2014. Seven centers were selected. All children were tested using the Quantiferon TB gold in tube (QTR) test and the tuberculin skin test (TST). For TST, an induration diameter ≥10 mm was considered positive. Results: We included 456 children living in contact with a PTM+ patient. The results for TST and QFT were available for 319 children. The mean age of the children was 6.7 years (standard deviation = 3.9). The sex ratio (Male/Female) was 1.26, and 15.8% (50) did not have a Bacilli of Calmette & Guerin (BCG) vaccination scar. Among the children, 46.1% (147) and 43.4% (138) were positive for QFT and TST, respectively, and 6.1% (19) have received isoniazid preventive therapy. Fifty-one children progressed to TB and received antitubercular treatment. We analyzed and compared our results between children who progressed to TB and those who did not progress to TB. Finally, we discuss our methodology and results in relation to the literature.

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Objective/background: Tuberculosis (TB) is one of the most common infectious diseases in Iran and around the world. Diagnosis of this disease in many cases is difficult and often requires the use of paraclinical methods. Current diagnostic methods are either too slow or lack enough sensitivity or specificity. Several mycobacterial antigens are involved in the complex interaction with the immune system of the host. They can be helpful for mycobacteria diagnosis. Antigen 60 (A60) is a thermostable antigen found in the cytosol of Mycobacterium bovis and Mycobacterium tuberculosis. This antigen is used in ELISA systems design for diagnosis of tuberculosis. The aim of this study is purification of A60 from bacterial cytoplasm and to evaluate the efficiency of this antigen and compare it with the production human tuberculin and standard human tuberculin. Methods: Using gel filtration chromatography with a sepharose 4B column, A60 was purified from other bacterial components. A60 was recognized by agar gel immunodiffusion with anti-BCG and anti-A60 antiserum, where it formed an immunoprecipitation line with anti-BCG antiserum and anti-A60 antiserum. Molecular weight components of the A60 were obtained using electrophoresis. Results: Seven fractions were obtained by chromatography. In analyzing with dot blotting, both the cytoplasm and cell wall of BCG had A60. This test showed that the first fraction creates maximum color intensity and as a result, the highest amount of A60 was in fraction one. In agar gel immunodiffusion, the cytoplasm sample and all fractions obtained from chromatography showed a positive reaction with anti-A60 antiserum, and fraction one had the most among sediment of the other fractions. Molecular weight components of the A60 were identified to be approximately 35 kDa, 38 kDa, 40 kDa, and 65 kDa. Conclusion: Results of reactions of the injected A60 and standard human tuberculin shows the effectiveness of this antigen in comparison with standard human tuberculin. Detection of antibody in the serum of patients is a rapid and repeatable method. A60 with 89% sensitivity and 94% specificity could be an appropriate matter for the diagnosis of tuberculosis. Because this method can be performed without radioactive materials or advanced and expensive equipment, it will provide results quickly.

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Objective: Avian tuberculosis is one of the most important infections affecting most species of birds. Several mycobacterial species have been identified causing avian tuberculosis, and the organisms confirmed most frequently are Mycobacterium avium and Mycobacterium genavense. Any species of birds can be infected with M. avium. Generally, domesticated fowl or captive wild birds are affected more frequently than those living in the wild. M. avium can not only infect all species of birds, but can also infect some domesticated mammals to cause disease, usually with localized lesion. In immunocompetent individuals, M. avium complex isolates produce localized soft tissue infections, including chronic pulmonary infections in the elderly and cervical lymphadenitis in children, but rarely any disseminated disease. In patients infected with HIV and AIDS or in other immunocompromised individuals, M. avium complex isolates frequently cause severe systemic infections. The importance of avian tuberculosis and the risk of its zoonotic spread motivated our interest to determine the drug susceptibility testing of M. avium subsp. avium isolates from naturally infected domestic pigeons to avian tuberculosis. Methods: Based on their clinical signs, 80 pigeons suspected with avian tuberculosis were subjected to the study. Out of the 51 identified isolates, 20 M. avium subsp. avium were subjected to the test. Drug susceptibly testing was performed according to the guidelines by Centers for Disease Control and Prevention and using proportional method. Results: In the drug susceptibility testing, all isolates were resistant to streptomycin, kanamycin, ethionamide, and thiophene carboxylic acid hydrazide. Additionally, 3, 2, and 1 isolates were susceptible to isoniazid, rifampin, and ethambutol, respectively. To date, no study has documented the drug susceptibility testing of M. avium isolates from infected birds to avian tuberculosis. Pigeons are extensively kept in urban and rural areas for homing and racing purposes; thus, they can infect people and farm animals exposed to their droppings containing pathogenic M. avium, and the severity of drug resistance of these isolates indicate lethality in immunocompromised individuals and incurable lymphadenitis in immunocompetent individuals. Conclusion: We suggest drug susceptibility testing for more nontuberculous mycobateria, particularly M. avium complex isolated from infected birds and humans, as well as molecular basics of drug sensitivity in order to detect resistance genes of pathogenic M. avium subsp. avium.

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Objectives/Background: Efforts to control the global burden of tuberculosis (TB) epidemic have now been jeopardized by the rapid evolution of drug-resistant Mycobacterium tuberculosis (MTB), which are resistant to one or more anti-TB drugs. Multidrug resistant (MDR) TB in Ethiopia may be more prevalent than previously appreciated; thus, up-to-date national drug resistance studies are critically needed. Therefore, this meta-analysis aimed, first, to determine pooled prevalence of MDR TB among newly diagnosed and previously treated TB cases, and second, to measure the association between previous anti-TB exposure and acquisition of MDR-MTB infection. Methods: PubMed and Embase databases were consulted. Studies that reported the prevalence of MDR TB among newly diagnosed and previously treated TB patients were selected. Studies or surveys conducted at a national or subnational level, with reported MDR-TB prevalence or sufficient data to calculate the prevalence, were considered for the analysis. Two authors searched and reviewed the studies for eligibility and extracted the data in predefined forms. Forest plots of all prevalence estimates were performed, and summary estimates were also calculated using random effect models. Associations between previous TB treatment and MDR-MTB infection were examined through subgroup analyses stratified by new and previously treated patients. Results: We identified 16 suitable studies, and found an overall prevalence of MDR TB of 1.7% (95% confidence interval 1.2–2.3%) among newly diagnosed and that of 14.1% (95% confidence interval 10.9–17.2%) among previously treated TB patients, and the observed difference was statistically significant (p <.01). For the past 10 years, the overall MDR-TB prevalence showed a stable time trend. There was an odds ratio of 8.1 (95% confidence interval 7.5–8.7) for previously treated TB patients to develop an MDR-MTB infection compared with newly diagnosed cases. Conclusion: The MDR-TB prevalence remains high, especially in previously treated TB cases. Previous TB treatment was the most powerful predictor for MDR-MTB infection. Hence, strict compliance with anti-TB regimens and improving case detection rate are urgently needed to tackle the problem.

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Background: Early detection for tuberculosis (TB) and rifampicin-resistant TB (RRTB) is crucial for proper control of this disease. WHO recommended the use of the GeneXpert assay at district level to cover these two public health demands? A study evaluated the diagnostic impact of the GeneXpert assay in detecting TB and RRTB. Odd results were observed in this study in the form of discordance between the GeneXpert assay and the conventional culture and drug-susceptibility testing (DST). Aim of the study To assess the molecular diagnostic validity of the GeneXpert assay when results do not match phenotypic results given by DST. Methods: Pulmonary TB patients with recently detected sputum positive for acid-fast bacilli (AFB) were recruited from random geographical clusters (18 out of 36 primary healthcare districts in the middle five governorates in Iraq) during a 1-year period (November 2013–October 2014). Sputum samples from all enrolled patients were sent for GeneXpert assay testing, culture, and DST. Genotype mycobacterium (GM) from Hain Lifescience (Nehren, Germany) was used to detect non-tuberculosis mycobacteria (NTM) whenever suspected. Those with discordant results regarding the status of RRTB between GeneXpert assay and DST were retested with the line probe assay (LPA). Simple frequency distribution was used to describe study results. Results: Four-hundred ten patients were enrolled, all of whom were culture positive. Only two patients were found negative for TB on GeneXpert assay who were then diagnosed as NTM by LPA (GM). Out of the 408 patients, discordance between GeneXpert and DST regarding the status of rifampicin susceptibility was observed in 17 cases (4%). Nine patients were RR on GeneXpert but rifampicin susceptible (RS) on DST. LPA agreed with GeneXpert assay for all nine cases. Eight patients were RS on GeneXpert but RR on DST. Here, LPA disagreed with GeneXpert assay only in one patient who was found to be RR by LPA. Conclusion: GeneXpert assay is a valid molecular test for TB and RRTB regardless of its discordance with conventional culture and DST.

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Objective/background: There is a paucity of published data on the effect of tuberculosis (TB) chemotherapy on nitric oxide (NO) synthesis and metabolism in newly diagnosed and relapsed patients with or without multidrug-resistant TB (MDR-TB). Methods: The pattern of NO response in 140 patients with pulmonary TB, including 74 with MDR-TB (1st group) and 66 without MDR-TB (2nd group) has been studied and compared with the NO status of 30 healthy donors (3rd group). Patients comprised those with newly diagnosed pulmonary TB (Subgroups 1B and 2B) and recurrent or relapsed TB (Subgroups 1A and 2A). The NO status was assessed by measuring inducible NO synthase (iNOS), nitrites, and nitrates levels. This was measured prior to treatment initiation and 2 months after the prescribed chemotherapy. Results: Increased levels of NO indices were found in patients with TB when compared with healthy controls—1st group: iNOS, 231.6 ± 6.65pmol/min/mgB; nitrites, 5.626 ± 0.15 μmol/L; and nitrates, 62.89 ± 1.42 μmol/L (Subgroup 1A: iNOS, 208.40 ± 8.26pmol/min/mgB; nitrites, 5.027 ± 0.17 μmol/L; and nitrates, 59.29 ± 1.79 μmol/L and Subgroup 1B: iNOS, 260.4 ± 8.56pmol/min/mgB; nitrites, 6.371 ± 0.19 μmol/L; and nitrates, 67.36 ± 2.03 μmol/L); 2nd group: iNOS, 286.3 ± 5.92pmol/min/mgB; nitrites, 6.747 ± 0.17 μmol/L; and nitrates, 72.02 ± 1.43 μmol/L (Subgroup 2A: iNOS, 260.9 ± 14.12pmol/min/mgB; nitrites, 5.686 ± 0.20 μmol/L; and nitrates, 66.26 ± 1.89 μmol/L and Subgroup 2B: iNOS, 293.7 ± 6.13pmol/min/mgB; nitrites, 7.059 ± 0.19 μmol/L; and nitrates, 73.72 ± 1.71 μmol/L) versus healthy controls (iNOS, 81.03 ± 2.36pmol/min/mgB; nitrites, 3.83 ± 0.093 μmol/L; and nitrates, 37.98 ± 1.30 μmol/L). After 2 months of chemotherapy, a significant decrease in NO indicators was observed in the patients with TB, particularly in those without MDR-TB—1st group: iNOS, 114.9 ± 3.2pmol/min/mgB; nitrites, 4.21 ± 0.13 μmol/L; and nitrates, 46.65 ± 1.04 μmol/L (Subgroup 1A: iNOS, 125.3 ± 4.5pmol/min/mgB; nitrites, 4.42 ± 0.14 μmol/L; and nitrates, 49.38 ± 1.30 μmol/L and Subgroup 1B: iNOS, 102 ± 3.53pmol/min/mgB; nitrites, 3.93 ± 0.13 μmol/L; and nitrates, 43.26 ± 1.50 μmol/L) and 2nd group: iNOS, 91.4 ± 2.53pmol/min/mgB; nitrites, 3.67 ± 0.09 μmol/L; and nitrates, 35.65 ± 1.06 μmol/L (Subgroup 2A: iNOS, 106.7 ± 5.2pmol/min/mgB; nitrites, 4.04 ± 0.19 μmol/L; and nitrates-40.53 ± 1.83 μmol/L and Subgroup 2B, iNOS, 86.7 ± 2.59pmol/min/mgB; nitrites, 3.56 ± 0.1 μmol/L; and nitrates, 34.22 ± 1.19 μmol/L). The decline in NO activity was less prominent in patients with recurrent TB and MDR-TB, which suggests lower level of immunologic and reparative processes in such patients. Conclusion: In patients with pulmonary TB, significantly higher levels of NO activity were observed as compared with the levels in healthy individuals. In patients with recurrent TB and MDR-TB, significantly lower levels of NO indicators were observed in comparison with patients with newly diagnosed pulmonary TB. After 2 months on chemotherapy, a significant decrease in iNOS activity and NO metabolites was observed in patients with pulmonary TB, but the decrease in NO indicators was manifested mostly in the newly diagnosed pulmonary TB patients and patients without MDR-TB as opposed to patients with recurrent TB and MDR-TB, which suggests lower levels of immunologic and reparative processes in such patients. Therefore, the levels of nitrites and nitrates as well as iNOS activity may serve as additional diagnostic criteria to differentiate MDR-TB from nonresistant TB in patients with relapsed and newly diagnosed TB. Easily assessed NO-related markers can also serve as predictors of treatment outcome because patients with drug-susceptible strains had lower NO output approaching levels found in controls.

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Objective/Background: Interferon gamma (IFN-γ) plays a key role in protective immune response against Mycobacterial infection. IFN-γ excretes its antimycobacterial effectors mechanisms by activation of macrophages and dendritic cells via interaction with its receptor complex, that is, a ligand-binding subunit [IFN-γ receptor (IFNGR)1] and an accessory subunit (IFNGR2) on the cell surface. It has been shown that individuals with complete or partial IFNGR1 receptor deficiency are highly susceptible to infection by nontuberculous mycobacteria (NTM), Mycobacterium tuberculosis, and some Salmonella species. In the present study, we aimed to study the IFNGR1 T-56C single nucleotide polymorphism (SNP) in pulmonary patients that were infected with rapid grower mycobacterium. Methods: Sputum specimens from suspected nontuberculosis pulmonary patients (n = 95) were digested and decontaminated using 4% NaOH method. Molecular identification of mycobacterium was then performed by hsp65 genes using polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP). Finally, the host genomic DNA from confirmed patients with rapid-grower mycobacterium (n = 20) and control subjects (n = 20) were screened for SNPs of IFNGR1 (T-56C) by PCR-RFLP. Results: Out of 95 NTM patients, 20 (21.0%) were infected with rapid grower mycobacterium (RGM). The frequency of Mycobacterium chelonae (n = 12) was more than Mycobacterium fortuitum (n = 8), but the differences were not statistically significant. Interestingly, 18 patients (90%) had CC genotypes, whereas the remaining two had TC genotypes. The frequency of CC genotypes in the control group was <10% (p <0.05). Conclusion: There is a significant association between SNP of IFNGR1 at –56 and susceptibility to rapid grower infection.

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Mycobacterium bovis is mainly detected in cattle throughout the world. This bacterium is considered the main causative agent of tuberculosis in man and animals. M. bovis is also reported to be endemic in badgers and in farmed and feral deer. The disease caused by M. bovis is a slow progressive disease with clinical signs not apparent until late in the disease process. key factors for effective control of tuberculosis includes rapid detection, adequate therapy, and contact tracing to halt further transmission. However, in order to locate the source and route of transmission of M. bovis infection, a thorough epidemiological analysis is routinely carried out, that could lead to the control of disease in the herds and avoid economic losses. Recent developments in DNA technology and molecular biology have led to methods for the rapid detection of mycobacterium DNA. Among a number of described molecular methods, IS6110 fingerprinting is the recommended standard primary genotyping method and the most widely used worldwide. In this study, we used restriction fragment length polymorphism analysis with probes derived from the insertion element IS6110, the polymorphic GC-rich sequence (PGRS), and the direct repeat (DR) sequence which proved to be a useful method for differentiating M. bovis strains. A total of 13M. bovis samples from infected cattle in the West Azerbaijan Province of Iran were included in the study. The samples were submitted to the Tuberculosis Reference Laboratory at the Razi Vaccine and Serum Research Institute, Karaj. All isolates were cultivated on Lowenstein Jensen media with pyruvate (pyruvic acid), and then identified according to The World Organization for Animal Health (OIE) recommendations. The extracted genomic DNA samples of the isolates in the study were subjected to IS6110 primers, digested with restriction enzyme PvuII, and hybridized by oligonucleotide probes PGRS and DR. Polymorphic banding patterns obtained after hybridization discriminated the M. bovis strains and a database of strain types was established. Based on our results, the 13 isolates showed five different DNA patterns with a PGRS probe and similarly five patterns were obtained with the DR probe. PP-1 pattern was found almost among all the isolates while a distinct DNA pattern PP-3 was seen specifically from the West Azerbaijan Province.

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Objective/Background: Tuberculosis (TB) is a major global health problem and poses immense threats to many populations. The association between tobacco smoke and TB has already been studied. Water-pipe smoking has become an increasing problem not only in Middle Eastern countries but also globally as it is considered by users as being safer than cigarettes. The presence of high levels of toxic substances in water-pipe smoke may be predisposing factors that enhance the incidence of pulmonary disorders in water-pipe smokers. For example, uncontrolled macropinocytosis occurs in alveolar epithelial cells following exposure to water-pipe smoke, which may predispose individuals to pulmonary infection. In this work, we studied the effects of water-pipe condense (WPC) on the internalization of Mycobacterium bovis (bacillus Calmette–Guérin [BCG]) by macropinocytosis in Type II alveolar epithelial cells (A549). Methods: A549 cells were treated by WPC (4 mg/mL) for 24 h, 48 h, 72 h, and 96 h, respectively. The effect on cell proliferation was studied using a methylthiazolyldiphenyl-tetrazolium bromide (MTT) reduction assay. Cells were exposed to fluorescein isothiocyanate (FITC)–dextran (1 mg/mL; control) and FITC–BCG (multiplicity of infection, 10) for 20min at 37°C before their collection and the uptake of BCG–FITC was determined by flow cytometry. Similar experiments were performed at 4°C as a control. Results: WPC (4 mg/mL) after 72 h (1.4 ± 0.2-fold, p <0.05) and 96 h (1.6 ± 0.2-fold, p <0.05) hours increased the uptake of BCG–FITC. No effect on BCG–FITC uptake was observed at 24 h or 48 h. WPC also significantly increased the uptake of FITC–dextran (2.9 ± 0.3-fold, p <0.05) after 96 h. WPC also significantly decreased cell proliferation after 24 h (84 ± 2%), 48 h (78 ± 3%), 72 h (64 ± 2%, p <0.05), and 96 h (45 ± 2%, p <0.05). Conclusion: WPC exposure increased epithelial cells' permeability and death and enhanced their capacity for macropinocytosis. Our in vitro data suggest possible harmful effects of WPC on the ability of lung epithelial cells to phagocytose mycobacteria. Further studies will be conducted to understand the mechanism of action of WPC.

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Objective/background: Nontubercular mycobacteria (NTM) is not known to be associated with fistula-in-ano. Methods: A total of 311 consecutive fistula-in-ano patients operated on over 2 years were analyzed. The histopathology of fistula-in-ano tract lining was performed in all the cases and other tests [real-time polymerase chain-reaction (RT-PCR), Gene Xpert, mycobacterial culture] were completed in patients with a high index of suspicion of having mycobacterial disease. Results: Two patients had histopathological features suggestive of mycobacterial disease. Out of these, one patient had NTM and another had Mycobacterium tuberculosis (MTB) on RT-PCR. Four patients had normal histopathology features but tested positive on RT-PCR (two for NTM and two for MTB). Therefore, a total of six patients tested for mycobacterial disease (three for NTM and three for MTB). Mycobacterium culture was studied in two patients (both NTM) but was negative. Five out of six patients (two NTM and three MTB) presented with delayed recurrences after operations (6–18 months after complete healing). Conclusion: Nontubercular mycobacteria can cause fistula-in-ano. It could be an undiagnosed contributory factor in fistula recurrence. Mycobacterial disease (both tubercular and nontubercular) may be associated with delayed recurrence of fistula. RT-PCR is a highly sensitive test and can differentiate between NTM and MTB. It should perhaps be performed in all recurrent and refractory cases.

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Objective/Background: Mycobacterium avium subsp. paratuberculosis is a slow growing, Gram-positive, and acid-fast bacillus. It is the causative agent of the chronic enteritis disease of ruminants called Johne's disease. Most of the infected animals are young. However, most clinical cases belong to adult cattle aged 3–5 years. Due to a prolonged incubation period, identification of subclinically infected animals is one of the most crucial problems in Johne's disease. The aim of this study was to detect M. avium subsp. paratuberculosis in cattle using indirect absorbed enzyme-linked immunosorbent assay (ELISA) and culture for positive samples in Alborz Province, Iran. Methods: Briefly, 384 blood samples were taken from cattle of five areas of Alborz Province. Blood samples were tested by indirect absorbed ELISA (Razi paratuberculosis kit, Razi Vaccine and Serum Research Institute, Karaj, Alborz, Iran). Soluble antigens of Mycobacterium phlei were used to remove nonspecific antibodies in the bovine serum. Before transferring serum samples of cattle to the plate that coated with (MAP) antigens, serum samples of cattle were diluted and pre-incubated with a dilution buffer containing M. phlei antigens. During this project, we cultured fecal samples of cattle who displayed positive results for ELISA test. Anti-ruminant immunoglobulin G and Horse Radish Peroxidase (HRP) were added to all microwells. After washing, the substrate solution tetra-methyl benzidine (TMB) was added to eliminate the excess conjugate. The microplate was read using a spectrophotometer (ELISA reader, Bio Rad-Model 620) at 450nm in the Razi Vaccine and Serum Research Institute (Karaj, Iran). All ELISA-positive samples were cultured in media tubes (3 tubes of Herrold's egg with Mycobactin and 1 tube of Herrold's egg without Mycobactin for every sample). Results: In total, 3.19% of cattle serum samples showed significant antibodies titer to infection with M. avium subsp. paratuberculosis in all the areas of sample source, whereas 96.81% serum samples were negative. Of the 12 ELISA-positive samples, six samples showed growth in the media. Conclusion: Because there is no treatment or cure for Johne's disease, detection of infected cattle and subsequent culling is very important for preventing infection in other cattle. Fecal culture is a standard method for the identification of the disease. However, in Johne's disease, due to prolonged incubation and shedding of the disease, the probability of isolating the responsible agent is very low. To identify the infection, the indirect absorbed ELISA method is used for eradication. This technique is considered as one of the most reliable for identification of the disease worldwide due to its ease of use and low cost. However, for confirmation of ELISA-positive results, culture method has been recommended.

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Bovine tuberculosis (TB) is an important zoonotic disease that is caused by Mycobacterium bovis. Eradication efforts in developed countries have reduced the prevalence of this disease significantly. TB can be difficult to diagnose based only on the clinical signs; therefore, it is usually diagnosed in the field with the tuberculin skin test and diagnostic blood tests, including the lymphocyte proliferation assay, the interferon (IFN)-γ assay, and enzyme-linked immunosorbent assay. The aim of this study was to compare the tuberculin and IFN-γ tests. A total of 110 animals were evaluated by tuberculin skin test (TST) and IFN-γ assay; the culture was selected as a gold standard. The animals were selected randomly from 700 cattle on dairy farms, aged 3–5 years and suspected of having TB. Ten cattle were positive using the TST and nine were positive by IFN-γ assay. All nine positive samples in the IFN-γ assay were positive in culture too. The observed errors in IFN-γ assay were less due to laboratorial tools. It is suggested that all positive samples in TST are also positive by IFN-γ too.

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Objective: Tuberculosis (TB) is a zoonotic infectious disease common to humans and animals that is caused by the rod-shaped acid-fast bacterium Mycobacterium bovis. Rapid and sensitive detection of TB is promoted by specific antigens. Virulent strains of the TB complex from M. bovis contain 16 regions of difference (RD) in their genome that encode important proteins, including major protein of Mycobacterium tuberculosis 64 (MBT-64, which is a primary immune-stimulating antigen encoded by RD-2. In this study, we cloned, expressed, and purified MPT-64 as a potent M. bovis antigen in a prokaryotic system for use in future diagnostic studies. Methods: The antigenic region of the Mpt64 gene was investigated by bioinformatics methods, cloned into the PQE-30 plasmid, and expressed in Escherichia coli M15 cells, followed by isopropyl β-d-1-thiogalactopyranoside induction. The expressed protein was analyzed sodium dodecyl sulfate polyacrylamide gel electrophoresis and purified using a nickel-affinity column. Biological activity was confirmed by western blot using specific antibodies. Results: Our data verified the successful cloning of the Mpt64 gene (687-bp segment) via the expression vector and purification of recombinant MPT-64 as a 24-kDa protein. Conclusion: These results indicated successful expression and purification of recombinant MPT-64 protein in a prokaryotic system. This protein can be used for serological diagnosis, improved detection of pathogenicity and non-pathogenicity between infected cattle, and for verification of suspected cases of bovine TB.

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Objective/Background: With the introduction of novel molecular techniques that rely on rifampicin (RIF) susceptibility, resistance to isoniazid (INH) or other first-line drugs remains undetected. Such patients are prescribed first-line antituberculosis therapy and are on RIF monodrug therapy during the continuation phase, which may lead to therapeutic failure and emergence of multidrug resistance. We aimed to study INH resistance among RIF-susceptible Mycobacterium tuberculosis (MTB) isolates from retreatment patients. Methods: The Drug Susceptibility Testing data for four first-line drugs (streptomycin [SM], INH, RIF, and ethambutol [EMB]) using BACTEC MGIT 960 (Becton Dickinson, Franklin 124 Lakes, NJ ,USA) and for two drugs (INH and RIF) using line probe assay was analyzed retrospectively at the Department of Microbiology, National Institute of Tuberculosis and Respiratory Diseases (New Delhi, India). Results: We analyzed 4910 drug susceptibility results performed using the BACTEC MGIT960 liquid culture system from 2009 to 2015. We found that 969 (19.7%) isolates were sensitive to all four first-line drugs, 3941 (80.3%) isolates were resistant to one or more drugs, and 3041 (61.9%) isolates were resistant to both RIF and INH with or without resistance to any other drug (multidrug resistant). Monodrug resistance to SM and EMB was observed in 94 (1.9%) and 8 (0.16%) isolates, respectively. RIF resistance without INH resistance was observed in 22 (0.44%) isolates. There were 776 isolates sensitive to RIF, but resistant to INH. Among these, INH resistance with EMB and/or SM was observed in 367 (7.47%) isolates, whereas 409 (8.3%) isolates were resistant to INH alone. The results of line probe assay from 2012 to 2015 were also analyzed, and the resistance to INH alone among all isolates with valid results was found to be 9.32% (1462/15,676). More than 75% of these isolates harbor mutations in the kat G gene associated with high-level resistance. Conclusion: INH resistance among RIF-susceptible isolates was present in 10–15% of the total cases. Among these cases, the use of RIF susceptibility alone will fail to detect INH resistance. Since higher rates of failure, relapse, or acquired resistance are linked with INH resistance, rollout of techniques focusing on RIF resistance must, therefore, be accompanied by strict monitoring for better management of patients.

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Objective/background: World Health Organization estimates that approximately one-third of the global community is infected with Mycobatcerium tuberculosis (MTB). Various molecular epidemiology methods were developed and found very promising for assessing the genetic diversity among MTB complex strain. The two major tools restriction fragment length polymorphism (RFLP) and spoligotyping were commonly used in various studies. Some Indian studies raise the question about the utility of IS6110-RFLP in India due to the presence of zero and low copy number of IS6110 element in MTB isolates. In this short study, we attempt to evaluate the usefulness of IS6110-RFLP in genotyping of MTB isolates in North India. Methods: We conducted a short study involving 26 MTB isolates collected from Sawai Madhopur, Rajasthan, India. IS6110-RFLP analysis was performed as previously described method. In brief, the procedure involve MTB DNA digestion (PvuII restriction enzyme), electrophoresis on 1% agarose gel, transfer of DNA fragments on positively charged nylon membrane, hybridization with digoxigenin-labeled IS6110 probe, and detection by digoxigenin nucleic acid labeling and detection kit. Results: IS6110-RFLP analysis of 26 MTB isolates showed presence of IS6110 element in varying range from 0 to 17 copies. Out of the 26 MTB isolates, two (7.8%), three (11.5%), and 21 (80.8%) showed zero, low, and multiple copy numbers, respectively. The isolates, which had IS6110 element, showed 22 different RFLP patterns. Two clusters of two isolates each were found, and 20 isolates showed unique RFLP pattern. The two clusters of isolates had 11 and 13 copy numbers of IS6110. Conclusion: In this study, we found that the majority of isolates were having multiple copy numbers of IS6110 element and showed a very diverse pattern. These results showed that the IS6110-RFLP analysis is still a promising genotyping method and has good discriminatory power to differentiate strains of MTB isolates in India.

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Objective/background: Mycobacterium tuberculosis (MTB) is a known cause of refractory and recurrent fistula-in-ano. Histopathology of fistula tract and acid fast bacillus (AFB) smear of the pus are the standard procedures employed to diagnose MTB. However, they have some drawbacks. Nontubercular mycobacteria (NTM) has also been detected to cause fistula-in-ano and these methods cannot differentiate between MTB and NTM. Secondly, as these methods have low sensitivity, they could possibly be missing out MTB patients. Real-time polymerase chain reaction (RT-PCR) has high sensitivity in detecting mycobacteria. The aim of the study was to compare the sensitivity of RT-PCR, histopathology, and AFB smear in detecting MTB in fistula-in-ano. Methods: The histopathology and RT-PCR of tissue (fistula tract) was done along with AFB smear and RT-PCR of the pus was done in all the cases as per the availability of the specimen. The histopathology, AFB smear and RT-PCR was done by same pathologists in all the cases and all the patients were operated by a single surgeon. Results: A total of 286 samples were tested in 161 patients of fistula-in-ano who were operated over a period of 1year. The mean age was 38.6 ± 10.5 and male/female ratio was 153/8. Histopathology and RT-PCR of tissue (fistula tract) was done in 131 patients and 141 patients respectively. AFB smear and RT-PCR of pus (fistula) was done in 14 patients. Overall, MTB was detected in total of 17/161 (10.63%) patients. Out of these, MTB was detected in tissue (fistula tract) in 1/131 (0.76%) by histopathology and 14/141 (10%) by RT-PCR tissue. In pus samples, AFB smear was negative in all cases (0/14), whereas RT-PCR detected MTB in four of 14 (28.6%) patients. In 17 patients detected to have MTB, four-drug antitubercular therapy (ATT) was recommended. ATT was started in 15 patients. Nine of 17 patients completed 6 months ATT and were cured. Six of 17 patients are currently taking ATT. Two patients did not take ATT; both of these have persistent symptoms of pus formation. Out of nine cured patients, two patients did not start ATT for 2 months after detection. Only after the symptoms (persistent pus discharge) continued, did they start ATT and were subsequently cured. Conclusion: RT-PCR is significantly more sensitive than histopathology and AFB smear in detecting MTB in fistula-in-ano. The routine practice of doing only histopathology and AFB smear in fistula patients might be missing a significant number of MTB cases and could be responsible for many recurrences in fistula patients. RT-PCR should preferably be done in all fistula cases and at least in refractory and recurrent fistulas.

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Objective/background: To evaluate the Kudoh swab method for improving laboratory diagnosis of tuberculosis (TB) in Gambia. Methods: A total of 75 sputa (50 smear positive and 25 smear negative) were examined. Sputum samples were collected from leftover routine samples from the Medical Research Council Unit, Gambia TB Diagnostic Laboratory. The samples were processed using the standard N-acetyl-l-cysteine-NaOH (NALC-NaOH) methods currently used and Kudoh swab method. These were cultured on standard Lowenstein Jensen (LJ) and Modified Ogawa media, respectively, and incubated aerobically at 36 ± 1°C for mycobacterial growth. To determine if the decontamination and culture methods compared could equally detect the Mycobacterium tuberculosis complex (MTBC) highly commonly isolated in Gambia, spoligotyping was done. Results: In total, 72% (54/75) of MTBC were recovered by both LJ and Modified Ogawa methods. The LJ method recovered 52% (39/75) and Modified Ogawa recovered 56% (42/75) of the MTBC, respectively. Spoligotyping showed Euro-American 35% (19/54), Indo-Oceanic 35% (19/54), Mycobacterium africanum (West African type 2) 26% (14/54), Beijing 2% (1/54), and M. africanum (West African type 1) 2% (1/54). Conclusion: The Kudoh method is simpler and cheaper than the NALC-NaOH method. There was no significant difference in recovery between the methods. The Kudoh method is ideal in overburdened TB laboratories with poor resources in developing countries. The predominant lineages were Euro-American and Indo-Oceanic, followed by M. africanum (West African type 2).

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Background/Objectives: The genus Mycobacterium contains over 140 species comprises pathogenic and nonpathogenic strains. Nontuberculous mycobacteria (NTM) causing clinical disease have become increasingly common and more diverse. Widespread features of NTM infection can make the diagnosis difficult. Precise species-level detection can aid in distinguishing environmental contamination from actual infection and, furthermore, can assist in making a choice of antimicrobial therapy. Materials and methods: Mycolic acids extracted from saponified mycobacterial cells were examined by high performance liquid chromatography (HPLC) in addition to phenotypic tests for the identification of 20 clinical isolates of mycobacteria at Masoud Laboratory during 2014–2015. Results: Mycobacterium abscessus (8 isolates), Mycobacterium tuberculosis (6 isolates), Mycobacterium intracellulare (3 isolates) and Mycobacterium fortuitum (3 isolates) were identified in these isolates. The phenotypic tests also confirmed the identity of the clinical isolates. Conclusion: Our results showed that HPLC is a more reliable, rapid, simple, easy-to-perform, cost-effective, and specific identification method compared with other identification procedures like phenotypic tests.

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Objective background: Mycobacterium simiae has been identified as the most prevalent slow growing mycobacteria that has been isolated among Iranian patients. Due to an importance of molecular approaches in the treatment of nontuberculosis mycobacteria, this study aimed to evaluate molecular drug susceptibility testing against the first-line (rifampin and isoniazid) and second-line (ciprofloxacin–amikacin and kanamycin) treatment in different subtypes of M. simiae. Methods: The study involved all patients presenting to our referral tuberculosis center from March 2014 to August 2016, with confirmation of M. simiae. For all sputum samples, after digestion and decontamination, followed by DNA extraction, polymerase chain reaction-restriction fragment length polymorphism was performed using the hsp65 gene. Furthermore, susceptibility to rifampin, isoniazid, ciprofloxacin, amikacin, and kanamycin were evaluated using rpoB, inhA, katG, gyrA, and rrs genes, respectively. Results: In total, 60 cases (58.33% men and 41.66% women) of M. simiae infections were identified and all were confirmed as subtype I. All the patients were resistant to the first line-tuberculosis drugs, while 88.33% and 91.66% of cases were susceptible to ciprofloxacin and both amikacin and kanamycin, respectively. Conclusion: Regarding the given result, first-line tuberculosis drugs should be excluded from the treatment regimen of M. simiae patients. In addition, subtype I seems to be the most or even the only isolated M. simiae subtype among all Iranian patients. While both amikacin and kanamycin drugs showed better efficacy in the treatment M. simiae, the most susceptible antibiotic is still indeterminate.

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Objective/background: To study whether interleukin (IL)-10 gene polymorphism is associated with multi-drug resistant tuberculosis (MDR TB) during the intensive phase of standard chemotherapy. Methods: The study comprised 170 individuals in Kharkiv region of Ukraine including 74 patients with pulmonary MDR TB (Group 1), 66 patients without MDR TB (Group 2), and 30 healthy donors (Group 3). Serum level of IL-10 was evaluated by enzyme-linked immunosorbent assay (pg/L). Measurements of serum samples were conducted before or during the initial days after hospital admission and after 2 months on antituberculous therapy. Investigations of IL-10 gene polymorphism were performed using restriction analysis of the amplification products of specific regions of the genome. The method of investigation (for the sets real-time) — an allele-specific PCR using intercalating coloring Sybr Green. Polymorphism G1082A of gene IL-10 rs1800896 were genotyped with amplification-refractory mutation system-polymerase chain reaction. Results: In Group 1, the level of IL-10 was (38.01 ± 0.78) pg/L, compared with 43.88 ± 0.70 in Group 2, and 50.25 ± 1.26 in Group 3 (p <0.05 among the groups). In Group 1, 56 (75.68 ± 4.99%) patients had heterozygote GA genotype, 11 (14.86 ± 4.14%) patients had homozygote AA genotype, and seven (9.46 ± 3.40%) patients had homozygote GG genotype. In Group 2, 41 (62.12 ± 5.97%) patients had homozygote AA genotype, 17 (25.76 ± 5.38%) patients had heterozygote GA genotype, and eight (12.12 ± 4.02%) patients had homozygote GG genotype. In Group 3, 17 (56.67 ± 9.05%) healthy donors had homozygote GG genotype, seven (23.33 ± 7.72%) healthy donors had heterozygote GA genotype, and six (20.0 ± 7.30%) healthy donors had homozygote AA genotype (p <0.05 among the groups). Following 2 months antituberculous therapy treatment, there was a significant increase in IL-10 levels in Group 1 (44.58 ± 0.78) and Group 2 (50.59 ± 0.99; p <0.05 between the groups), when compared to the beginning of therapy and after 2 months (p <0.001). Conclusion: Compared to the healthy control group, patients with TB had significantly lower levels of IL-10. This coincided with a greater frequency of heterozygote GA genotype in Group 1 and homozygote AA genotype in Group 2. Further studies are warranted to determine whether a higher number of patients without MDR TB have a causal immunogenetic relationship with IL-10 gene polymorphisms than patients with MDR TB. Standard 2-month TB therapy results in reversal of inflammation characterized by increased IL-10 to a level comparable to that in healthy donors. IL-10 is an immune correlate of treatment outcome and can help to identify a better strategy for TB management. TB chemotherapy may have an immunomodulatory effect of an anti-inflammatory nature.

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Aims and objectives: Despite the successes in managing drug-susceptible TB, drug-resistant (DR) tuberculosis is a major challenge to the effectiveness of National Tuberculosis Program in Armenia, placing the country in the list of 18 high-burden countries for MDR-TB in the WHO European Region. Estimated burden of MDR-TB in 2012 was 9.4 (7–12) and 43 (38–49) among retreatment TB cases. A total of 92 laboratory confirmed cases had been reported to the WHO (57 new and 35 previously treated) out of 511 cases tested for MDR-TB. GenoType MTBDRsl is a new molecular kit designed for rapid identification of the resistance to the second-line antituberculosis drugs with a single strip. The aim of this study was to identify the mutation that confers resistance to ethambutol (EMB) in Mycobacterium tuberculosis in comparison with the phenotypic drug susceptibility test (DST). Ethambutol is used in M/XDR-TB regimens only if it is susceptible by DST results. Methods: A set of 173 drug resistances TB isolates during 2011 and 2012 period, being either acid fast bacterium positive or negative but culture positive resistant to isoniazid, rifampin, or both according to the GenoType MTBDR plus assay were consecutively tested, using the GenoType MTBDRsl (MTBDR plus version 1.0 and MTBDRsl version 2.0 Hain Lifescience, Nehren). embB gene analysis and the results were compared to phenotypic EMB testing (DST). The DNA preparation method used as described recommended by the manufacturer. Results: Genotypic analysis identified mutations at codon 306 of the embB gene in 20.8% (36/173) and miss band of wild type in 12.71% (22/173) of the resistant isolates in comparison to only 14.45% (25/173) of those that were phenotypically resistant to EMB by DST MGIT liquid media. Mutation locus were identified in embB MUT1B and embB MUT1A 8.67% (15/173) and 12.13% (21/173), simultaneously. Phenotypic retesting in MGIT demonstrated that 45 (306 of the embB gene and miss band of wild type together) genotypically resistant isolates were phenotypically resistant to EMB. This implies that 58.62% (33/58) of EMB resistance had been phenotypically missed by routine laboratory procedures. EMB resistance was closely linked to multidrug resistance (MDR); 70.69% (41/58) of the EMB-resistant isolates were resistant to both isoniazid and rifampicin. Conclusion: Implementation of more accurate diagnosis of EMB resistance may enhance patient management in Armenia, as standardized treatment of MDR-TB with second-line drugs is currently dependent on the outcome of the EMB resistance test.

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Objective/Background: Mycobacterium avium subsp. paratuberculosis (MAP) is an etiological agent of Johne's disease in ruminant including cattle, sheep and goats. This disease is considered an economically important disease in cattle. Animals with paratuberculosis shed viable MAP, particularly in their milk and feces. MAP may be involved in the development of Crohn's disease in humans through the consumption of contaminated milk and dairy products. Common methods of pasteurization are not enough to kill all MAP present in the milk and the bacterium has been isolated from raw milk, pasteurized milk and cheese samples. The purpose of this study was to evaluate two different methods for detecting MAP in milk and milk products. We analyzed the commonly usedMethods: such as culture and molecular biology for identification of MAP. Methods: For this study, 50 milk samples from cows with suspected Johne's disease located in two dairy farms and 10 commercially available pasteurized milk and cheese samples from the market in Karaj city, Iran were selected. Following Ziehl–Neelsen staining of milk samples, direct microscopic detection of MAP was performed. All milk samples were centrifuged, and the concentrated samples were decontaminated using hexadecyl pyridinium chloride. The decontaminated milk suspensions were washed three times by centrifuging, and the collected filtrates were cultivated on Herrold's egg yolk medium enriched by Mycobactin J. Finally, identification and confirmation of isolates to MAP was performed using IS900-nested polymerase chain reaction (PCR). Results: According to the obtained results by culture and PCR methods, none of the pasteurized milk and cheese samples showed the presence of MAP. However, 10% of the tested raw milk samples collected from suspected cattle showed the presence of MAP by both culture and PCR methods. Conclusion: Culture and PCR methods are reliable for identification of MAP from milk samples.

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Objective/Background: Johne's disease, also called paratuberculosis, is a very important chronic infectious disease of ruminants worldwide. The causative agent is an acid-fast bacterium, Mycobacterium avium paratuberculosis (MAP). Finding a precise method for detecting MAP is essential for the control and eradication of this disease in small ruminant herds. Methods: In this study, appropriate samples were obtained from the ileum, cecum, colon, and mesenteric lymph nodes of 10 suspected naturally infected goats. Each sample was divided to two parts: the first part was stored at −20°C for bacteriologic culture and the second part was placed in 10% neutral formalin for molecular and histopathologic examination. To isolate MAP, the double incubation method was used for decontaminating the tissues and Middlebrook 7H9 broth-based culture associated with OADC (oleic acid, albumin, dextrose, and catalase) supplement with/without mycobactin J were used. Polymerase chain reaction (PCR; IS 900) was performed for media with positive acid-fast staining. Results: Acid-fast staining was positive in 40% of ileum samples, 50% of cecum samples, 40% of colon samples, and 50% of lymph node samples with mycobactin J and in 60% of ileum samples, 60% of cecum samples, 40% of colon samples, and 40% of lymph node samples without mycobactin J. All samples were confirmed by IS 900 PCR assay. Diffuse granulomatous enteritis with multibacillary lesions and paucibacillary multifocal lymphadenitis associated with calcification were diagnosed histopathologically. Conclusion: MAP detection in intestinal content and in tissues is quite necessary for the diagnosis, control, and eradication of this disease in small ruminant herds.

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Objective/background: Tuberculosis (the white plague) is regarded as one of the most widespread infectious diseases and continues to be a leading cause of death and the most prominent public health problem worldwide. It is caused by Mycobacterium tuberculosis complex, which refers to a group of seven species; one of them known as Mycobacterium bovis—the cause of bovine-type tuberculosis—has an exceptionally wide host range. It infects cattle, humans, goats, cats, dogs, buffalo, and deer. Many susceptible species, including man, are spillover hosts in which infection is not self-maintaining. The objective of this study is to investigate the role of infected slaughtered cattle in spreading tuberculosis to those who work in abattoirs. Methods: Three hundred slaughter cattle in some abattoirs of the Baghdad governorate were examined grossly. Tissue samples were taken from lesions that had appeared on lymph nodes, lung, liver, spleen, peritoium, and intestines. A routine examination was performed: (1) smear for Ziehl Neelsen acid-fast stain; (2) cultured: each sample was cultured on Stone-brink with sodium pyrovite and on Lowenstein media which contain glycerol; and (3) incubated at 37°C for 4–10 weeks, to observe the characteristic features of bacterial colonies. Biochemical tests, nitrate reduction, urea analysis, tween 80 lysis, and catalase test were employed to isolate and identify the bacteria. Pieces from tissue samples were kept in 10% formalin for histopathological investigation. Tuberculin tests and X-rays were conducted for 186 workers who were in contact with slaughtered cattle in the same abattoirs, with an age range of 15 years to 60 years. Sputum samples were collected from all workers in clean and sterile containers, and subjected to the same routine examination. The collection of samples was carried out under strict and sterile conditions and the sputum was kept in 50% oxalic acid for 20 min before culture on media to avoid the contamination. Results: Gross examination of cattle carcasses showed tubercle in four of them that was distributed in lymph nodes and different organs especially in lungs, livers, and in one case tubercle appeared on the peritoneum and intestines. A histopathological study revealed different lesions with an accumulation of lymphocytes and macrophages in lymph nodes and organs. Four isolates of M. bovis were diagnosed and identified by routine examination that indicated the percentage of infection in slaughtered cattle was 1.33%. The result of the workers' examinations clarified that only one of the workers had a positive result for the tuberculin test, whereas three of them had positive results in X-ray and routine examination. Three isolates were obtained from workers (1.6%); two of these isolates were diagnosed as M. bovis and the other as M. tuberculosis. Conclusion; The main conclusion of this study is that two workers were infected with cattle's strain which confirms the role of slaughtered cattle in the transmission of this dangerous, chronic, and zoonotic disease to man.

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Objective/Background: Mycobacterium tuberculosis, the etiologic agent of tuberculosis, causes large-scale morbidity and mortality, particularly in developing countries. In recent years, there has been a significant increase in the drug-resistant ability of M. tuberculosis, triggering a major public health crisis. A detailed analysis of the evolution of the mycobacterial genome helps to better understand the genotype–phenotype relationship in this bacterium. Different strain typing methods have already revealed the worldwide diversity of mycobacterial isolates. Therefore, DNA-fingerprinting tools have been developed to improve tuberculosis case detection and control. Molecular typing techniques allow to detect and follow the spread of individual strains of the M. tuberculosis complex (MTC), complementing conventional epidemiological methods. Among these techniques, restriction fragment length polymorphism (RFLP) has been considered the standard method for genotyping of MTC. The aim of this work was to isolate M. tuberculosis from rodents in cattle farms contaminated with MTC located in the city of Booin-Zahra, Iran. Methods: A total of 100 samples were collected from the rodents in the contaminated farms and analyzed for the presence of Mycobacterium by growing the samples on Lowenstein–Jensen medium. All isolates were further identified by RFLP and DNA hybridization studies. Results: As much as five samples showed the presence of Mycobacterium and these were subjected to PCR-16SrRNA, PCR-IS6110, and RD Typing (RD1, RD4, RD9, and RD12) methods. Further differentiation was performed with PvuII digestion (RFLP) and DNA hybridization using the polymorphic guanine/cytosine-rich repetitive sequences (PGRS) probe. The PGRS probe results classified two of the isolates as belonging to one cluster, whereas the remaining isolates were classified as belonging to different clusters. An analysis of the obtained genetic pattern and a comparison of these patterns with the genetic pattern of other infected farms allowed us to record the similarities and difference. The results indicated the transmission of Mycobacterium from infected rodents to the cows located in the same farm. Conclusion: These results highlight the possible danger of transmission of Mycobacterium among animals.

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Background/objective: Mycobacterium bovis, responsible for bovine tuberculosis is a member of Mycobacterium tuberculosis complex. This bacterium is responsible for infection in wide range of hosts. Bovine tuberculosis is well known as Zoonotic tuberculosis in human and is transmitted via consumption of unpasteurized milk, contaminated meat products and also by ingestion of Mycobacteria from the environment. The objectives of the present study was to compare the genomic pattern of M. bovis obtained from human subjects in Zanjan province, with those of atypic cattle in Iran by Restriction fragment length polymerization and DNA hybridization methods. Methods: DNA was isolated from 2 M. bovis strains isolated from suspected patients by van Sooligen method. Finger printing methods using RFLP and DNA hybridization with probes DR and PGRS was performed. The obtained patterns were compared with the genomic pattern of 161 M. bovis strains isolated from infected cattle lymph nodes, present in the Tuberculin reference Laboratory at Razi vaccine and Serum Research Institute, Karaj. Results: Comparisons of the genetic pattern of the 2 M. bovis strains from Zanjan province with 16 distinct patterns obtained with PGRS probe and 20 patterns with DR probes from 161 M. bovis isolates indicated no correlation of the patterns of Zanjan isolates with those present at Razi Tuberculin Reference Laboratory. Conclusion: With respect to the age of patients, absence of epidemic and lack of cluster in the mentioned province and other provinces, our results indicate recurrence of the infection due to M. bovis isolates which were present in Zanjan province in previous years. These isolates had no association with the bovine tuberculosis isolates present in Razi Tuberculin Reference laboratory.

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Introduction: Mycobacterium bovis has a broad host range, and it is the principal agent responsible for tuberculosis (TB) in bovine, domestic and wild mammals. M. bovis also infects human, causing zoonotic TB through ingestion, inhalation and, less frequently by contact with mucous membranes and broken skin. Zoonotic TB was formerly an endemic disease, usually transmitted to man by consumption of raw cow's milk. It is indistinguishable clinically or pathologically from TB caused by M. tuberculosis. Objective: The aims of this study were, to isolate and identified M. bovis from raw milk samples by different methods, and evaluate the virulence of M. bovis in laboratory animals (Rabbit). Materials and methods: To conduct the study, ninety three cow's milk samples were collected from farms around Baghdad governorate. The decontamination of milk samples was firstly carried out, then samples were subjected to routine tests which include, direct smear for Ziehl Neelsen acid fast stain, culture, each sample was cultured on Lowenstein Jensen media with Sodium pyruvite (All cultures incubated on 37°C for 4–10 weeks with continuous observation), and biochemical testes as Nitrate reduction test, Niacin paper strip test and pyrazinamidase test, were employed to diagnose and identified the bacteria. Beside molecular assay was used to confirm the identification of the isolates by Polymerase Chain Reaction (PCR) using specific primers for M. bovis. The virulence of these isolates were investigated through inoculate it in group of laboratory animals consist of 8 rabbit in addition to other group of 4 animals as control (inoculate with Phosphate Buffer Saline). The animals were scarified after 6 weeks of inoculation, post- mortem examination was carried out, smears were taken from lesions, and tissue samples were collected from lymph nodes and different organs. Results: The results revealed five isolates of M. bovis in direct smear by acid fast Ziehl-Neelsen stain, while eight isolates observed by culture, the colonies appeared with characteristic feature of cream color, rough, and with irregular edge. The molecular assay using PCR technique confirmed the diagnosis of eight positive isolates in smears and culture. The virulence of these isolates were investigated through the pathological effects appeared in inoculated rabbit which showed lesions scattered mainly in lymph nodes and different organs as lung, liver, spleen and kidney when compared with control group which were naive. Beside the infiltration of mononuclear cells in the internal organs particularly in the lungs. The result of histopathological examination clarified the virulence of M. bovis isolates, and its impact on tissue and organs of the rabbit. Conclusion: Our study conclude the presence of M. bovis isolates in milk in high percentage pause important source of tuberculosis infection for human being.

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Objective/background: Humans have been in a constant battle with tuberculosis (TB). Currently, overuse of antibiotics has resulted in the spread of multidrug-resistant Mycobacterium tuberculosis (MDR), leading to antibiotic ineffectiveness at controlling the spread of TB infection in host cells and especially macrophages. Additionally, the Mycobacterium tuberculosis (Mtb) has developed methods to evade the immune system and survive. With the discovery of nanoparticle (NP)-based drugs, it is necessary to research their anti-mycobacterial properties and bactericidal mechanisms. In this study, we synthesized mixed metal oxide NPs and tested their ability to inhibit Mtb growth into macrophages and investigated the cytotoxic effects of NPs in THP-1 cells. Methods: Silver (Ag) NPs and zinc oxide (ZnO) NPs were synthesized by chemical reduction and chemical deposition in aqueous solution, and the diffraction light scattering, scanning electron microscopy, transmission electron microscopy, and ultraviolet–visible light-absorption spectra were used to identify NP properties. Ag and ZnO NPs were mixed together at a ratio of 8_ZnO/2_Ag and diluted into Löwenstein–Jensen medium followed by the addition of bacteria and incubation for 28 days at 37°C. The toxicity of NPs to THP-1 cells was assessed by MTT test, and macrophages were infected with Mtb for 4 h at 37°C under 5% CO₂. Results: Nano-sized particles were estimated at ˜30–80nm, and the initial concentration of Ag NPs and ZnO NPs were estimated at ˜20 ppm and ˜60 ppm. The minimal inhibitory concentration ratio of 8_ZnO/2_Ag NPs against Mtb was detected at ˜1/32 of the initial concentration. Ag NPs in the range of concentrations exhibited no anti-Mtb effects, whereas ZnO NPs showed potent antibacterial activity at ˜1/128 of the initial concentration. ZnO NPs at all concentrations showed cytotoxic activity, whereas 100% of THP-1 cells remained viable in the presence of Ag NPs at ˜1/32 and ˜1/64 of the initial concentrations. However, at ratios of 8_ZnO/2_Ag, ˜39.94% of the cells at ˜1/16 of the initial concentration remained viable, with 100% of THP-1 cells at ˜1/32 of the initial concentration remaining viable. Conclusion: Although Ag NPs exhibited low cytotoxicity, they were unable to inhibit Mtb growth in vitro. ZnO NPs exhibited strong anti-Mtb activity and inhibited bacterial growth, but exhibited high cytotoxicity to human macrophage cells. By mixing Ag and ZnO NPs at a ratio of 8_ZnO/2_Ag, we acquired a mixture that exhibited potent antibacterial activity against Mtb and no cytotoxic effects on THP-1 cells, resulting in inhibition of both in vitro and ex vivo Mtb growth [Figure 1],[Figure 2],[Figure 3], [Table 1],[Table 2],[Table 3].{Figure 1}{Figure 2}{Figure 3} {Table 1}{Table 2}{Table 3}

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Tuberculosis (TB) is one of the most common zoonotic infectious diseases in the world. Identification of Mycobacterium isolates is essential for proper treatment of TB. The aim of this study was to identify Mycobacterium isolates collected from TB patients in Alborz Province, Iran, by region of differentiation (RD)-typing. Fifty samples from tuberculosis patients were cultured in pyruvate and glycerinated Lowenstein–Jensen medium. DNA was extracted from the isolates by the van Solingen method and subjected to polymerase chain reaction (PCR)-16SrRNA, PCR-IS6110, and RD-typing with primers RD1, RD4, RD9, and RD12, respectively. Out of 50 isolates, only one isolate appeared negative in IS6110-PCR and was considered nontuberculosis complex. The remaining isolates gave PCR products of approximately 543bp, 245bp, 146bp, 172bp, 235bp, and 369bp with 16s-rRNA, IS6110-PCR, RD-1, RD-4, RD-9, and RD-12 PCR, respectively. PCR-restriction fragment length polymorphism of oxyR pseudogene confirmed the results. All isolates except one from Alborz Province appeared positive for Mycobacterium tuberculosis. Based on the obtained results, all isolates except one were identified as M. tuberculosis. The only negative isolate appeared 93% and 97% similar to Nocardia or Mycobacterium sp. (Mycobacterium neoaurum), respectively, based on sequencing and alignment of 16s-rRNA and hsp65. Accurate identification of Mycobacterium isolates is of utmost importance for proper and immediate treatment of TB patients. In this study, RD-typing appeared to be a suitable method for correct identification of M. tuberculosis isolates.

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Objective/background: Tuberculosis has long been recognized as a zoonotic disease and remains one of the most life-threatening diseases worldwide. In spite of the successful treatment of this disease, the World Health Organization considers it as a disease with the highest priority in 1993, and it is now the second leading cause of infectious mortality. Since the identification of Mycobacterium tuberculosis complex species is of special importance, the aim of this study was to identify the species from the submitted mycobacterial isolates of tuberculars in Khorasan Razavi sent to the cell reference laboratory of Razavi Institute by the molecular method. Methods: Today, various molecular methods are used as a useful and quick tool to identify the isolates. In this research, we used polymerase chain reaction (PCR) test on the 16sr RNA gene to determine if the isolates belonged to the Mycobacterium genus. Then another PCR test of IS6110 performed to confirm that all of isolates are belonging to M. tuberculosis complex. Finally RD typing method was used to differentiate the complex members, and the member species of the complex was identified by this method. Conclusion: Using molecular identification of 100 submitted isolates in Khorasan Razavi province, it was found that all isolates are M. tuberculosis. The results showed that the sanatorium in this province was not infected with M. bovis and other members of the complex. This is either due to the widespread use of pasteurized milk and nonuse of milk contaminated with M. bovis or due to the lack of regular tuberculosis control and eradication programs in the city.

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Objectives/background: The region of differences (RDs) polymorphisms is a potential molecular epidemiology method to distinguish origins of Mycobacterium tuberculosis. To date, 68 RDs have been identified in M. tuberculosis. This study was designed to determine the frequency of RD deletions in M. tuberculosis strains that were isolated from patients with pulmonary tuberculosis who were referred to the National Research Institute of Tuberculosis and Lung Disease for diagnosis and treatment. Therefore, highly polymorphic regions (RD1, RD150, and RD181) among M. tuberculosis strains isolates were investigated. Methods: A total of 250 M. tuberculosis isolates were identified by conventional and molecular methods. Subsequently, spoligotyping and RD typing (RD1, RD150 and RD181) were performed to genotype these strains. Results: The most frequent spoligotype belonged to Haarlem (n = 85, 34.0%) followed by CAS (n = 54, 21.6%), T1 (n = 27, 10.8%), and Beijing (n = 28, 11.2%) lineages. Deletion in RD181 was identified only among the Beijing lineage ([Figure 1]).{Figure 1} Conclusion: As we found a deletion in RD181 in the Beijing strains only, we propose to use this marker as an identification tool for genotyping of the Beijing strain.

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Objectives/Background: Dihydrofolate reductase (DHFR) is one of the validated drug targets in Mycobacterium tuberculosis (Mtb) infection. DHFR inhibitors have been used to treat various life-threatening diseases such as cancer, malaria, and several bacterial infections. However, all clinically effective DHFR inhibitors are non-selective, and inhibit both human and pathogenic DHFRs more or less to a similar extent. The crystal structure of various DHFRs complexed with nicotinamide adenine dinucleotide phosphate and different inhibitors is available in the protein data bank. The crystal structures are validated and have been used for new drug designing. M. tuberculosis DHFRs and human (h) DHFRs show 26% structure similarity, but their active sites are not identical and this characteristic forms the basis of this study. Because most of the reported inhibitors of M. tuberculosis DHFR are pteridine based and nonselective in nature, that is, they inhibit both microbial and host DHFRs, this study aimed to design and develop selective nonpteridine M. tuberculosis DHFR inhibitors. Method: In the ternary complex of methotrexate with M. tuberculosis DHFR, whose structure is also available in the protein data bank, the side of the aminopterin ring is accessible to the solvent; additionally, a glycerol “A” molecule is found in a depression nearby. This glycerol molecule interacts with the side chains of Trp22, Asp27, and Gln28, which form a pocket in M. tuberculosis DHFR; by contrast, glycerol is absent in h-DHFR. In the h-DHFRs (complexed with folate or N-(4-carboxy-{-[(2, 4-diamino pteridine-6-yl methyl)-amino]-benzoyl amino} -butyl) pthalamic acid (COP)), the site is well packed with three hydrophobic residue side chains, Leu22, Pro26, and Phe31, which correspond to Leu20, Arg23, and Gln28, respectively, found in M. tuberculosis DHFR. Therefore, compounds with side chain, which could mimic the binding mode of glycerol to protein, may bind to M. tuberculosis DHFR selectively. Such a derivative should be sterically and chemically hindered from forming a complex with h-DHFR. This assumption forms the basis of this study and this understanding has been used for designing selective inhibitors of M. tuberculosis DHFR. Results: A number of novel nonpteridine-based molecules have been identified after the virtual screening of three databases (MDPI, NCI and inhouse databases). The best molecules identified after screening the three databases have been synthesized and tested for antitubercular activity. The results are promising and require further work in this direction. Conclusion: Structure based drug design can be used as an effective tool for the design of new cheiocal entity. Number of novel agents have been identified as antitubercular agents whose mechanism of action needs to be ascertained.

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Introduction Mutations in embB gene, especially those in ethambutol resistance-determining region (ERDR), are known as “hot spots”. These mutations have frequently been reported in EMB-resistant M. tuberculosis isolates, using the Sequence analysis as a precise and effective method. The aim of this study was to detect mutations in embB gene associated with resistance to ethambutol in Mycobacterium tuberculosis strains isolated from TB patients in the west of Iran (2014–15). Material and methods This study was performed on smear-positive patients of the west side of Iran, in the TB research center located in Kermanshah city, during 2014–15. Out of 1069 strains of Mycobacterium tuberculosis, 50 strains with pulmonary TB were selected and evaluated (22 men and 28 women; aged between 23 and 86; median age: 54.5 years). Results: Mutation in the embB gene was detected in all of the seven EMB-resistance isolates and five (42.71%) cases of them were MDR. The most frequent substantiation of amino acid occurred at the codon 306 in six (64.85%) of the EMB-resistant isolates. The Met306Val substitution resulting from an A → G transition was detected in three (42.85%) EMB-resistant isolates; and the Met306Ile substitution, due to a G → A transition was also detected in three (42.85%) resistant isolates. No mutations at the embB gene were detected in susceptible strains. Conclusion: Our results were similar to those that were regularly reported in earlier studies. The only mutations in the EMB-resistant isolates were found in embB 306 and 406 codons. Substantiation amino acids at codon 306 were the most frequent. The data indicated that embB 306 mutations are sufficient to induce ethambutol resistance, and detection of these mutations is advisable to be considered in the development of rapid molecular tests. Sequence analysis of the ERDR in the embB gene is adequate for rapid detection of EMB resistance, and mutations in the codon 306 are good predictive markers for resistance to EMB.

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Introduction: Although tuberculosis (TB) is curable, the rate of failure and mortality is high in comparison to other infectious diseases worldwide. It has been shown that majority of TB patients leave treatment before completing the therapeutic regimen. The aftermath of incomplete regimens might result in drug resistant-TB bacilli (DR-TB), relapses, and death. For this reason, proper knowledge about the disease and associated risk factor is crucial to decreasing TB cases among the general population. In the present study, we aimed to investigate the associated factors and competing events among pulmonary tuberculosis patients. Materials and methods: Based on a cohort study, associated risk factors and competing events from 2366 confirmed TB patients that referred to the National Center for Tuberculosis for diagnosis and treatment (2005–2015) were collected and analyzed. Results: Our results showed that gender, age, marital status, TB contact, drug adverse effect, and HIV positive, imprisoned, significantly affect the relapse cases, drug resistance, and mortality rate (P-value <0.05). Conclusions: Use of competing risks model with competing events can provide a better way to understand the associated risk factors co-related with outcome of the pulmonary TB process, especially among DR-TB patients.

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Introduction: Since an accurate test for detection of Mycobacterium tuberculosis early infection is urgently needed, this study was designed for development of an efficient screening test in diagnosis of tuberculosis infection. Materials and methods: In the present study, two recombinant proteins CFP-10, ESAT-6 were tested as antigens for the diagnosis of recent tuberculosis. The proteins were produced in Escherichia coli, purified and tested in indirect ELISAs with sera from 63 subjects with positive clinical results. Also, 56 sera from healthy persons were tested as controls. The results were compared with molecular and culture. Results: The levels of antibodies against M. tuberculosis antigens in patients with tuberculosis were significantly higher than those in healthy subjects. Among 63 patients, 58 were positive for ESAT-6, 54 for CFP-10. Conclusion: Altogether, the role of M. tuberculosis recombinant proteins, as a suitable candidate for early diagnosis of tuberculosis infection was supported in this study. However, these strongly offer the potential of mixture or fusion of these recombinant proteins for better sensitivity and specificity.

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Objective/background: Lack of rapid and accurate diagnostic testing is a critical obstacle to global tuberculosis (TB) control. Sensitivity of sputum smear microscopy (SSM) is not optimal; however, it remains the most prevalent tool for TB confirmation in poor countries. As a part of passive case finding of TB detection, this study was conducted to determine the clinical performance of PURE TB-LAMP assay using liquid culture medium as the gold standard. Methods: Centre Antituberculeux de Yopougon is one of the 17 intermediate Tuberculosis centers in Côte d’Ivoire. A standardized questionnaire was submitted to patients with signs and symptoms consistent with tuberculosis by a trained caregiver. After obtaining signed consent forms, sputum samples were collected according to National TB Control Programme guidelines (spot-morning). SSM after Ziehl–Neelsen staining and TB-LAMP assay were blindly performed on the first sample. Samples transported to Institut Pasteur de Côte d'Ivoire were decontaminated according to the N-acetyl-L-Cystein method. In Mycobacteria Growth Indicator Tube (MGIT), 500 mL of pellets were inoculated and incubated in the MGIT 960 system. MPT64 antigen was detected in positive cultures. Results: Of the 500 patients enrolled, 469 (232men and 239 women) patients were included. The mean ages of men and women were 36.9 (15–86) and 37.3 (15–37.3) years, respectively. There were 56 (12.2%) HIV-infected patients, including 14 women. Clinical isolates of M. tuberculosis complex were detected for 157 (33.5%) patients. Compared with culturing, the overall sensitivity and specificity of SSM were 86% (95% confidence interval [CI] = 81–91) and 96% (95% CI = 94–98), respectively. The overall sensitivity and specificity for TB-LAMP was 92% (95% CI = 0.88–0.96) and 94% (95% CI = 0.91–0.97), respectively. Positive likelihood ratios for TB-LAMP and SSM were 15.3 and 21.5, respectively, and negative likelihood ratios for TB-LAMP and SSM were 0.09 and 0.15, respectively. Among the 469 patients, active tuberculosis was detected using TB-LAMP assay and SSM in 162 (34.5%) and 147 (31.3%) patients, respectively. Conclusion: For accurate diagnostic of pulmonary TB, TB-LAMP could be used as a tool of the first intention.

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Referral hospitals in sub-Saharan Africa concentrate large numbers of tuberculosis (TB) and multidrug-resistant TB (MDR-TB) patients, failed by community TB services. We have previously shown, from enhanced screening and through autopsy studies, a significant burden of missed TB infections at the University Teaching Hospital, Lusaka, Zambia, with many patients dying or being discharged without treatment. With minimal TB isolation facilities and minimal political will to invest in broader screening and isolation, the risk of nosocomial transmission is likely to be extremely high. Studies from other hospitals in low burden settings and in South Africa have shown that next generation sequencing (NGS) is a very powerful tool for rapidly sequencing whole TB genomes and comparing them to confirm or rule out nosocomial transmission. The established platforms for NGS analysis, such as Illumina, are very expensive, immobile, and require regular maintenance, making them a costly inclusion on a research proposal or programmatic intervention grant in Africa. MinION nanopore sequencing has changed the NGS landscape with cheap portable sequencers, rapid simple library preparation (15 min), and automated real-time analysis tools. The application of highly portable MinION nanopore sequencing technology for the monitoring of nosocomial TB infection will be discussed. Preliminary data from our pediatric pneumonia study will demonstrate the detection of TB in induced sputum from children admitted to the University Teaching Hospital.

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