Aim and objectives: The surveillance of genetic diversity and genotypes of Mycobacterium tuberculosis (MTB), such as Beijing, Beijing B0/W148 genotypes, T (T1), LAM (LAM 9), U, X, Manu 2, H4, is important for Belarus to evaluate mycobacterial population shift under vaccination and demographic changes and the use of new anti-TB drugs. The aim was to find out the origin of multidrug resistant tuberculosis (MDR) upsurge in Belarus by investigation of M. tuberculosis genetic diversity and prevalence of Beijing genotype and its subtype B0 /W148. Methods: There were 241 isolates of MTB processed in the study, of which 108 MTB were isolated in Minsk and Minsk region, 83, 21, 20 - in Mogilev, Gomel, Grodno regions, 9 – in Brest region, 1 culture - in Vitebsk; 7 were isolated from males with no fixed abode. The gender distribution of studied MTB was as follows: 172 cultures (71.4 ± 3.4%) from males and 69 isolates (28.6 ± 5.44%) from females. An express multiplex real time PCR method with dual hydrolysis probes was used to differentiate Beijing and non-Beijing genotypes of M.tuberculosis. Standard PCR with primers INS1, Rv2665R, W139F2T was applied to identify lineage B0/W148 of Beijing genotype. NGS (both Illumina MiSeq and Oxford Nanopore MiniION) was used to characterize genome of M.tuberculosis genotype Beijing lineage B0/W148. Results: The study provides evidences of Beijing genotype dominance among circulating M.tuberculosis in patients with pulmonary tuberculosis in Belarus. The proportion of Beijing genotype in patients with primary diagnosed TB was 61,7% (29/47). The frequency of Beijing genotype isolation in Minsk, Mogilev, Gomel and Grodno regions was 51.85 ± 9.62, 66.27 ± 10.38, 85.71 ± 15.93, 55 ± 23.28% respectively. The lineage B0/W148 accounted for 22.82 ± 5.41% of all isolates and 37.93 ± 8.29% of the Beijing genotype isolates. The genetic clone B0/W148 was revealed in Minsk, Mogilev, Gomel, and Grodno regions, where its proportion among isolates of the Beijing genotype accounted for 44.64 ± 13.22, 23.64 ± 13.48, 33.33 ± 24.33, 72,73 ± 33.59%, respectively. The genetic variant B0/W148 was characterized by a high level of transmission (53.22%) mainly among the male population (p <0.05), unemployed, disabled people of the 2nd group, pensioners, by dominant spread in Minsk and Grodno regions (p < 0.05), a higher frequency of drug resistance (all strains are either pre-XDR or XDR) and to a wider range of anti-TB drugs. Sequenced genome of of M.tuberculosis strain 5005 belonging to genotype Beijing lineage B0/W148 (biosample in GenBank - SAMN14150054), sized 4418311 nt, was downloaded to GenBank - accession number CP049108.1. Conclusion: Epidemiology of tuberculosis in Belarus is characterized by spread of M. tuberculosis belonging to genotype Beijing (61.7 ± 7.1% of newly diagnosed patients) that has high degree of association with pre-XDR tuberculosis (p <0.05) and ineffective treatment (primary and secondary (p <0.05)).

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Aims and objectives: Apart from Single Nucleotide Polymorphism (SNP) variations, MTBC genomes evolve by insertion, deletions, duplications, recombination, and variations of repeated sequences (phages, Insertion sequences, VNTR, CRISPR…), likely under both neutral and Darwinian selection. Methods: Spoligotyping, the typing of the CRISPR Locus using the classical Kamerbeek et al. 1997 technique has been used worldwide to produce a very large knowledge of Mycobacterium tuberculosis genetic diversity and has allowed the deciphering of MTBC population structure. It also remains, in many countries, a basic though useful molecular technique to get some clues on the very evolutionary history of a patient isolate and a first step towards genomic epidemiology. New techniques such as Whole genome sequencing, that produce Short Reads Archives (SRA) need to be linked to ancient knowledge, but must also be used to go beyond them. Results: We tackled the computationally difficult task to reconstruct the CRISPR Locus of Mycobacterium tuberculosis complex using Short Reads Archives (SRA) using a de Bruijn graph approach, since current available methods were not able to allow an efficient reconstruction of the locus. We believe that such an application will be interesting for the clinicians and epidemiologists since it both performs an equivalent of spoligotyping and goes beyond. Conclusions: Using this new software, designated as “CRISPR-builder-TB”, we show that the use of SRA allows to infer new subtle and previously hidden characteristics on the MTBC genomes and to dig into the complex history of tuberculosis with more details. This software is freely available on GitHub with adequate tutorials.

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Aims and objectives: Bioinformatics and computational tools are increasingly used in a wide variety of scientific studies. Genotyping, genomics and whole genome sequencing (WGS) methods allowed the generation of high amounts of data that are difficult to analyze manually. The purpose of this review is to establish a potential list of available databases and bioinformatics tools/resources related to Mycobacterium tuberculosis complex (MTBC), the causative agent of tuberculosis (TB). Methods: At present, a total of 36 tools with freely-accessible links were recovered from publications indexed in PubMed or Google Scholar. Following keywords were searched: “Mycobacterium tuberculosis AND database”, “Mycobacterium tuberculosis AND web tool”, “Mycobacterium tuberculosis AND tool”, “Mycobacterium tuberculosis AND bioinformatics”. Resources found were classified into several thematic sections/categories such as biological databases, drug resistance, or prediction/classification tools. Among recently available resources (published in 2019), one can mention COMBAT-TB-NeoDB (https://neodb.sanbi.ac.za/browser/), CPLP-TB (http://cplp-tb.ff.ulisboa.pt/), DeepAMR (http://www.robots.ox.ac.uk/~davidc/code.php), SecProMTB (http://server.malab.cn/SecProMTB/index.jsp), SITVIT2 (http://www.pasteur-guadeloupe.fr:8081/SITVIT2), SNP-IT (https://github.com/samlipworth/snpit), StackTB (https://stacktb.cs.sfu.ca/), and TBProfiler (http://tbdr.lshtm.ac.uk/). Results: Although several tools/resources have been developed over time, many are either not regularly updated, or not made available permanently. We argue in favor of sustainable development of resources in order to assist the scientific community and policy makers to take timely concerted action for TB control. At “Institut Pasteur de la Guadeloupe”, we intend to build a newer generation of SITVIT databases including classical genotyping, epidemiologic and demographic data, information on drug resistance, as well as WGS data. Several thousands of complete genome sequences are today available for MTBC strains on public sequence repositories (such as GenBank, DDBJ, or ENA). Thanks to recently developed tools, data extracted from available complete sequences would be juxtaposed and stored for comparative and molecular epidemiological studies. Conclusion: Although, the present non-exhaustive list is certainly not “revolutionary”, it would nonetheless foster the construction of integrated pipelines/tools related to specific biological questions concerning MTBC. This list will be made available soon through Wikipedia; and in the meantime it is already available for consultation via link: http://www.pasteur-guadeloupe.fr:8081/SpolSimilaritySearch/info.jsp.

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Introduction. Globally, an estimated 10.0 million (range, 9.0–11.1 million) people fell ill with tuberculosis (TB) in 2018, a number that has been relatively stable in recent years [1]. Despite the obvious differences in the virulence and transmissibility of different genotypes [2], the pathogenesis of tuberculosis, especially in the later stages, is still poorly understood [3]. At the same time, some researchers suggest that the ability of Mycobacterium tuberculosis to form biofilms in vivo [3] is an important aspect of pathogenesis TB that should be used in the development of new generation vaccines [4]. Previously, we suggested that in the long treatment of TB, a specific lung microbiota can develop in the caseous contents of tuberculosis foci, forming a polymicrobial biofilm, resistant to anti-tuberculosis therapy and contributing to the survival of M. tuberculosis [5]. Here, we present results of the metagenomic analysis of 14 tuberculomas and discuss the results obtained. Methods. The study recruited 13 patients undergoing planned surgical treatment at the Irkutsk Regional TB Hospital, Russia. DNA was isolated from caseous contents of 14 tuberculomas obtained during surgery. Two samples were obtained from one patient at two time points separated by six months. Next-generation sequencing of DNA samples was performed at the NextSeq 550 platform (Illumina, USA). The genomic DNA library was prepared by Nextera XT kit (Illumina). High Performance Sequence Analysis was completed on the NextSeq 550 platform (Illumina, USA) with NextSeq 500/550 v2.5 sequencing reagent kits (Illumina, USA). The NGS short sequencing reads (fastq files) were deposited to the NCBI (PRJNA659860). The assembly of short reads pertaining to mycobacterial DNA from the total pool of metagenomic sequences was carried out with the Magic-BLAST tools [6]. Mapping of short reads to genome of the reference strain M. tuberculosis H37Rv (NC_000962) was performed using Burrows-Wheeler Aligner [7]. The extraction of sequences from the mapped genome with the calculation of the coverage of each position was performed with SamTools [8]. Taxonomic classification of high-throughput sequencing reads from metagenomic whole genome sequencing was performed by using Kraken 2 [9]. Results. Among 5167 bacterial reads, a total of 105 families were identified. Of them, 68 taxa belonged to Gram-negative bacteria, and 35 to Gram-positive. At the same time, the number of reads of Gram-negative and Gram-positive taxa was very similar - 2430 and 2746, respectively. This was probably due to the presence of 1801 reads from the family Mycobacteriaceae (M. tuberculosis). The microbial content of the tuberculomas was estimated as 10⁴-10⁸ bacterial genomes per 1 gram of sample. The vast majority of reads (over 95%) were attributed to the human DNA. We have subdivided the samples into two types of bacterial communities: (i) paucibacillary bacterial community with a predominance of TB (63%-96%) and (ii) low-bacterial community with a predominance of non-mycobacterial taxa (13%-38%). Staphylococcaceae, Pseudomonadaceae, Eggerthellaceae, Pasteurellaceae, and Acetobacteraceae prevailed among non-mycobacterial communities. Conclusions. It can be unambiguously concluded that the microbiota of about half of the tuberculomas did not have a dominant taxon or it differed from M. tuberculosis. Acknowledgments. This work was supported by Russian Foundation for Basic Research (Grant No. 19-515-55009).

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Aims and objectives: Tuberculosis (TB) is estimated to have caused the deaths of 1 billion people in the last 200 years. Methods: In the first step, the microplate alamar blue assay (MABA) was used for the detection of seventy-eight clinical isolates from Golestan Regional Tuberculosis Reference Laboratory (GRTRL) and the results were compared with the proportion method. In second step, the MABA was used for the susceptibility testing of 35 clinical isolates and the results were compared with the proportion method. Results: For the MABA, the sensitivity was 100 (90.97-100), specificity was 74.36 (57.87-86.96), positive predictive value (PPV) was 79.59 (65.66-89.76), negative predictive value (NPV) was 100 (88.06-100). The concentrations of isoniazid (INH) and rifampicin (RMP) ranged from 1.0-0.01 μg.mL and 2.0-0.03 μg.mL, respectively. For the proportion method, the INH concentration was between 1.0-0.03 μg.mL and the RMP concentration was between 2.0-0.06 μg.mL. For the MABA to RMP, the sensitivity was 100 (89.11-100), specificity was 100 (29.24-100), PPV was 100 (89.11-100), NPV was 100 (29.24-100), and for the MABA to INH, the sensitivity was 84.38 (67.21-94.72), specificity was 66.67 (9.43-99.16), PPV was 96.43 (81.65-99.91), NPV was 28.57 (3.67-70.96). Conclusion: We found the high accuracy between the MABA-RMP and the proportion method. In fact, the rapid and low-cost MABA assay is inexpensive and alternative assays for the detection of RMP resistant tuberculosis in low-income countries.

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Aims and objectives: Extra-pulmonary tuberculosis (EPT) previously considered as rare would seems to be progressing thus constituting, at present, a real public health problem. Methods: Our study aims to determine frequency and characteristics of PET diagnosed in the medical microbiology department of CHU Mustapha (MMDCM), Algiers. A retrospective descriptive study was conducted over 3 years (2012, 2013 and 2014) identifying cases of PET diagnosed in the MMDCM. Results: From January 2012 to December 2014, 3529 samples were received at MMDCM and seeded in culture on Löwenstein -Jensen: 1814 from pulmonary origin and 1715 from extra-pulmonary sites. Of the 153 positive cultures, 74 were extra-pulmonary, ie 48.4%. Pleural tuberculosis was the most reported form (39%). Peritoneal and pericardial tuberculosis were also reported at 6.75% and 2.70% respectively. The neuro-meningeal, osteoarticular and lymph node forms accounted for 5.4%, 10.8% and 9.45%, respectively. We noted a slight predominance of women. Conclusions: The EPT rate determined during our study reflects the progress of this infection. There is also a diversity of clinical forms, which should encourage systematic bacteriological confirmation of suspected cases, to ensure regular and continuous monitoring.

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Aim and objectives: Mycobacterium tuberculosis (MTB) is a causative agent of tuberculosis (TB) which remains as an endemic disease in Iran. One of the most significant steps for control of TB is the fast and accurate diagnosis of patients. Examination of sputum smears is the primary method for TB diagnosis, and the culture method remains as the gold standard. Recently, a molecular assay that named Xpert MTB/RIF assay (Xpert), was introduced for rapid diagnosis of TB. The aim of this study was to evaluate the performance of the Xpert assay for diagnosis of TB in Golestan province. Methods: The TB suspected cases which referred to Tuberculosis Reference laboratory, Gorgan, Iran, from March 2018 to February 2019 were included to this study. The sputum specimens were examined using smear microscopy after Ziehl–Neelsen staining and graded according to the International Union Against Tuberculosis and Lung Disease scale (scanty, 1+, 2+, 3+ and 4+). The Xpert analysis was performed according to the manufacturer's instructions and the provided semi-quantitative results for positive specimens (very low, low, medium and high) were documented. The specimens were cultured into Lowenstein-Jensen (LJ) culture media after decontamination by Petroff's method. Sensitivity and specificity of Xpert and smear microscopy were calculated using a culture as reference standard. Results: Of 102 presumptive TB cases, 17 (16.7%) had culture proven TB. The set of 16 (94·1%) culture-confirmed TB cases were equivalently detected using smear microscopy and Xpert. Compared to the culture method, specificity of Xpert and smear microscopy was 100% (85/85) and 97.6% (83/85), respectively. Both tests had a sensitivity of 94.1% (16/17). When the semi-quantitative results of Xpert were compared with smear microscopy, there was 92.3% agreement for specimens with medium/high results, with those that had 1+ to 3+ scales. Conclusion: The rapid and accurate laboratory diagnosis of TB is one of the most important steps for TB control programs. In this study, the Xpert was effectively used to early and accurate diagnosis of TB in the study area. In the other hand, correlation between the Xpert semi-quantitative results and smear microscopy showed that Xpert could help in evaluation of patient's transmission potential. The Xpert can simplify patients' access to early and accurate diagnosis, and decreasing morbidity associated with diagnostic delay. Although Xpert showed high overall sensitivity and specificity for diagnosis of MTB in sputum samples, its accuracy must be evaluate for other clinical specimens in Iran.

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Aims and objectives: To establish a Mycobacterium tuberculosis genome database in collaboration with Asian countries, and to utilise the data for genotypic drug susceptibility and testing molecular epidemiology Methods: M. tuberculosis isolates collected in each participating country based on drug resistance and/or prevalence survey was collected as bacteria or genomic DNA, and whole genome sequencing was performed using Illumina sequencers. As a meta-data, the minimum inhibitory concentration (MIC) and conventional drug susceptibility testing (DST) data were collected for each isolate. Results: Approximately 5,000 genome data has been collected from Japan, China, Korea, Taiwan, Philippines, Vietnam, and Mongolia. Approximately 75% of isolates belonged to lineage 2.2.1. Base on the MIC and mutations/indels data, some confident mutations/indels were identified. With the data, the sensitivity of genetic DST for pyrazinamide was increased from 60.1% to 97.2% without losing specificity. Conclusions: Genome database will be a basis of genotypic DST and molecular epidemiology. However, the confidence of meta-data including MIC and other clinical information will be the key factor of genome database. In addition, genome data solution programme/software will be of great importance.

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Aim and Objective: Previous studies reported the implications of host-immune cell signalling pathways contributing towards survival of mycobacteria. Among the few, abrogation of PI3K-AKT-mTOR pathway has been shown to impact bacterial viability in macrophages. However, the intricate molecular mechanisms associated with such outcome at the juncture of host-pathogen interaction remain elusive. Methods: We have used novel AKT inhibitor against MTB infected macrophage and tested its survivability and its molecular mechanism of killing inside macrophage. Result: In the present work mouse peritoneal macrophages infected with pathogenic strain of M.tb (H37Rv) exhibited increased phosphorylation of AKT. Interestingly, suppression of AKT activity by a novel small molecule inhibitor during mycobacterial infection promoted phagolysosome fusion, apoptosis and autophagy, altogether contributing towards reduction in bacterial count in vitro. At the molecular level, we noticed that, small molecule inhibitor to AKT treatment deciphered the requirement of phosphorylation of AS160, Bad and mTOR by phosphorylated AKT in infected macrophages. Additionally, inhibition of phosphorylated AKT hampered nuclear translocation of NF-kB resulting in decreased cytokine expression. Conclusion: Taken together, our inhibitor can be utilized as an adjunctive therapy, as it shows high efficacy in decreasing bacterial count in combination with standard drugs to TB and also prevent the development of antibiotic resistance as host pathways are being targeted.

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Aim and objectives: Tuberculous pleural effusion (TPE) is one of the most common forms of extrapulmonary tuberculosis. Patients with tuberculous or malignant pleural effusions (MPE) frequently have similar clinical manifestations and pleural fluid profile. New biomarkers for the differential diagnosis of TPE are required. Objective: We sought to determine of whether cytokine profiles in the pleural effusion of patients were suitable as tools for the differential diagnosis of TPE. Methods: 30 patients with TPE, 30 patients with MPE, 14 patients with empyema and 14 patients with parapneumonic effusion were enrolled consecutively from the Masih Daneshvari Hospital, Tehran, Iran between Dec 2018-Dec 2019. The levels of interleukin (IL)-6, IL-18, IL-27, CXCL-8, CCL-1 and IP-10 were determined in pleural effusions by ELISA along with measurements of adenosine deaminase (ADA). Results: The levels of all analytes measured except IL-18 were higher in TPE compared with non-TPE subjects (all p < 0.01). The best predictors of TPE were combined ADA.IL-27 (optimal cut-off value = 42.68 10³.U.ng/L², sensitivity 100%, specificity 98.28%, p ≤ 0.0001), ADA (optimal cut off value 27.5 IU/L, sensitivity 90%, specificity 96.5%, p ≤ 0.0001) and IL-27 (optimal cut-off value = 2363 pg/ml, sensitivity 96.7%, specificity 98.3%, p ≤ 0.0001). A high level of IL-6 (optimal cut-off value = 3260 pg/ml, sensitivity 100%, specificity 67.2%, p ≤ 0.0001), CXCL-8 (optimal cut-off value = 144.5 pg/m, sensitivity 93.3%, specificity 58.6%, p ≤ 0.0001), CCL-1 (optimal cut-off value = 54 pg/mL, sensitivity 100%, specificity 70.7%, p ≤ 0.0001) and IP-10 (optimal cut-off value = 891.9 pg/mL, sensitivity 83.3%, specificity 48.3%, p = 0.0001) were also predictive of TPE. Conclusion: High ADA.IL-27, ADA and IL-27 levels differentiate between TPE and non-TPE with improved specificity and diagnostic accuracy.

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Aims &Objective: Gender variations are common features of many physiological parameters. Thus sex differences in haemostatic and haematological parameters are expected, but how anti-tuberculosis therapy modulates these differences is unclear. We hypothesized that anti-tuberculosis therapy will induce further changes in the level of these parameters when compared between male and female tuberculosis (TB) subjects. Aims and Objectives: This comparative follow-up study was designed to compare the levels of haemostatic and other haematological parameters between male and female TB subjects at pre-treatment, and followed up with a repeat comparison at post-intensive phase treatment and post-continuation phase treatment, to ascertain the differences at each phase of treatment. Methods: A total of sixty TB subjects of both genders (35 males and 25 females) were enlisted in this study. They were recruited before initiation of therapy and followed up post-intensive phase and post-continuation phase treatment and samples collected and analysed. Whole blood was used for measurement of total (TWBC) and differential white cell count, platelet count (PLT) and packed cell voume (PCV) while P-selectin (P-SEL), platelet activating factor (PAF), platelet factor-4 (PF-4), GP IIb/IIIa complex, thrombopoietin (TPO) were assayed using ELISA test kits. Results: At pre-treatment, P-selectin (ng/ml), PCV (l/l), neutrophil-lymphocyte ratio (NLR) and platelet count (x10⁹/l) were significantly higher in male compared to female TB subject (P=0.044, 0.022, 0.040 and 0.039). At post-intensive phase treatment, P-selectin (ng/ml), TPO (pg/ml), total white blood cell count (x10⁹/l), neutrophil count (x10⁹/l), lymphocyte count (x10⁹/l), monocyte count (x10⁹/l), eosinophil count (x10⁹/l) and monocyte lymphocyte ratio (MLR) was significantly higher in male compared to female TB subjects (P=0.046, 0.030, 0.022, 0.044, 0.029, 0.036, 0.042 and 0.036). Conversely, the mean platelet count (x10⁹/l) was significantly higher in females than males (P=0.022). At post-continuation phase treatment, the median P-selectin (ng/ml) was significantly higher in male compared with female TB subjects (P=0.028). Conclusion: Sex differences in haemostatic and haematological parameters vary with different phases of anti-tuberculosis treatment. But P-selectin displays a consistent higher level in male TB subjects at all phases of treatment.

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Aims and Objective: The SAR COVID2 virus devastated the world in the very short time. The virus not only effected the individuals, but also the economy and infrastructure of laboratories working in other infectious diseases. The synergy between patients with other air borne diseases like tuberculosis and COVID-19 is not very clear, but it have been shown that few patients expired having two diseases at the same time. Now the question comes out for safety of laboratory personal and necessary precaution for those that handle the specimens from patients who got both infection (tuberculosis &COVID-19). Results: The investigation should that most of peripheral TB laboratories (85%) in Iran are using biological safety cabinet type 1 or 2; therefore they are not using open bench for making smear. Small number of general hospital (30%) use the open bench for smear preparation at the peripheral levels. It is advisable that after smearing they kept the smear for drying in the cabinet with using UV light for 1-2 hours; then they proceed with fixation and staining. May be it is also advisable to handle the specimens, with proper clothing also. Conclusion: It looks that with pandemic of COVID-19, even TB laboratories need re-vision of their protocol as far as safety of personal is concerned. Also, the open benching for smearing should be avoided in all other countries.

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Aims & Objective: Interferon gamma (IFN-γ) plays 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. 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 Mycobacterium infected pulmonary patients (n = 200) 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). The host genomic DNA from confirmed patients with Mycobacterium infection in 150 mycobacterium and control subjects (n = 100) were screened for SNPs of IFNGR1 (T-56C) by PCR-RFLP. Results: Out of 150 mycobacterial infected patients, 140 patients (90.3%) had CC genotypes, whereas the remaining ten had TC genotypes (6.6%). 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 Mycobacterium infection.

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Aims &Objective: SARS-CoV-2 spreads when you cough, sneeze, talk, sing, laugh, or breathe out rapidly. Therefore, to protect the individuals or health-care workers, the WHO and CDC have given their standard precautions to use the different types of masks depending on the load of SARS-CoV-2 in their working area. We made a mask that not only protect ourselves from SARS-CoV-2 through respiration, but also give us a protection through inactivation of attached SARS-CoV-2. Methods: The spray made by modified molecule of macrocycle (Ghanavi, United States patent; 10147951). The clothing was sprayed and after using in contaminated area was observed under scanning electron microscopy. The virus load was counted using RTPCR. Result: the Load of virus showed reduction up to 64 fold (94%) in sprayed clothing. The inactivation of COVID-19 were observed under scanning electron microscopy Conclusions: By this technology, the spread and transmission of virus from contaminated to clean area can be reduced or controlled.

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Aims and Objective: The rapid growth of Mycobacteria (RGM) is a member of Non-tuberculous Mycobacterium that are environmental and opportunistic pathogens whose role in human disease is increasingly recognized. RGMs are highly found in various natural and man-made environmental resources. They can be opportunistically transmitted to humans through contaminated water sources and cause their infectious pulmonary and extrapulmonary infectious diseases. Misdiagnosis of this type of mycobacterium can lead to destructive results. As a result, improper treatment Faced with it can lead to recurrent infections as well as drug resistance. Methods: The aim of this study was to review the existing studies on the prevalence of RGMs between 1990-2017 in worldwide. Data on the prevalence of RGM infection were collected from databases such as PubMed, Web of Science, Cochrane Library, Embase, Scopus, and other scientific databases. Results: In Asia, 776 cases were reported. Among these, the M. furtuitum 23.32% (181,776), and M. chelonae 18.04%(140,776) were the most prevalence, respectively. Also, 105 cases reported in Africa, with the highest prevalence cases being M. fortuitum 46.67%(49,105), M. neoaurium 14.29%(15,105), and M. septicum 8.57% (9,150), respectively. On the other hand, 1363 cases were reported throughout Europe. Among these, the M. furtuitum 30.37% (414,1363), M. chelonae 18.86%(257,1363), and M. peregrinum 13.94%(190,1363) were the most prevalence, respectively. In the United States, 237 cases of RGM were reported, which the highest prevalence was related to the genus M. chelonae 25.32%(60,237), M. mucogenicum 23.63%(56,237) and M. furtuitum 22.36%(53,237), respectively. Also, in Australia 120 cases were observed, with the highest prevalence in M. fortuitum 49.17%(59,120), M. septicum 21.67%(26,120), and M. chelonae 8.33%(10,120), respectively. Conclusion: Given the increasing prevalence of RGMs in different environments, it seems necessary to implement more infection control strategies, appropriate diagnostic methods and control guidelines for NTM diseases. It can be said that we need to increase the quality of diagnostic reference methods and more accurate measures to prevent and control RGM infections.

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Background: Tuberculosis (TB) worldwide re-emergence constitutes a major public health problem with multi-drug resistant (MDR) strains becoming a greater threat. Genotyping and drug susceptibility of Mycobacterium tuberculosis complex (MTBC) are important in order to improve the control of the disease. This study sought to determine drug susceptibility and genetic diversity of MTBC in three West/Central African countries, namely Cameroon, Nigeria and Ghana located in the Gulf of Guinea. Methods: A collection of 503 isolates (158 from Cameroon, 202 from Nigeria and 143 from Ghana) obtained from culture using Lowenstein–Jensen medium and Middlebrook 7H9 Broth were included in the study and some isolates tested for drug susceptibility using the proportion method or Xpert MTB/RIF. Isolates were further subjected to IS6110 typing, large sequence polymorphisms and spoligotyping. Results: The Cameroon sub-lineage was the most prevalent genetic family [150/202 (74.3%) in Nigeria, 45/102 (44.1%) in Ghana and 54/150 (36%) in Cameroon] and SIT 61 the largest cluster with 111/202 (55%), 43/150 (28.6%) and 22/102 (21.6%) isolates in Nigeria, Cameroon and Ghana respectively. MDR was observed in 29/202 (14.4%) cases in Nigeria, and HIV positivity [37/202 (18.3%) patients] was associated with rifampicin (RIF) [9/37 (24.3%)] resistance (p=0.012), as well as gender (p=0.009). Equally, gender (p=0.038) and age (p=0.015) had significant associations with the LAM10_CAM family in Nigeria. In Cameroon, 13/147 (8.8 %) isolates were resistant to RIF, and 6 (35.3%) of the 17 UgandaI sub-lineage were RIF resistant (OR, 9.58; 95%CI, 2.74-33.55, p=0.001).). RIF resistance was equally associated (OR, 0.18, 95%CI, 0.05-0.69; p=0.023) with previously treated patients [(4/14 (28.6%)] compared to new ones [9/133 (6.8%)]. No sub-lineage was associated with MDR in Ghana Other sub-lineages such as Haarlem, Ghana, West African 1 were recorded in the three countries. Conclusion: RIF resistance in TB/HIV co-infected patients and its association with a sub-lineage calls for a reinforced surveillance strategy to curb drug resistance development in the Gulf of Guinea.

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Aims & objectives: Resistance to TB drugs is a severe obstacle to effective TB control globally. Timely detection of multidrug-resistant TB (MDR-TB) is crucial to avoid poor cure rates and further development and transmission of increasingly drug-resistant strains of M. tuberculosis. Molecular rapid tests are recommended by WHO for the early detection of mutations associated with drug-resistance. When resistance to rifampicin and/or isoniazid is detected, additional DST should be performed for additional drugs. Optimally all drugs used in the therapy of MDR-TB should be tested, and regimens include only drugs to which the patient's isolate has documented susceptibility. The recent introduction of new anti-TB drugs made more effective and shorter treatment of MDR-TB possible. An example is the WHO recommended all-oral bedaquiline-containing MDR-TB regimen (WHO TB treatment handbook 2020) which contains bedaquiline, levofloxacin/moxifloxacin, clofazimine, ethionamide, ethambutol, isoniazid (high dose) and pyrazinamide for 4 months followed by 5 months of treatment with levofloxacin/moxifloxacin, clofazimine, ethambutol and pyrazinamide. The use of rapid diagnostic tests and more effective MDR-TB treatment regimens has the potential to improve treatment outcomes and reduce transmission of MDR-TB. Results: The introduction of new drugs and WHO recommendations for the therapy of MDR-TB put increased demands on TB-laboratories around the world to update their diagnostics algorithms to make sure they reflect new treatment regimens. If possible, DST should be performed for all drugs used in modern MDR-TB therapy. It is a challenge for TB laboratories to introduce DST for several compounds not earlier tested. On the other hand, DST of obsolete drugs such as the second line injectables, and especially kanamycin and capreomycin which are no longer recommended for use should be discontinued. Standardized protocols with critical concentrations for solid or liquid culture-based assays are needed to enable a successful re-focusing of DST at clinical TB laboratories. The possible added value of MIC determinations, as an alternative to just testing a single critical drug concentration, should be considered especially for the new drugs where we have more limited experience. Irrespectively of the method, there is a strong need for relevant drug-susceptible and drug-resistant control strains to ensure high quality when a new DST method is introduced. It is also of importance to develop and implement QC/EQA methods for all drugs tested. The need for knowledge transfer and training should not be neglected. Conclusions: Extended DST is initially a task of the NRL. In high drug-resistant TB burden areas, there might be a subsequent need for decentralization. To meet the demands of rapid detection of drug resistance there is a need for increased use of molecular tests. Such tests promise to be increasingly useful, but they can not be initiated for new drugs until we have a sufficient understanding of which genetic markers need to be examined to predict clinically relevant drug resistance in a reliable way. Research is ongoing to determine this for the new drugs and I expect to see such tests developed in the not so far future.

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Aims & objectives: Potentially transformative new tuberculosis (TB) drugs like bedaquiline are undergoing roll-out, however, this is largely in the absence of programmatic drug susceptibility testing (DST). We lack information on how susceptibility changes during treatment in patients on bedaquiline-containing regimens, especially in at risk patients who have complex treatment histories, are in programmatic rather than trial environments, and have a delayed treatment response (defined here as sustained culture-positivity). Methods: Serial isolates from 51 patients with drug resistant (DR-)TB who were culture-positive after ≥4 months of a programmatically administered bedaquiline-containing regimen, were collected. Bedaquiline phenotypic DST in MGIT 960 (1μg/ml), targeted deep sequencing (Rv0678, atpE, pepQ) and whole genome sequencing was done on paired isolates (pre-bedaquiline initiation, post-four-month). Results: 24/51 (47%) patients were phenotypically and genotypically resistant (39% acquired resistance). Excluding one patient with an unknown history, prior clofazimine exposure was associated with bedaquiline-resistance [21/24 (88%) bedaquiline resistant cases had prior clofazimine vs. 12/26 (46%) susceptibles; p=0.002]. Diverse combinations of single nucleotide polymorphisms (SNPs) and indels were in the Rv0678 promoter region and the Rv0678 and atpE genes. Examples of newly described resistance associated variants (RAVs) include Rv0678 -8 T/G and atpE 223 C/T. RAVs were not in defined hotspots and sometimes occurred concurrently with atpE RAVs. Discussion: The rate of bedaquiline resistance acquisition in this population is alarmingly high and associated with prior clofazimine exposure. The diverse RAVs pose challenges to molecular test development. This study highlights the existence of a potentially infectious pool of bedaquiline-resistant patients present under programmatic conditions in a resource-constrained setting and illustrates the danger of starting patients with complex histories on a novel drug without routinely available DST.

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Aim and objectives: Despite the availability of sufficiently effective treatment regimens, prevention and control, the widespread transmission and prevalence of resistant variants of Mycobacterium tuberculosis, which do not respond to any of the commercial drugs, remains a serious health problem for both developed and developing countries. Multidrug-resistant (MDR) strains present new threat to health security. This situation is of global concern due to increased human mobility, partly illegal and spontaneous. Patients infected with drug resistant strains are hard or impossible to be cured, and their treatment is more toxic and expensive. The urgent need of new antimycobacterial agents and development pathways is becoming more and more apparent. Methods: In this study, we synthesized new compounds containing 2-aminobutanol and camphane moieties and evaluated them with the reference strain and clinical M. tuberculosis isolates from a high-burden country, Russian Federation. All four compounds were obtained by simple synthetic procedures as pure diastereoisomeres and characterized by mass spectrometry, nuclear magnetic resonance, melting points and elemental analysis. The cell lines HEP-G2, HaCaT, HEK293 and CCL-1 were used to study the cytotoxicity and metabolism of tested compounds. The MTT colorimetric test was used to determine the average inhibitory concentrations of 50% (IC50). The CFU test was used to assess the long-term antiproliferative effect of the compounds. In vitro antimycobacterial activity of all compounds was tested by resazurin microtiter assay (REMA). All compounds were tested against reference strain H37Rv and 11 clinical isolates. Results: The isolates represented different clusters within clinically/epidemiologically important Beijing genotype (B0/W148, 94-32, Central Asian Outbreak, ancestral sublineage) and differed in phenotypic drug resistance (one extensively drug resistant, six multidrug resistant, two polyresistant, and two susceptible to all tested drugs). The most efficient compound showed a wide range of MIC that varied from 3.1 μg/ml (H37Rv, ancestral Beijing, most of B0/W148 isolates) to 100 μg/ml (other isolates). Conclusions: To conclude, this study highlighted a key importance of testing new anti-TB compounds not only with reference laboratory strain H37Rv that belongs to the phylogenetically marginal branch of M. tuberculosis, but on the clinical drug-resistant isolates circulating in the countries with high burden of MDR-TB. Acknowledgements. This study was supported by the Russian Science Foundation (grant 19-15-00028). Partial support was received from the Bulgarian National Science Fund (grant KP-06-H39-7).

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Aim and objectives: Emergence of multi-drug resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis strains requires use of novel compounds. New drugs require long-term treatment regimen and, consequently, long-term drug selective pressure gives more time to mycobacteria to develop resistance. At the same time, new compounds are expensive and, albeit efficient, they are not routinely used in the Russian Federation which situation with tuberculosis is characterized by high and increasing prevalence of multidrug resistant strains. Bedaquiline is a novel and promising anti-TB drug. Substitutions in the M. tuberculosis atpE gene prevent interaction of the drug with its target, ATP synthase and were associated with bedaquiline resistance. Mutations in two other genes involved in intermediary metabolism and respiration were also linked to the bedaquiline resistance: pepQ coding for probable cytoplasmic peptidase and atpC coding for probable ATP synthase epsilon chain. Mutations in the repressor gene mmpR (Rv0678) lead to increased expression of the efflux genes mmpL5 and mmpS5, providing export of bedaquiline. Finally, mutations in Rv1979c coding for possible conserved permease involved in transport of amino acids across the membrane were also shown in bedaquiline-resistant isolates. Perchlozone is a new thiosemicarbazone that is similar to thiacetazone and approved for treatment of MDR-TB in Russia. It is a prodrug activated by monooxygenase EthA and its target is FASII dehydratase complex HadABC. A cross-resistance to TAC and PCZ was shown by in vitro experiments and was mediated by both ethA and hadA mutations. On the other hand, EthA is known to activate second-line drugs ethionamide (ETH) and prothionamide (PTH) and ethA or ethR mutations were described as ETH/PTH resistance mechanism. Linezolid is other important anti-TB drug used for treatment of MDR/XDR-TB. The drug binds to the ribosome at the peptidyl-transferase center, and in vitro and clinical studies described mutations in the 23S rRNA and L3 ribosomal protein genes in resistant strains. Russia is a recognized hot spot for MDR-TB. TB incidence is decreased in the European Russia and is very high in Siberia and Far East, however, MDR TB is high and increasing in all parts of the country. For example, in Northwest Russia, TB incidence is 31.3/100,000 and MDR rate in primary TB - 27.4%. Kaliningrad is the westernmost Russian region and has a unique geographic position, separated from the mainland Russia by Lithuania, Poland and the Baltic Sea. The region is characterized by high TB incidence (38.8/100,000) and very high rate of MDR among newly diagnosed TB patients (30.5%). The M. tuberculosis population structure is dominated by the Beijing genotype. In this study, we aimed to gain insight into molecular basis of M. tuberculosis resistance to bedaquiline, perchlozone and linezolid in the Kaliningrad region in Russia. We used whole-genome sequencing of multiple consecutive isolates from patients spanning long time period in order to gain comprehensive and quantitative view on the real-time mycobacterial adaptation to the human host. This study included patients admitted to the clinical tuberculosis dispensary in Kaliningrad whose treatment regimen included second-line, repurposed and new anti-TB drugs, including BDQ, PCZ and LZD. All samples used in this study were coded and lacked personal information about patients. All personal data were treated anonymously and, according to the regulations of the St. Petersburg Research Institute of Phthisiopulmonology, no additional ethical approval was required. Methods: M. tuberculosis isolates were recovered from sputum at the 1-3 months intervals. M. tuberculosis drug susceptibility testing for first- and second-line drugs was performed using modified proportion method on Middlebrook 7H9 liquid culture and Bactec MGIT 960 system (Becton Dickinson, Sparks, Md.) according to the manufacturer's instructions. The laboratory is externally quality assured by the System for External Quality Assessment “Center for External Quality Control of Clinical Laboratory Research” (Moscow, Russia). Whole genome sequencing was performed at the MiSeq platform (Illumina). The fastq files were aligned to the complete genome of the reference strain H37Rv (NC_00962.3) using Geneious 9 program (Biomatters, New Zealand) and additionally checked with PhyReSE online tool (https://bioinf.fz-borstel.de/mchips/phyresse/). Results: Mutations in Rv0678 (mmpR) were most frequent in BDQ resistant strains and different types of mutations were found (non-synonymous substitutions, frameshift, multi-codon indels). In some cases, different mutations coexisted and heteroresistance was observed in all cases, i.e., simultaneous presence of wild type and mutants alleles (which correlates with previous studies). Only in one patient, atpE mutation emerged in M. tuberculosis isolate during treatment. Analysis of genes associated with PCZ resistance (ethA, ethR, and hadABC) identified three mutations in ethA and one mutation in hadA. The ethA frameshift mutation (genome position 4327363 CT>C) was detected in isolates from patients who did not receive previous PCZ treatment but were all phenotypically resistant to PTH or ETH. Thus we assumed that this mutation emerged as PTH/ETH resistance mutation. No mutations in rrl, rplC, rplD genes associated with LZD resistance were found. Conclusions: To conclude, mutations in Rv0678 present a main mechanism of bedaquiline resistance in the Russian setting; development of resistance during treatment is not rare as bedaquiline resistance mutations emerged in 40% of patients. Furthermore, development of BDQ resistance 2-3 months after stop of treatment is possible, due to long half-life of the drug. With regard to PCZ resistance, presence of cross-resistance mutations to both PCZ and ETH/PTH suggests that these mutations should be assessed before inclusion of PCZ in the treatment regimen. A large prospective study of the more diverse M. tuberculosis collection is warranted to elucidate the development of resistance to these novel drugs in the Russian setting taking into consideration the population structure of M. tuberculosis population.

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Aims and objectives: Selection pressure, such as drug treatment or host microenvironment, drives the evolution of M. tuberculosis in vivo. The emergence of drug resistance by the accumulation of mutations leading to drug target modifications, drug transport or activation pathways inactivation, is the leading cause of treatment failure. Besides that, secondary mutations involved in bacterial adaptation, fitness-compensation, and resistance level were identified in the genome by microevolution studies. The genotype-phenotype correlation is complicated also by the partial cross-resistance between first- and second-line drugs: rifampicin and rifabutin acting on beta-subunit of RNA-polymerase in a slightly variable manner, or isoniazid and ethionamide possessing different activation pathways by InhA and EthA, respectively. Genome-wide association studies (GWAS) of a large number of sequenced clinical isolates allows for the identification of minor determinants of resistance, co-evolution markers, and its epistatic interactions. Such type of data could lead to an increase in molecular tests sensitivity and treatment schemes personalization. Various approaches are used for such analysis; most of them analyze the impact of a single mutation, omitting the mutation interactions due to calculation complexity. Methods: Whole-genome sequencing data of 2212 clinical isolates of M. tuberculosis with resistance data were downloaded from SRA. Identification of single nucleotide variations (SNVs) was performed using BWA-MEM and C-Sibelia scripts using AL123456.3 as reference. The SNVs database handling and calculations were performed with custom Python scripts. Fisher test analysis was performed as for individual mutations, also for mutation combinations. Pairs of mutations were joined using logical AND function. Thus, n^2/2 records were analyzed for correlation with resistance, where n – total number of mutations in the database. Resistance to the rifampicin, isoniazid, and ethionamide was analyzed. Results: A novel approach for GWAS was developed: we studied the correlation of simultaneous occurrence of mutations pairs and phenotypic resistance for all possible pairs. We identified several SNVs associated with resistance in combinations with other mutations, and its statistical significance was below the threshold when analyzed separately. Thus, Rv0175 coding for a membrane-associated protein with an unknown function was associated with rifampin resistance and intergenic mutation in Rv1829-Rv1830 – with isoniazid resistance. Previously described, as associated with MDR and XDR phenotype, frameshift glpK mutation was found to affect the resistance to rifampin and ethionamide, but not to isoniazid. However, in the case of rifampin, it was also identified from the individual mutation GWAS, in case of ethionamide – only in combination with other mutations. In addition to glpK, novel SNVs in rv0083 gene and promoter regions of rv1451 and whiB4 genes were identified as associated with ethionamide resistance, synergistically acting in combinations with other mutations. Conclusions: Fisher exact test for mutation combinations allows for the identification of genetic determinants missed by conventional GWAS approach. Further evaluation of novel resistance determinants using other sets of clinical isolates data from various world regions is needed. Acknowledgements This study was supported by the Russian Foundation for Basic Research (project 20-015-00463).

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Aims and objectives: Vaccine is one of the most effective tools available to prevent infectious diseases and their complications. One of the most controversial vaccines for its therapeutic effect on COVID-19 is the BCG vaccine. The BCG vaccine has been used worldwide since 1921. The first BCG strain was introduced in 1947, but mass production and nationwide prescribing began in 1984. The BCG vaccine is able to stimulate the immune system's response to a large number of antigens that are effective in causing infectious diseases. Involved mechanisms include activation of NK and NKT memory cells and an increase in 1β-IL against viral infections. Results: Due to the prevalence of COVID-19 virus and the need for research to become more aware of the virus and the process of prevention, control and treatment, there is hope of the effectiveness of the BCG vaccine in recovering diseases. Studies show that tuberculosis infection, both active and latent, increases the severity and progression of the disease in people with COVID-19. The BCG vaccine is likely to be able to reduce the cytokine storm after exposure to SARS-COV-2, resulting in mild COVID-19 and early recovery. Children also seem to be protected against COVID-19 compared to adults. In late adulthood, mechanisms such as the reduction of regulatory T cells can help exacerbate COVID-19. Conclusions: Despite all the studies, the claim that the BCG vaccine is effective against COVID-19 needs further investigation worldwide. Any incorrect information and lack of accurate tests can lead to misuse of the vaccine and this can lead to the deprivation of children in need of vaccine in some countries due to its lack. As a result, it is better to conduct more comprehensive studies to obtain definitive results.

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Aims & objectives: The aim of this work was to evaluate the performance of mfloDx® MDR-TB assay kit on DNA extracted from 100 pulmonary sputum samples. mfloDx® MDR-TB is a qualitative test that can identify bacteria belonging to the Mycobacterium tuberculosis complex (MTC) as well as robustly identify single-nucleotide variants of the most common mutations in multidrug resistant M. tuberculosis DNA. The current assay can detect the most prevalent mutations causing resistance to Rifampicin (RIF) and Isoniazid (INH). Methods: Genes of interest were selectively enriched and targeted using amplifiable padlock probes (PLP). PLPs that recognize mutation or wild type were ligated upon specific recognition of the correct target sequence. Once PLPs were circularized, they underwent rolling circle amplification. Polymers resulting from this amplification were monomerized and subsequently visualized on disposable cassettes. The developed bands allowed for accurate genotyping of eleven resistance-related mutations, as well as respective wild type alleles and MTC identification. Results: From 100 samples; 81 were successfully genotyped, 16 were negative for MTC, two were inconclusive and one ran out during analysis. Among the genotyped samples; 46 were resistant to both RIF and INH, eight samples were resistant only to INH, three samples were resistant only to RIF. The remaining samples were susceptible to both drugs. Clinical sensitivity and specificity were calculated based on phenotypic drug susceptibility test results (MGIT culture) as reference. Sensitivity was 89.6% for RIF and 92% for INH resistance. Specificity was 95.5% for RIF and 89.5% for INH susceptibility. Conclusions: In conclusion, mfloDx® MDR-TB offers a reliable and affordable rapid drug-susceptibility testing for MDR-TB with potential implications in early detection of drug-resistance in resource-limited, high burden TB and MDR-TB settings. It enables clinicians to ensure the timely administration of effective drug therapy which will not only reduce the risk of treatment failure but also limit the further development and spread of dug resistant TB.

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Aims & objectives: Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), is amongst the most successful pathogens worldwide with a yearly incidence of approximately 10.0 million new cases and a death toll that is well above million people. Even though this is an ancient pathogen affecting humankind since its dawn, as a bacterial species it displays a very low mutation rate and shows a strictly clonal population structure owing to the lack of lateral gene transfer. This latter aspect imposes a limitation on the pathways by which drug resistance can emerge in this species: emergence of resistance must occur through de novo mutations affecting genes that are associated with the action mechanism of a given drug. Methods: The mutations and mechanisms leading to resistance to the drugs that are part of the frontline armamentarium have been extensively studied and the extent of its spectrum is relatively well understood. However, newer drugs such as bedaquiline and delamanid have been introduced to TB treatment in a very recent past. These were the first two drugs to become available in approximately 40 years and, as such, the toxicity and side effects associated with these drugs still pose several challenges. From a mechanism of action standpoint, these drugs act in entirely novel ways when compared with drugs that were already used for TB treatment. Bedaquiline not only targets ATP synthase subunit c but also appears to constribute to the dissipation of the proton motive force due to its proposed activity as a H⁺/K⁺ ionophore. Since its introduction, one concerning aspect pertains frequent reports of the quick emergence of resistance to this drug, which is thought to be mainly mediated by mutations in three genes: atpE, Rv0678, and pepQ. Whereas atpE codes for bedaquiline target it is noteworthy the importance of loss-of-function mutations in Rv0678 that lead to the derepression of the MmpS5-MmpL5 efflux pump and for which bedaquiline is a suitable substrate. This latter mechanism is also associated with cross-resistance to clofazimine and to pre-existing resistance to bedaquiline by M. tuberculosis strains predating the introduction of this drug to TB treatment. Results: Delamanid, a nitroimidazooxazole, is a pro-drug that requires metabolization to an intermediate metabolite that inhibits the synthesis of methoxy-mycolic acids and keto-mycolic acids. This activation step is mediated by the deazaflavin(F₄₂₀)-dependent nitroreductase (Ddn) and it is the loss a function of this enzyme that constitutes the main mechanism of resistance acquisition to delamanid. Other gene products susceptible to mutagenesis and part of the F₄₂₀ metabolism have been implicated in delamanid resistance (fbiABC and fgd1). Conclusions: Several genomic variants occurring in these genes have been reported yielding different resistant levels however, owing to the restricted usage of these drugs, many of these are reported from in vitro studies. The scenario portrayed by clinical strains is somewhat different and the mutational spectrum associated with resistance more limited. Besides covering the mechanisms of action and drug resistance this talk will discern between clinically relevant mutations vs those generated in vitro and the phylogenetic distribution of different polymorphisms in genes associated with resistance.

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Resistance to first-line anti-tuberculosis (TB) drugs continues to threaten global TB control with over 500 000 cases of rifampicin resistant (RR)-TB being reported in 2018. Treatment of RR-TB requires the use of toxic drugs often leading to adverse events, poor adherence and poor treatment outcomes. In order to address the toxicity of treatment and to improve treatment outcomes the World Health Organization has promoted the use of injection free regimens which include new and repurposed drugs: bedaquiline (BDQ), linezolid (LZD) and clofazimine (CFZ). In 2017, South Africa rolled out the inclusion of BDQ in the treatment of all pre-XDR- and XDR-TB cases as well as patients experiencing adverse events from kanamycin. In late 2018, South Africa included BDQ into three injection free regimens for treating RR-TB. This was done before routine drug susceptibility testing for BDQ could be implemented. Resistance to BDQ occurs primarily through mutations in Rv0678 (mmpR), pepQ and atpE. Understanding which mutations confer resistance and why they arise in a patient will be critical for early diagnosis as well as for preventing the emergence of resistance thereby ensuring successful treatment. Collation of the mutations identified in Rv0678 shows that BDQ resistance is largely driven by small insertions/deletions which disrupt the function of the repressor thereby leading to over expression of the efflux pump mmpL5 responsible for extruding BDQ from the cell. Understanding the association between gene mutation and phenotypic resistance remains critical for the development of new molecular diagnostics to rapidly diagnose resistance thereby affording the clinician the opportunity to adjust the regimen. Analysis of serial isolates collected from patients receiving BDQ (Access program pre-2017) has shown the emergence of mutations in Rv0678, atpE and pepQ during treatment in patients who remain culture positive after three months of treatment. Using targeted deep sequencing it is now possible to track the emergence of resistant sub-populations at the sub-phenotypic level (i.e., micro-heteroresistance) thereby affording the opportunity to study the association of the emergence of resistance with drug regimens, MIC distributions, PK and PD data and treatment outcomes. Our analysis showed that mutations conferring resistance occurred soon after the introduction of BDQ into the regimen and that clones harbouring these mutations were subsequently selected. Most surprising was the observation that different resistant sub-populations emerged during the period after the cessation of BDQ in the regimen. This demonstrates the continuing selective pressure as a result of the long half-life of BDQ. Although the origin of the distinct resistant clones is unknown, we hypothesize that resistance evolved independently in spatially separated lesions subsequently rupturing into the airways. A surprising observation was that concomitant emergence of different variants of the rpoC gene suggesting a compensatory mechanism to overcome the fitness cost of Rv0678 mutations.

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Aims and objectives: Like a mistral, the SARS COVID2 virus has devastated the world in a span of 6 months infecting 29 million and leaving behalf a trail of one million dead. In its trajectory it has joined forces with another old but far more side-tracked airborne disease, tuberculosis. The synergy so far does not have a clear face even in vulnerable countries like India which face a “double whammy” of TB and COVID-19. One out of 3 TB patients in the world is Indian whereas India may outstrip the USA soon in the COVID affected count. Methods: Vulnerable settings in India do not have the luxury of social distancing. The strict lockdowns in vulnerable settings may paint different pictures depending on community behaviour. Ten people huddled for days in a 100 sq. ft room may perpetrate transmission of TB disease. Juxtaposed is the finding of a 60% drop in TB notification. The implications of this paradox will be discussed in the light of overcrowded living conditions, pollution and highly prevalent comorbidities like T2DM and HT. Results: T-cell exhaustion in COVID-19 patients may increase susceptibility to TB and promote development of active TB in LTBI. Scarred lungs and pulmonary fibroses are sequalae of both diseases and could result in devastating disabilities. Modelling studies suggest a 6% increase in TB deaths overt extension of care pathways and treatment completion rates dropping to an abysmal 25%. The deviation of TB diagnostic infrastructure to COVID-19 diagnosis is advantageous as a single step, rapid test on a platform familiar to most laboratories in a country. However the reduction in TB diagnostic ability is likely to have a negative impact on TB. If coinfections of TB and COVID-19 do coexist in significant proportions then clinical management of patients would require a strong algorithm eg. lopinavir / ritonavir interact with both RFP and BDQ. What is less known is the co-occurrence of TB and COVID-19 both in the active and the latent (TB) / asymptomatic form. The approaches to develop a single test for dual detection of the 2 diseases is sorely needed but its technical development is challenging. Conclusions: Socio-financial and nutritional support to communities is an essential arm of mitigation. The COVID-19 pandemic at its best has also highlighted the importance of non-medical approaches of prevention especially in the realm of urban planning. The role of BCG, however ecological the evidence, is also a road worth exploring.

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Aims and objectives: The emergence and spread of multidrug resistant (MDR) Mycobacterium tuberculosis (M. tuberculosis) strains is a critical global health problem. In order to shorten the course of treatment and its effectiveness, it is essential to gain an in-depth insight into the genetic diversity, drug resistance mechanisms and host-pathogen interactions of drug resistant M. tuberculosis. Methods: Between 2014 and 2018, 606 MTBC strains were isolated from 13,892 suspected pulmonary tuberculosis (TB) patients in Tehran, Iran, including 16 MDR-TB cases. A combination of phenotypic (conventional drug susceptibility testing, MIC, Verapamil effectiveness) and genotypic methods (whole-genome sequencing and qPCR) was employed for the identification of additional drug resistances, the role of efflux pumps and strain-to-strain genetic distances as a marker for recent transmission events. Finally, the consequences of mutations in genes encoding the beta subunit of RNA polymerase (rpoB) for host-pathogen interactions was investigated by means of THP-1-derived macrophages infection and Cytokines and Chemokines Real-Time RT² Profiler PCR Array. Results: MDR and extensively drug-resistant (XDR) TB cases were almost exclusively infected by lineage 2/Beijing strains (14/16, P < 0.001). Out of the 16 MDR/pre-XDR/XDR isolates, 6 (37.5%) and 3 (18.8%) isolates demonstrated a significant decrease in rifampicin (RIF) MIC and isoniazid (INH) MIC due to the Verapamil exposure (64 μg/mL), respectively. More importantly, it was found that the administration of Verapamil profoundly decreased the MIC of Bedaquiline (BDQ) among most of the studied clinical isolates. Moreover, the efflux pump genes of Rv2938, Rv2936, Rv1145, Rv1146, Rv933, Rv1250, Rv876, Rv2333, Rv2459, Rv849, and Rv1819 were overexpressed in the presence of anti-TB drugs, showing the contribution of these efflux pumps to the overall resistance phenotype. Finally, the macrophages immune responses induced in response to infection with drug-resistant M. tuberculosis (RMR strain) may not be substantially different from drug susceptible M. tuberculosis (H37Rv strain), but the severity of the responses was different in some cases specially IL-6, IL-8, CNTF, IL-13, TNFSF10 and CD40LG in response to RMR and H37Rv strains. In addition, RMR strain showing significantly higher intracellular bacillary growth compared to H37Rv strain. Conclusions: The crucial finding is that almost all of our MDR/pre-XDR/XDR strains are of the Beijing lineage (14/16) and more than half of them are probably from recent transmission (8/14). Our results clearly showed that efflux systems, besides spontaneous mutations, play a role in the development of INH/RIF resistance. In addition, Verapamil was very successful at the phenotypic level specially in response to BDQ. Finally, the results initially demonstrate that the M. tuberculosis carrying the rpoB-S450L can modulate macrophage responses to mediate bacterial survival.

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Aims and objectives: Multidrug-resistant TB( MDR-TB) is resistant to isoniazide and rifampin simultaneously. In addition to isoniazide and rifampin, extensively drug-resistant TB (XDR-TB) is also resistant to fluoroquinolones and at least one of the three injectable drugs : amikacin, kanamycin or clarithromycin. Considering different treatment protocols, having some clues in chest computed tomography (CT) scan to differentiate between the two can be helpful clinically. The aim of this study is to compare chest CT findings of MDR-TB and XDR-TB. Methods: In this cross-sectional study, TB patients who referred to Masih Daneshvari Hospital (Tehran, Iran) between 2013 and 2020 were enrolled. TB was diagnosed by sputum smear, sensitive molecular and microbial tests. Based on different types of drug resistance, the patients were divided into two groups: MDR-TB and XDR-TB and chest CT scan findings were compared. Results: The frequency and characteristics of cavitary lesions in MDR-TB and XDR-TB patients were similar (P > 0.05). The frequency of small and large nodules, tree in bud pattern, ground glass opacity, bronchiectasis, cicatricial emphysema, mediastinal lymphadenopathy and pericardial effusion did not differ significantly in the two groups (P>0.05). Parenchymal calcification was more common in the XDR-TB (P=0.01). Conclusions: MDR-TB and XDR-TB have almost similar CT scan findings. However, we found parenchymal calcification and left pleural effusion more frequently in XDR-TB group. Overall, it can be inferred that chest CT scan is not a reliable discriminating tool in this regard.

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Aims and objectives: Sarcoidosis and tuberculosis (TB) are two granulomatous inflammatory diseases with several common symptoms. The aim of the present study was to compare the serum levels of biomarkers including interleukin-4 (IL-4) and IL-13, calcium (Ca), hemoglobin, sedimentation rate, and lymphocyte-to-neutrophil ratio between patients with pulmonary TB, patients with sarcoidosis, and control group. Methods: This case–control study was performed on patients referred to the Masih Daneshvari Hospital, Tehran, from April 2017 to 2018. In this study, 24 newly diagnosed patients with active pulmonary TB, 34 patients with pulmonary sarcoidosis, and 30 healthy individuals as the control group were enrolled. Demographic data, erythrocyte sedimentation rate (ESR), the ratio of neutrophil-to-lymphocyte (NLR), serum Ca level, hemoglobin (Hb), and IL-4 and IL-13 were compared between the study groups. Receiver operating characteristic (ROC) curve analysis, sensitivity, and specificity were also calculated using SPSS 16.0 software. Results: The mean age was 47.71 ± 10.88 and 55.25 ± 21.58 years in the sarcoidosis and TB. The mean ESR in sarcoidosis patients was 21.45 ± 13.37 mm/h and 41.4 ± 17 mm/h in the TB group. The percentage of peripheral blood lymphocytes in sarcoidosis and TB patients was 28.02 ± 12.20 and 21.41 ± 12.49, respectively, which was significantly higher among patients with sarcoidosis. NLR was also 2.4 ± 1.6 and 4.4 ± 2.9 in sarcoidosis and TB patients, respectively, which showed a significant difference among the groups. Regarding the evaluation of the level of IL-4 and IL-13 in patients, it is worth noting that IL-4 in patients with sarcoidosis was 90 pg/ml compared to 20 pg/ml for TB patients (P < 0.001). There was no significant difference in the levels of IL-13 in the TB and control groups, which varied between 20 and 80 pg/ml (P = 0.35). However, its value was significantly higher in patients with sarcoidosis (P = 0.01) than in the healthy control group and TB (P = 0.01). The ROC curves showed that the diagnostic cutoff of ESR level, Ca, NLR, and Hb could be valuable due to the area under the curves. The cutpoint of 34 mm/h for ESR had a sensitivity of 86% as well as 80% specificity to distinguish TB from the sarcoidosis. Conclusions: Serum levels of the biomarkers indicated a stronger immunological background in sarcoidosis using NLR, Ca, ESR, and Hb.

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Aims & objectives: The basis of antibiotic resistance in Mycobacterium tuberculosis (MTB), unlike Enterobacteriaceae, is the mutation in its chromosomal genes such as katG (Gene ID: 885638, causes isoniazid resistance) and rpoB (Gene ID: 888164, rifampin resistance). Evaluation of whole genome sequence of the standard strains of H37Rv in gene bank revealed the absence of integrons, plasmids and transposons. There are few reports on these genetic elements in clinical strains of MTB isolated from the patients. In this study, as a hypothesis based on the genetic composition differences between H37Rv and clinical isolates, and probably geographic differences between clinical strains genomic, we designed a study on a probably presence of a few genes in Iranian clinical strains. Methods: Previous studies of our research group showed that there is a new fragment in our clinical strains of MTB that was first recorded in the GenBank (Accession: MF279142.1). During extensive bioinformatics and gene bank (insilico) studies, it was found that this fragment might be a part of an integrase, belonging to a probably integron, plasmid, phage or transposon inside or outside the chromosome. Existence of its complete gene in different coding sequences was carefully investigated. A few genes including kleE, pmaB, sul, and suf, surrounding this fragment were amplified by using Mycobacterium abscessus plasmid and other non-tuberculosis mycobacteria as templets by PCR. Specific primers based on the aforementioned strains were designed. PCR reactions were optimized with various amplification programs. Bands were purified and were sequenced by ABI system apparatus. Sequencing results were analyzed by Mega, Chromas, and Basic Local Alignment Search Tool programs. Results: Bioinformatics analysis of sequencing results of purified 463bp amplicon revealed that the studied fragment was belonging to gene encoding dihydropterate synthase of Mycobacterium fortuitum but not in H37Rv and the other MTB strains in Gene Bank. It was confirmed that this new fragment there are in 30% of our clinical MTB strains. Conclusion: As the results, presence of a part of suf gene was reported for the first time in clinical isolates of Mycobacterium tuberculosis. Further experiences are under investigation to find the complete gene, and to examine whether it belongs to a larger genetic structure.

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Aim and objectives: Tuberculosis (TB) is one of the most lethal global infectious diseases. Despite the availability of much higher levels of technology in health and medicine, tuberculosis still remains a serious global health problem. Mycobacterium tuberculosis has the capacity for prolonged survival inside macrophages by exploiting host metabolic and energy pathways and perturbing autophagy and apoptosis of infected cells. Results: The mechanism (s) underlying this process are not completely understood but evidence suggests that mycobacteria subvert the host miRNA network to enable mycobacterial survival. The role of miRNAs in TB immune escape mechanisms and the potential for miRNA-based TB therapeutics has been described. Further validation studies are required to (i) elucidate the precise effect of TB on host miRNAs, (ii) determine the inhibition of mycobacterial burden using miRNA-based therapies and (iii) identify novel miRNA biomarkers that may prove useful in TB diagnosis and treatment monitoring. Circulating exosomes have been used as diagnostic biomarkers in various diseases. Recently, the serum-derived exosomes were isolated from TB patients and expression of miR-484, miR-425, and miR-96 was examined. Conclusion: They found that the expression of miR-484, miR-425, and miR-96 were significantly increased in serum of TB patients which correlated with the TB infection level. In conclusion it is well stablished that exosomal miRNAs have diagnostic potential in active tuberculosis. The diagnostic power may be improved when combined with conventional diagnostic markers.

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Aims and objectives: Treatment of patients with drug-resistant tuberculosis is time-consuming and requires expensive drugs with more toxicity. Proper and timely identification of drug-resistant genotypes and determining the minimum inhibitory concentrations (MICs) alongside with the molecular basis of resistance can minimize the risk of further resistance development. Methods: Multidrug-resistant Mycobacterium tuberculosis (MDR-MTB) strains from different provinces were identified at Tehran Regional Reference Laboratory for Tuberculosis. Drug susceptibility testing for the first and second-line anti-TB drugs were performed and compared to the whole genome sequencing (WGS) results. Results: Simultaneous resistance to antibiotics included in each of the fluoroquinolones and second-line injectable drug families was observed in most of the MDR isolates. Sequencing was able to detect resistance to anti-TB drugs with a proper sensitivity. The most prevalent lineage among the Iranian MDR strains was Beijing. Conclusions: Mutations in drug resistance-related genes were mostly in accordance with the phenotypic drug susceptibility testing results. Drug-resistant tuberculosis can be identified quickly using molecular techniques. However, genetic background of resistance for some antibiotics were remained for more investigation.

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Aims and Objectives: The incidence of nontuberculous mycobacterial (NTM) disease continues to rise, especially in developed countries. While NTM can affect almost any organ system, pulmonary infection is more prevalent than disseminated disease and can occur in patients with COPD, non-cystic fibrosis (CF) bronchiectasis, and CF. Mitochondria are the powerhouse of the cell, but it is unknown what role they play in the host response to NTM infection. Using electron microscopy, we have previously shown that mitochondrial morphology is altered upon infection with Mycobacterium avium intracellulare (MAI). Thus, we hypothesize that mitochondrial damage leading to changes in mitochondrial function and dynamics impair host response to infection with MAI and Mycobacterium abscessus (MAB). Methods: We infected MH-S cells (murine alveolar macrophages) and bone marrow-derived macrophages (BMDM) with clinical strains of MAI or MAB (multiplicity of infection [MOI] of 25) for 6 and 24 hours. We determined the expression of mitochondrial DNA fragments mt79 and mt230 that are associated with mitochondrial DNA damage using RT-qPCR; we calculated the fold change of these fragments relative to GAPDH and determined a ratio of these fragments. We also determined the expression of key mitochondrial quality control genes, including peroxisome proliferator activated receptor gamma coactivator (PGC)-1α, transcription factor A mitochondrial (TFAM), and mitofusin (MFN)-2, using RT-qPCR. Results: We found that in MH-S cells, the mt79:mt230 ratio was increased at 6 and 24 hours for both MAI and MAB relative to controls infected with PBS. Furthermore, expression of genes involved in mitochondrial biogenesis, PGC-1 α and TFAM, were both decreased at 6 and 24 hours. MFN2 was also decreased at both 6 and 24 hours for MAI and MAB. These data were consistent in BMDM, with a more significant decrease in these genes noted at 24 hours. Conclusions: These data suggest that there is an increase in mitochondrial damage as indicated by the mt79:230 ratio when macrophages are infected with NTM. The infected macrophages exhibit decreased mitochondrial biogenesis and also have impaired mitochondrial quality control, including mitochondrial fusion and fission. The functional impact of these impaired mitochondria on host response to NTM infection needs to be further explored as there may be a role for pharmacologic interventions, like PGC-1 α activators, that can augment bacterial killing in patients infected with NTM.

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Aims and objectives: Mycobacterium abscessus complex consisting of M. abscessus subsp. massiliense, M. abscessus subsp. bolletii and M. abscessus subsp. abscessus, have now emerged as clinically important NTM pathogens in patients with cystic fibrosis (CF). Culturally, these organisms are difficult to isolate from sputum amongst mixed populations of extremely rapidly growing Gram-negatives, particularly Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Achromobacter spp. Our objective was to develop a highly selective medium, not requiring decontamination of CF sputum but which exploited M. abscessus complex's resistance to several antibiotics. Methods: ASA medium was formulated employing Glucose, 16g/l; Agar, 20g/l; Yeast extract, 30g/l; Peptone, 6.8g/l; Chloramphenicol, 50mg/l; Ceftazidime, 32mg/l; Colistin, 24mg/l; Trimethoprim, 21.33mg/l; Sulfamethoxazole, 106.67mg/l and Novobiocin, 50mg/l. Mycobacterium abscessus complex organisms consisting of M. abscessus subsp. massiliense (n=2), M. abscessus subsp. bolletii (n=2) and M. abscessus subsp. abscessus (n=2) [circa 10⁶ colony forming units (CFU)] were plated onto ASA medium and cultured aerobically for 2 weeks at 30^oC. In addition, the following non-NTM organisms were also plated onto ASA medium, as above, Gram-positive: Enterococcus faecalis, Staphylococcus aureus (n=6), Staphylococcus aureus (mucoid), MRSA (Hospital-associated), MRSA (Livestock-associated cc398); Gram-negative: Achromobacter sp. (n=4), Burkholderia gladioli, Burkholderia multivorans, Escherichia coli (n=2), Pseudomonas aeruginosa (n=7), Salmonella enterica subsp. enterica serotype Nottingham, Salmonella enterica subsp. enterica serotype Typhimurium & Stenotrophomonas maltophilia. Following incubation, growth was assessed semiquantitatively, as +++, ++, +, few or no growth. Results: ASA medium supported the prolific growth (+++) of all M. abscessus complex organisms, but inhibited all non NTM organisms from growing (no growth). Conclusions: ASA medium is highly selective, which allowed for the proliferation of all members of the Mycobacterium abscessus complex, but successfully inhibited all other bacterial flora in CF sputum. This medium shows potential in isolating M. abscessus complex organisms from polymicrobial specimens/environments.

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Aims & objectives: Bovine tuberculosis (BTB) and brucellosis are communicable diseases that require epidemiological surveillance in real-time as a key strategy to control and eradicate outbreaks. Thus, our purpose was to design an epidemiological tool to monitor BTB and brucellosis in Colombia. Methods: We collected the epidemiological data from the epidemiological bulletins of the Colombian Agricultural and Livestock Institute (ICA) between 2004-2019. Data analysis was conducted using multiple correspondence analysis (MCA) and a Chi-square test in Factominer and Factoextra packages executed in the R software (v3.6). We designed a user-friendly dashboard with epidemiological information using the ArcGIS platform coupled with the Esri's Map-Viewer tool. Results: Our final dataset included 529 BTB and 739 brucellosis cases reported in Colombia for 15 years. The variables location and case number for both diseases were significantly associated. Our tool's analysis showed that Cundinamarca and Valle del Cauca provinces have been epidemic areas for bovine BTB and brucellosis, respectively (p-value <0.001). Furthermore, increased case detection was reported in 2012 for bovine BTB and brucellosis (p-value <0.001) during 2019. We implemented a user-friendly dashboard for the mapping of both diseases during the observation period to focus on program control actions. Conclusions: We expect that our GIS-based tool will support disease control strategies, tracking disease hotspots, and identify trends over time to provide useful information for animal health authorities tailoring new strategies for control programs. Dashboard availability: https://arcg.is/1uvSLv

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Aims & objectives: The Venezuelan socio-political crisis has boosted massive exodus of Venezuelan citizens due to the shortage of medicines and spread of some preventable infectious diseases. We aimed to assess the impact of tuberculosis (TB) and HIV burden, health expenditure and the cost of illness under the framework of Colombian-Venezuelan migration flow focused on Norte de Santander, Santander, and La Guajira provinces. Methods: A retrospective study was conducted including TB and HIV data between 2009-2018. A database was made based on the records from the Colombian Surveillance System (SIVIGILA), official reports of the World Health Organization, Indexmundi, Global Health Observatory, IHME HIV Atlas and UNAIDS. Disability metrics in terms of DALYs (Disability Adjusted Life Years) and YLDs (Years Lived with Disability), were compared between both countries. Interactive maps were carried out by using ArcGIS program and the official migration data of Venezuelan citizens. We performed a phylogenetic analysis by retrieving pol sequences based in HIV cases from Venezuela and Colombia by using MEGA X and HIV Alamos database. Results: TB country profiles from Colombia and Venezuela were identical in terms of disease burden in 2017 and 2018. However, the Colombian public health settings reported an increase in the TB incidence above national average (22.1 cases per 100,000 inhabitants) in Santander, Norte de Santander and La Guajira provinces during the last years. Similar situation was observed regarding TB multidrug-resistant, prolonged hospital stays (150 days) and low-testing rates for cases of multidrug-resistant TB (67%) and HIV/AIDS (60%). We identified an underfunding for HIV/AIDS control programs and patient care. Our DALY analysis showed an increased disability in HIV/AIDS patients (362.35 for 2017). Moreover, our phylogenetic analysis shows three defined clusters, which indicate specific linages through specific areas, and shared cluster in the Colombo-Venezuela border. Conclusions: This study suggests that the massive migration and program underfunding in Venezuela might exacerbate the dual burden of TB and HIV in Colombia, especially towards the Colombo-Venezuelan border.

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Aims and objectives: Because the world is mobilized with all its might to fight the COVID-19 epidemic, control of common but deadly diseases such as tuberculosis may be neglected. With a conservative estimate and modeling working, we can show that if too much attention to the COVID-19 continuing it can leads to a 25% reduction in global TB diagnosis by 3 months and TB mortality will increase by 13% and this brings us back to the level of TB mortality we had 5 years ago. Between 2020 and 2025, 1.4 million TB deaths could be recorded as a direct consequence of COVID-19. Therefore, ensuring the continuation of basic services and operations to deal with long-standing health problems like tuberculosis or other similar diseases to protect the lives of people is very important. Methods: This study is a case series has been performed in three hospital centers in Tehran where respiratory and infectious patients were treated. We report on eleven cases of patients who were either unaware of their tuberculosis or of their COVID-19 and after visiting a medical center, they found out that they have both of them. Results: Of the eleven patients in this study, seven were male and four were female, with a mean age of 56.6 years (minimum age 27 years and maximum age 91 years). Five patients were previously diagnosed with tuberculosis and six patients were initially admitted with a diagnosis of COVID-19. Seven were Afghans and four were Iranians. Ten patients had respiratory tuberculosis and one had both pulmonary and spinal tuberculosis. Fever, productive cough, and hemoptysis were present in all of them. After clinical suspicion, spiral lung CT-scan, COVID-19 RT-PCR assay, smear and culture of sputum for mycobacterium tuberculosis, were performed and finally simultaneous tuberculosis and COVID-19 co-infection was diagnosed. Two patients died in the first week of treatment after being admitted to the intensive care unit. However, both patients also had diabetes mellitus and hypertension. Nine other cases were transferred to the tuberculosis unit after 7 to 10 days of treatment in the COVID-19 ward. Conclusions: Tuberculosis and COVID-19 are both common infections of the respiratory system and have common signs and symptoms and these similarities may cause overlapping the two diseases. While experience with COVID-19 infection in TB patients is still limited, it is predicted that people with TB and COVID-19 may have poorer treatment outcomes, especially if TB treatment is postponed.

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Aims & objectives: Mycobacterium tuberculosis is the etiological agent of tuberculosis (TB), which is one of the ten most deadly diseases and the first cause of death by bacterial origin around the world. One of the causes that increase the problem are the M. tuberculosis resistant strains to conventional antibiotics, multi-drug resistant tuberculosis (MDR-TB) and extensively-drug resistant tuberculosis (XDR-TB); so it is necessary to develop new drugs with different mechanisms of action. M. tuberculosis is characterized by having a highly hydrophobic cell membrane that includes P-type ATPases that are membrane proteins which have been studied as drug targets, because they are involved in several cellular functions that allow cell viability; so the development of drugs as inhibitors of ATPase activity in pathogens is an interesting strategy. Antimicrobial peptides are molecules generated in the host innate response with effects on mycobacteria. CAP-18 is a cathelicidin involved in the formation of channels in the cell membrane and in bacterial lysis. Methods: In this study, the effect on the mycobacterial cell membrane ATPase activity of CAP-18 derived peptide with unnatural amino acids was assessed. Using bioinformatic tools, the amino acid sequence of CAP-18 with the best features of helical structure and antibacterial activity was determined; the corresponding peptide had changes for D-Lys and N-methyl-Gly in the sequence, generating the peptides CAP18-BD and CAP18-CD and the peptoid CAP18-BP. Results: In vitro assesament of the peptides and peptidomimetics were tested in plasma membrame from Mycobacterium smegmatis mc²155. The inorganic phosphate released (Pi) was quantified during the ATP hydrolysis reaction by the action of ATPases, using the Fiske and Subarrow method modified by Cariani et al., in the presence and absence of the peptidic sequences to be evaluated, where the basal ATPase activity in the absence corresponds to zero percent inhibition of these enzymes, and 100% of the residual ATPase activity, understood as the enzymatic activity present with or without peptide treatment. Conclusions: The results showed inhibition of basal ATPase activity and Na⁺/K⁺, Cu(I), Zn (II) pumps in the plasma membrane, which is associated with its mechanism of action, which suggests that CAP18-BD, CAP18-CD and CAP18-BP can be considered as a potential antimycobacterial compounds targeting cell membrane ATPases.

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Aims & objectives: Tuberculosis (TB) is the infectious disease that results in the largest number of deaths around the world caused by a bacterial infectious agent. According to the World Health Organization (WHO), there were 10 million new cases and 1.2 million deaths caused by TB in 2018. The problem increases due to drug resistance and HIV/AIDS coinfection, which is why it is necessary to develop new anti-TB compounds with different mechanisms of action. Antimicrobial peptides are molecules gener42ated in the innate immune response of the host with an effect on pathogens. This is the case of the cathelicidins present in mammals involved in the formation of channels in the bacterial cell membrane that leads to cell lysis. Methods: In this study, the antimycobacterial activity and the possible mechanism of action of peptides derived from CAP-18 with unnatural amino acids were evaluated. Using bioinformatic tools, the epitope of the native sequence with the best characteristics of helical structure, amphipathicity and antibacterial value was determined. The corresponding peptides, CAP18-B and CAP18-C, had changes from L-Lys to D-Lys and from L-Gly to N-methyl-Gly in the sequence, generating the peptides CAP18-BD, CAP18-CD and the peptoid CAP18-BP. Peptides and peptoids were synthesized using the Fmoc strategy, purified by RP-HPLC and characterized by ESI-MS, circular dichroism and H¹-NMR. The in vitro assesament of the designed sequences was carried out in Mycobacterium smegmatis mc²155 and Results: Mycobacterium tuberculosis H37Ra, obtaining a minimum inhibitory concentration increase from 2 to 6 times in the ranges from 1200 to 60 μg/mL, less than 10% hemolytic activity and no cytotoxic activity on U937 monocytes with LC₅₀ values at least 10 times higher than the MIC and synergy combined with isoniazid. Confocal microscopy showed the insertion of peptides and peptoids in Mycobacterium smegmatis mc²155 and transmission electron microscopy showed alterations in the cell membrane of Mycobacterium smegmatis mc²155 evidenced in the formation of blisters, microspheres, channels and cell lysis. Conclusions: The results suggest that analogous sequences derived from CAP-18 can be considered as potential antimycobacterial compounds.

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Aims & Objective: We studied the transmissibility in mice of two candidate vaccine M. bovis strains, M. bovis Δmce2 and M. bovis Δmce2ΔphoP, attenuated by gene knock out. The parental strain M. bovis UK (NCTC10772) and a M. bovis wild boar isolate (Mb04-303) were inoculated as controls. Methods: Groups of animals including 3 non-inoculated + 3 subcutaneously inoculated with every strain (1.10⁵ CFU) were placed in the same cage (in duplicate). Necropsy was performed at 60 and 90 days post inoculation (dpi) in search of visible tuberculosis lesions (VTL) in lungs and spleens. Organs were analyzed to determine the bacterial load. Results: None of the animals presented VTL and no M. bovis was detectable in the non-inoculated group at 60 and 90 dpi. M. bovis was isolated from spleen of all the inoculated mice and from lungs of mice inoculated with control strains. The candidate vaccines were not isolated from lungs. Conclusions: This study shows the absence of transmissibility up to 90 days. Recovery of viable control strains from lungs and spleen suggests that the administration route would not alter the capacity of virulent strains to colonize organs. Contrasting, the candidate vaccines were only isolated from spleen. Longer periods should be assayed to evaluate transmissibility of these strains in mice.

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Aims &Objective: Mycobacterium bovis is the causative agent of bovine tuberculosis. It is also a pathogen for other animals such as goats and humans. Molecular methods are applied to detect members of the M. tuberculosis complex in different clinical samples as milk. The Loop-Mediated Isothermal Amplification (LAMP) proceeds at a constant temperature using a strand displacement reaction. LAMP does not require a thermal cycler. It is characterized by its efficiency, rapidity, high yield of final product, robustness, sensitivity and specificity. The primers are specifically designed to recognize 6 distinct regions on the target gene. The amplified product can be detected by naked eye as a white precipitate or a color change of the reaction solution.: To detect species of the Mycobacterium tuberculosis complex in goat milk samples by IS6110-LAMP Methods: Twenty goat milk samples from animals reacting to the tuberculin test were studied by IS6110-LAMP, Touch-Down (TD) IS6110 and Rv2807 PCR. These goats had been slaughtered, their organs and milk cultured. The isolates obtained were typed by spoligotyping. DNA extraction was performed using 500uL of milk sample. It was incubated with sodium dodecyl sulfate / proteinase K and treated twice with phenol:chloroform:isoamylic alcohol and once with chloroform:isoamylic alcohol. Then NaCl was added and DNA was precipitated with isopropanol and washed with ethanol. The remaining ethanol was evaporated and the pellet resuspended in water. IS6110-LAMP reaction was performed in a 25uL volume containing Bst DNA polymerase, betaine, magnesium sulfate, dNTPs, and primers (F3, B3, FIP, BIP, LF, LR). SYBR Gold solution was added inside of the cup of the tubes that were incubated at 63°C during one hour. After that the tubes were shacked and a yellowish color indicated a positive reaction. The specificity of IS6110-LAMP was evaluated using DNA from different species of Non Tuberculous Mycobacterias (NTM) (Mycobacterium smegmatis, M. phlei, M. fortuitum, M. gordonae, M. intracellulare, M. porcinum, M. avium subsp. avium, M. avium subsp. paratuberculosis) and the close related Nocardia sp. TD IS6110 and TDRv2807 PCR were also performed with an initial step of 96°C for 3 min, followed by 8 cycles of 96°C for 1 min, annealing temperatures starting at 72°C for 1 min (decreasing by 1 °C/cycle), and 72°C for 1 min for extension. This step was followed by 30 cycles of 96°C for 1 min, 65°C for 1 min, 72°C for 2 min, and a final extension at 72°C for 8 min. The amplification products were 245 and 443 bp, respectively. Results: While 17/20 goats were confirmed by bacteriology, no isolates were obtained from milk culture. The 17 isolates were further confirmed as M. bovis by spoligotyping. Neither NTM nor Nocordia sp. templates amplified by IS6110-LAMP. The 40 % (8/20) of the samples were IS6110-LAMP positive. There was only one discordant result between IS6110-LAMP, TD-Rv2807 and TD-IS6110. Conclusion: IS6110-LAMP is a useful tool to detect species of the M. tuberculosis complex in goat milk samples that could be implemented in laboratories with low technological complexity.

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Aims and objectives: Mycobacterium avium subspecies paratuberculosis was first diagnosed and reported in imported cattle from England during 1962-1965. Further studies have shown that paratuberculosis is scattered throughout Iranian ruminants population. In spite of heavy economic loses; little genetic information is available. So, present study was carried out in order to obtain a better understanding of the MAP genetic diversity in Iranian isolates. Methods: During a 7-month period, 36 isolates of MAP was collected from milk and feces obtained from sheep, goat and cattle originating 10 provinces plus a further eleven archived MAP isolates and two MAP vaccine strains (MAP 316F & III &V) and two D4 reference strains (Weybridge and Turkish) were included and digested by restriction endonucleases PstI. Digested DNA was hybridised with a standard IS900 probe. Genomic fingerprints were scanned by camera and analysed using the software Gel pro analyzer & Bionumerix software package version 6.5 (Applied Maths Belgium). Dendrogram and Minimum Spanning Tree was draw using arithmetic averages (UPGMA) for Iranian isolates and vaccine strains. Results: ten RFLP (Pst I) types were detected and designated as B (bovine, ovine and caprine), and G (bovine, and caprine) and eight new type (three of them singletons and five cluster) in accordance with the study by pavlik et al 1999. The largest cluster (G strain) contained 45.8% of Iranian isolates (n = 22) then type II (B strain), represented by 11 isolates, recovered from all the three animals in six provinces. A single type was identified only in cattle from three provinces. The identical RFLP type was shown by MAP vaccine strains were different from the Iranian isolates. Two D4 reference strains had restriction pattern but did not hybridize to IS900 sequence. Dendrogram result showed B profiles close to G profile and MAP vaccine strains profiles (>98). The results of Minimum Spanning Tree also showed that all of the clusters and orphan were derived directly or indirectly from B profile with multiple hosts throughout the country. This pattern has been seen from throughout the world and multiple hosts. Conclusions: RFLP analysis showed that the Iranian isolates have perhaps originated from B specific profile but it still need to more future works using genotyping methods.

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Aims and objectives: Fluoroquinolones (FQs) are potent antibiotics against multidrug-resistant Mycobacterium tuberculosis (MDR-TB) and resistance to FQs is associated with higher patient mortality. Rapid and accurate drug susceptibility testing (DST) against FQs and injectable drugs is essential for successful treatment of MDR-TB. FQ resistance mutation detection is equivalent to culture-based DST for predicting MDR-TB treatment outcome. This study compared performance of GenoTypeMTBDRsl Version 1 and Version 2 for detection of resistance to FQs and injectable drugs among MDR-TB strains in Kuwait, a country with low incidence of MDR-TB. Methods: MDR-TB strains (n=93) cultured from 74 pulmonary and 19 extra-pulmonary specimens from 93 patients were analyzed by GenoTypeMTBDRsl Version 1 and Version 2 for FQ and injectable drug resistance-associated mutation detection. Repeat isolates from 22 patients, 50 pansusceptible isolates and M. tuberculosis H₃₇Rv (as control) were also tested. Results were confirmed by PCR-sequencing of gyrA, gyrB, rrs, embB and eis genes. Fingerprinting of isolates was done by spoligotyping. Results: Among 93 MDR-TB strains, GenoTypeMTBDRsl Version 1 with/without PCR-sequencing identified 10 isolates with gyrA mutation (A90V, n=2; D94G, n=4; D94A, n=2; D94N, n=1; D94Y, n=1), seven isolates with rrs mutation (A1401G) and 51 isolates with an embB mutation. GenoTypeMTBDRsl Version 2 with/without PCR-sequencing confirmed Version 1 results for gyrA/rrs genes and additionally identified two isolates with gyrB mutation (T500N, n=1; N538D, n=1) and one isolate with eis mutation (-10 G/A). Repeat isolates yielded expected results. No mutation was detected in 50 pansusceptible isolates. Spoligotyping identified 10 Shared International Types among 12 isolates with gyrA/B mutation. Conclusions: Our data show that GenoTypeMTBDRsl Version 2 is superior to Version 1 as it identified two additional isolates with gyrB mutations. Improved detection of FQ resistance by GenoTypeMTBDRsl Version 2 will help in proper management of MDR-TB patients in low MDR-TB settings. Supported by KURS grant MI01/18.

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Aims and objectives: Diabetic patients are considered at increased risk of infections, including tuberculosis. The purpose of this work was to analyze the epidemiological, clinical, diagnostic, therapeutic characteristics, aggravating factors and evolution in patients with pulmonary tuberculosis and diabetes. Methods: This retrospective study included 42 patients with pulmonary tuberculosis and diabetes, followed in Farhat Hached health center of Sfax and whose bacteriological diagnosis was confirmed in the hygiene laboratory of Hedi Chaker university hospital between 2009 and 2015. Results: Diabetes affects 10.8% of patients with pulmonary tuberculosis. The mean age was 55.5 years and the sex ratio were 1.2. Diabetes occurs before tuberculosis in 88% of cases. The majority of patients had type 2 diabetes, of which 23.8% required insulin treatment. Anti-diabetic treatment was modified in 21.4% of cases due to poor glycemic control. The diagnosis of pulmonary tuberculosis was based on clinical, radiological, histological data and microbiological analysis. Cough was the most frequent symptom (88.1%). Pulmonary tuberculosis patients with diabetes were more likely to have pulmonary cavities (66.7%) and apical lesions (76.1%). Smear was positive in 85.7%. After 2 months of treatment, only 50% of the positive smears became negative. Tuberculosis treatment lasted more than 6 months in 66.7%. Most of patients had a favorable evolution (90.5%), 4.7% of cases had relapsed and one patient died. Smoking (52%), family history of pulmonary tuberculosis (47.6%) and high blood pressure (30.9%) were the most frequent risk factors in these patients. Conclusion: The bidirectional association between pulmonary tuberculosis and diabetes due to the modification of the clinical presentation and evolution under treatment makes patients more fragile requiring a better medical care.

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Aims and objectives: Tuberculosis (TB) remains a health public issue in Algeria. In spite of the great efforts made for controlling the disease, the number of new reported cases exceeds the twenty thousand cases per year. One worthy of note epidemiological characteristics of TB is its seasonality. The seasonality of TB has been explored in some countries but no study has been conducted in Algeria. The aim of this study is to find out the seasonal pattern and the trend of TB incidence in Algeria. Methods: The monthly TB notification data from January 2008 to December 2017 extracted from the National Health Tuberculosis Register were examined to find out the seasonal pattern and trend of TB in Algeria. The seasonal autoregressive integrated moving average (SARIMA) model was used in analysing and predicting TB cases from 2008 to 2016. Cases registered in 2017 were used to assess the prediction accuracy of the selected models. The metrics mean square error (MSE) and mean absolute error (MAE) were applied to assess the better performance of prediction between the selected models. Results: From January 2008 to December 2017 there were 215,581 TB notified cases in Algeria with a yearly average 21,467 cases leading to an average annualized morbidity rate of 56,7 per 100,000 inhabitants. The monthly mean was 1796.5, the maximum number (2499) of reported cases was registered in April 2015 and the minimum (1202) in November 2009. The monthly TB data are normally distributed. The incidence of notified pulmonary TB cases decreased from 24 cases per 100,000 inhabitants in 2008 to 15 cases per 100,000 inhabitants in 2017; thus registering a drop of 37.6%, whereas the incidence of notified extrapulmonary TB cases increased from 27.5 cases per 100,000 inhabitants in 2008 to 35.8 cases per 100,000 inhabitants in 2017; thus registering an increase of 29.9%. The heatmap of TB incidence per quarter per province showed a significant temporal and geospatial variation. Time series analysis revealed seasonality with peaks in spring and summer and troughs in autumn and winter and showed that a (3,1,6)×(1,0,1)₁₂ SARIMA model offered the best fit to the TB surveillance data for the period 2008-2016. This model was used to predict TB cases for the year 2017, and the fitted data showed considerable agreement with the actual data. Conclusions: Our findings are optimistic for forecasting TB by means of SARIMA models and afford useful information to public health policy makers in formulating targeted prevention and preparedness measures.

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Aims and objectives: Commissioning and transferring services to new facilities can be a challenge and an opportunity for enhancing services for mycobacteriology laboratories. Challenges are especially prohibitive in low resource settings; however these create an opportunity to develop low cost solutions for maintaining quality reporting while staggering facility based services for laboratories. Since mycobacterial cultures require longer incubation times, commissioning of new facilities and decommissioning older facilities requires careful and time sensitive planning so that services run uninterrupted. We share our experience of successfully transferring reference and internationally accredited mycobacteriology services to a new biosafety level 3 facility, and highlight the need for planning, verification, and decommissioning of older facilities in a challenging setting.

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Aims and objectives: Multidrug resistant TB (MDR-TB) and extensively drug resistant (XDR) are responsible for significant obstruction to global control of tuberculosis (TB). Drug resistance in Mycobacterium tuberculosis mainly occurs through chromosomal mutations in genes encoding for drug target or drug activating enzymes. However, alternative mechanisms, such as efflux pumps may also be responsible for drug resistance. Methods: Insight into the mechanisms of drug resistance would help in limiting the disease through the use and development of better diagnostic and therapeutic tools. The importance of rapid and effective methods of identification of drug resistance in M. tuberculosis cannot be over emphasized. Traditional laboratory techniques are time consuming and cumbersome and not sufficiently sensitive or specific to fight this menace. Results: Though rapid molecular methods such as GeneXpert and line probe assays are vital tools in the fight against drug resistant TB, major advances in next generation sequencing (NGS) technology are allowing increasingly rapid and accurate sequencing of entire bacterial genomes, providing unprecedented depth of information. Conclusion: NGS has the ability to cause a revolutionary paradigm shift in the diagnosis of drug resistant tuberculosis and in understanding the evolution of drug resistance. However, certain challenges need to be overcome to enable the use of this promising technology in routine diagnosis and research, especially in resource limited regions of the world.

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Aim and Objective: In this study, five genes of Mycobacterium tuberculosis were used as DNA vaccine. Each gene was cloned in pcDNA3.1 Topo and later in pND mammalian expression vectors. Methods: All the clones were tested through animal cell lines. In animal trial studies, endotoxin-free DNA were used to inoculate eight week old female Balb/c mice in quadriceps muscles with 50 μg DNA/leg and 25 μg DNA intradermally at the base of tail. No boosters were given. The animals were divided into five groups including positive and negative control groups. Eight animals were used for hspx-pND vaccine, eight for cfp10-pND vaccine, and eight for equally mixed ag85a, b and c pND vaccines. Four animals were used as positive control group injecting Np-pND constructs while four animals were used for negative control group by giving normal saline. The animals were bled after three weeks interval till nine weeks post-vaccination. The antibody titers were confirmed by Western blot analysis and multiplex microbead immunoassay (MMIA). Result: The best humoral response was observed by hspx-pND vaccinated group. Similar but quantitatively less positive response was observed in case of equally mixed ag85a, b and c-pND vaccine group, whereas, cfp10-pND vaccinated animals showed poor immune response. Conclusion: The study concludes that all M. tb gene constructs were good in expression under in vitro and in vivo conditions and produced significant level of antibodies except cfp10-pND construct. The study proved that subunit based DNA vaccines alone or in combination with each or with BCG, can provide breakthrough in vaccines against TB.

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Aims and Objectives: The aim of this retrospective chart review was to determine the clinical characteristics and treatment outcome of the patients diagnosed with Tuberculous Meningitis (TBM) at a tertiary care hospital setting. Methods: This was a cross sectional study included children of either gender aged 1-18 years treated for suspected or confirmed diagnosis of TBM at the pediatric unit of Aga Khan University Hospital. The patients enrolled from January 2015 to June 2020 were identified through electronic medical record system. The data was collected on a predesigned paper-based questionnaire and results are presented as mean, standard deviation and interquartile ranges for continuous variables. Whereas, the qualitative variables presented as frequency and percentages. Results: There were 52 children included in the study with a mean age of 10.66 ± 6.21 years. Of these 27 (52%) were males and 25 (48%) females. Most of the patients 33 (63.5%) presented within ≤14 days of the onset of symptoms. Median length of stay at hospital was 10 days. The median time from admission to initiation of anti-tuberculous treatment (ATT) was 5 days however, the ATT was initiated before admission to hospital in 11 (21%). In 19 (35%) of patients. Cerebrospinal fluid (CSF) culture was positive for TBM in 5 (9%) patients. GeneXpert test performed on CSF sample in 40 (77%) children. Treatment was completed in half of the patients, seven each lost to follow up and died during treatment. A significant number of patients 33 (63%) developed mild to moderate disability in the initial phase followed by full recovery. Conclusion: The findings revealed that TBM is highly associated with morbidity and disability. Early initiation of treatment and diagnosis likely to play pivot role in outcomes.

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Aim and objectives: Adolescence is characterised by a dramatic increase in the risk of developing tuberculosis, a fact that has been appreciated since the early 20th century. The majority of the world's adolescents live in low and middle-income countries where tuberculosis remains common. It is increasingly apparent that adolescents lie at the heart of the global tuberculosis epidemic, but they have not yet been addressed as a distinct population in tuberculosis policy or within tuberculosis treatment services. Methods: We will review the epidemiology of Mycobacterum tuberculosis (M.tb) infection among 10-24 year olds globally and in high tuberculosis burden countries. Results: We will discuss the management of M.tb infection and disease, including HIV co-infection and multidrug resistant tuberculosis. Conclusions: In addition, we will highlight special considerations regarding the tuberculosis continuum of care during adolescents.

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Aim and objectives: To describe presentation and treatment course of children with cystic fibrosis and pulmonary NTM disease at Aga khan University in last 5 years Methods: A retrospective chart review of children diagnosed with cystic fibrosis at the Aga Khan University, Karachi and NTM Pulmonary disease between January 2015 to Dec 2019 will be conducted. We will evaluate clinical and laboratory profile of these children and present descriptive demographic and treatment-related statistics. Results will be presented as median age at diagnosis (IQR), median weight for age z scores at presentation and after treatment, sputum/PFTs/radiological findings as proportions (percentages) and treatment outcomes (sputum clearance, improvement in weight and PFTs, death). Conclusions: We will present clinical profiles of children with CF and NTM managed at Aga Khan University in the last 5 years and highlight challenges encountered in diagnosis and management of NTM in this population

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Aims and objectives: All public-private mix (PPM) facilities caring for tuberculosis (TB) patients in Lahore city, Pakistan, under four models: PPM1 (general practitioners), PPM2 (non-governmental organizations), PPM3 (private hospitals) and PPM4 (others). To assess the pre-treatment loss to follow-up (LTFU), defined as patients documented in the laboratory registers but not in the treatment registers of any PPM facility, among sputum smear-positive TB patients diagnosed during January–March 2015, and unfavorable treatment outcomes among patients registered for treatment and associated factors. Methods: This was a retrospective cohort study reviewing existing program records. Poisson regression was used to identify factors associated with outcomes. Data collection, variables and sources: The data were extracted from the TB laboratory registers and TB treatment registers maintained in the PPM facilities. The variables included type of PPM model, laboratory serial number, patient's name, age, sex, date of diagnosis and treatment start, smear grade, type of TB and treatment outcome. The data were collected using a structured proforma. Two electronic databases were created. Results: The two databases were then electronically matched for Results: Of 2473 patients diagnosed, 1590 (64%) were lost to follow-up before treatment. This was higher among males (68%) and the elderly (79%), and lower among 'high positives' (smear grading 2+ or 3+, 53%) and in the PPM1 model (34%). Of 883 patients started on treatment, 165 (19%) had unfavorable outcomes: 8% LTFU, 5% treatment failure, 3% died and 3% not evaluated. Previously treated patients (34%) and children (44%) had the worst outcomes. Conclusion: Pre-treatment LTFU was alarmingly high and requires urgent attention, including the development and institution of mechanisms for patient tracking using information and mobile phone technology, and making TB notification mandatory in the private sector.

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Aims and objectives: Mycobacterium bovis (M. bovis) is the causative agent of bovine tuberculosis; however, it also causes infection in a wide range of hosts including human beings. Johne's disease (JD) is characterized by chronic granulomatous enteritis predominantly observed in ruminants, caused by Mycobacterium avium subsp. paratuberculosis (MAP). Autophagy is an innate immune defense mechanism for the control of intracellular bacteria, regulated by diverse signaling pathways. It plays a key role in the innate immune defense mechanism for controlling intracellular mycobacterium. Nilotinib, a tyrosine kinase inhibitor (TKI), has been studied extensively in various tumor models; however, no information exists about the pharmacological action of nilotinib in bacterial infections. We hypothesized to investigate the effect of nilotinib on the regulation of host innate immune responses against Mycobacterial infection. In addition, to devise best possible therapeutic remedy towards achieving the goal of mycobacterial diseases treatment based on molecular mechanism both in vitro and in vivo, which may provide useful strategy to treat animal's tuberculosis. Methods: In the current study, we conducted both in vitro and in vivo experiments to investigate the role of nilotinib in the regulation of autophagy. Western blot, confocal microscopy, electron microscopy and immunohistochemistry were performed to investigate the induction of autophagy upon nilotinib treatment during M. bovis infection. Gross and histological analysis were conducted to observe the effect of nilotinib on the development of lung's lesions in mice infected with M. bovis. CFU assay was performed to determine the effect of nilotinib on the intracellular growth of M. bovis. Results: We found that nilotinib improved the host immune responses by inhibiting intracellular survival and growth of both M. bovis and MAP. Nilotinib also attenuated PI3k/Akt/mTOR axis for the regulation of autophagic degradation of intracellular mycobacterium mediated by inhibition of Abelson (c-ABL) tyrosine kinase. In addition, nilotinib promoted ubiquitination of intracellular bacteria through activation of E3 ubiquitin ligase parkin. Similarly, lung tissue sections of nilotinib treated mice also showed high intensity of parkin protein post M. bovis infection. From in-vivo experiment, we found that nilotinib effectively controlled M. bovis growth and the severity of disease in infected mice. Conclusions: Altogether, our data shows that nilotinib regulates protective host innate immune responses against intracellular pathogen both in-vitro and in-vivo and can be exploited as a novel host-directed therapeutic agent in the control of M. bovis and MAP infections.

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Aims and objectives: The control of pulmonary tuberculosis (PTB) mainly depends upon prompt diagnosis and effective treatment which can be hindered by co-infections affecting organs like gastrointestinal system and liver involved in drug bioavailability and metabolism. Among those, Helicobacter pylori (H. pylori) causes chronic gastritis leading to peptic ulcer and related gastric malignancies and liver malfunctions induced by Hepatitis C virus (HCV) co-infections increase the risk of anti-tuberculosis drug-induced hepatotoxicity. The rates of Hepatitis C virus and Helicobacter pylori infections seem to increase day by day in pulmonary tuberculosis patients which need proper investigation followed by timely control and prevention form disease. Methods: The hospital-based study was performed in the area of Southern Punjab, Pakistan. The Tehsil Headquarter hospital in the area, where the study was conducted contained TB Clinic. Inclusion and exclusion criteria of suspected patients of pulmonary tuberculosis included in the study. A total of 132 subjects were screened for PTB, HCV and H. pylori infections for finding the role of co-infection. The positive PTB subjects were screened for HCV and H. pylori infections. Both infections (H. pylori and HCV) were also found among TB patients. Results: A total of 52.27% were positive for PTB in which 50.72% and 17.39% cases were found in H. pylori and HCV infections respectively. H. pylori had increased rate of infection among PTB cases when compared with that of non-TB cases. HCV was also found to be associated with PTB when compared with non-TB cases. Non-significant gender and age-based differences were present, among study population. Among all PTB patients, HCV and H. pylori coinfections were also found in 5.8% patients in current study. Conclusions: Our study showed a high prevalence of HCV and H. pylori infections among PTB patients, which suggest that PTB patients must be screened for the two infections which might be helpful in the control of further complications during the treatment of PTB.

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The genus Mycobacterium comprises a broad spectrum of obligate microorganisms most notably the M. tuberculosis complex bacteria; potentially-pathogenic or opportunistic species gathered under the common name of M. avium-intracellulare complex, and finally saprophytic mycobacteria. In cattle farming, Mycobacterium bovis (M. bovis) is considered as the principle cause for bovine tuberculosis (bTB). Nevertheless, non-tuberculosis mycobacteria (NTM) are potential pathogens that are increasingly reported to interfere with contemporary intradermal tuberculin tests in veterinary medicine. Therefore, these neglected mycobacteria are capable to make hurdles for bTB diagnosis. In the Iranian environment, the extent and importance of this phenomenon in regard with the public health and economical aspects is just underestimated. This work was therefore intended to isolate and characterize the mycobacterial organisms potentially involved in causing positive reactions in tuberculination of Iranian cattle. Abattoir meat inspection and laboratory microbiological investigation of slaughtered heifers from a 2,000-head Simental cattle farm with 10% of the herd positive tuberculin reactors, failed to provide any evidence of M. bovis infection. No mycobacteria was detected in bacterial culture of specimens from feedstuff, water or even the visiting feral rodents of the farm. When the farm workers were included in assessments, 22 individuals were found tuberculin-positive with 5 of them also smear-positive in their sputa assessment. Molecular identification of the five isolates from workers through sequencing of the 16SrRNA proved their identity as Mycobacterium thermoresistibile (Mth). Removing of the carrier workers from the farm vicinity, successfully ended the issue of the constantly-positive tuberculin test in the herd with no return of the problem until now. We are convinced further investigations are essential in search for interference extent and importance of NTM species in cattle tuberculination scheme in Iran.

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Aims and objects: Bovine tuberculosis caused by Mycobacterium bovis is an important disease that has negative effects in public health and economic losses. Traditional controlling program in cattle is based on test and slaughter strategy, but false positive is disadvantage of tuberculin skin test. To overcome the limitation, there is a need of using proteins with high specificity. The objective of this study was to clone and express of virulent protein CFP-10 and ESAT-6 of Mycobacterium bovis AN5 for diagnosis of bovine tuberculosis. Methods: Briefly, full-length genes of cfp-10 and esat-6 were amplified by PCR technique. In parallel pET23a (+) and PCR product has been double digested by EcoRI and HindIII. After Ligation, transformation was done into competent E.coli DH5α. Then the cloned vector was transformed into E.coli BL21. Induction was performed by isopropyl-β-D-thiogalactopyranoside (IPTG). The expressed proteins almost entirely in the inclusion body form. To dissolution of expressed protein, urea 8M was used. Purification of recombinant proteins was done by Nickle-Resin. Urea eliminated by decreasing gradient. Delayed hyper sensitivity of two proteins beside bovine tuberculin, avian tuberculin and human tuberculin studied in guinea-pig model. Results: Protein expression was confirmed by Western Blotting (WB) using 6X His-tag antibodies. The results showed that CFP-10 and ESAT-6 molecules has been successfully cloned and expressed in prokaryotic system. Results showed that CFP-10 has satisfactory relative potency but it is not in range about ESAT-6. Conclusion: The recombinant CFP-10 can play a significant role in reducing the false positive in diagnosis of bovine tuberculosis by improving the specificity of the tuberculin skin test

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Aims and objectives: Tuberculosis (TB) remains a major challenge to public health. The disease burden caused by TB is falling globally, in all WHO regions, and in most countries, but not fast enough to reach the first, 2020, milestones of the End TB Strategy. By 2020, the TB incidence rate needs to be falling at 4–5% per year, and the proportion of people with Tuberculosis who die from the disease needs to fall to 10%. These deaths are in part due to late or missed diagnosis and could be prevented with appropriate treatment. The aim of this study was to identify new relevant cytoplasmic and conserved hypothetical protein biomarkers as TB diagnostic or therapeutic targets. Methods: TB Protein contents were prepared by detergent phase separation method. Two dimensional gel electrophoresis (2DE) was performed using the Ettan IPGphor 3 isoelecteric focusing (GE Healthcare). Comprehensive genomic and proteomic data were retrieved from MycoBrowser portal (www.mycobrowser.epfl.ch) followed by matrix assisted laser desorption ionization time-of flight Mass Spectrometry. The predictive of the MHC I peptide binding is carried out by the IEDB analysis resource (www.iedb.org). Results: At least six purified proteins with assigned putative functions were determined that corresponded to aldehyde dehydrogenase (Rv0147), iron regulated H-NS (Rv3597c), translocase SecE2 (Rv0379), methyltransferase (Rv3699), adenosylmethionin (Rv1392) and conserved hypothetical protein of Rv0443. Within the category of cytoplasm, membrane and conserved hypothetical patterns, six identified proteins were shown high expression during exponential growth in middlebrook 7H9 broth media. The MHC I peptide binding predictions of these protein spots on the SDS page gels were valued by the Immune Epitope Database tools. The IEDB-AR prediction result is given based on IC50nM value and percentile grad. Principally, a lower score, specified higher affinity. Peptides with IC50 estimation < 50 nM are regarded as high affinity. Conclusions: The output scores of the predicting peptides have been shown strong binding for each of identified peptides. Rank of the predicted output affinity of Rv0443 compared to the two others, Rv0147 and Rv0379, presumably leads to stronger binding affinity. The conserved hypothetical proteins, Rv3699 and Rv0443 displayed significant homology with several other TB proteins. These proteins can also be included in TB vaccine constructs or subunit vaccine mixtures combined with a potent adjuvant. We considered these protein profiles to represent reputed biomarker as a new TB vaccine or therapeutic strategies against TB.

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Aims and Objectives: In the present study we tried to improve anti-TB immunity offered by 2^nd generation rBCG30 vaccine. We attempted to bolster rBCG30 invoked anti-TB immunity in mice through modulation of the IL-10/STAT3 interaction mediated anti-inflammatory program. The study was aimed towards generating optimum activated APCs, early either after vaccination (with rBCG30) or infection (with Mtb), which can process and present mycobacterial antigens more efficiently to T cells. It was envisage that downmodulating IL-10/STAT3 signaling may improve immunity and protection offered by rBCG30 vaccine. Methods: A small molecule immunomodulator aimed at disrupting anti-inflammatory signaling orchestrated by IL-10/STAT3 axis was administered in rBCG30 immunized mice. The comparative expansion of pathogen permissive Alternatively Activated Monocytes/Macrophages/DCs (AAMs/AADCs) and Classically Activated Monocytes/Macrophages/DCs (CAMs/CADCs), the cells that resist establishment of successful Mtb infection, was profiled in both peritoneal and splenic compartment. To assess innate and adaptive T cell responses, splenocytes cytokine production was checked ex vivo using sandwich ELISA. We also checked polyfunctional T cell response, T cell memory response and comparative Th17/Treg cells expansion using flow cytometry. Finally, to assess the real time efficacy of the employed immunotherapeutic strategy, in vivo protection study was undertaken in BALB/c mice challenge with aerosolized Mtb (H₃₇Rv). Results: The small molecule mediated inhibition of IL-10/STAT3 signaling was resulted in improved pro inflammatory immune responses in both post-vaccination and post-infection immunotherapy schedules. The improved immune profile in rBCG30 vaccinated and immunotherapy administered mice was accompanied with enhanced expansion of CAMs/CADCs, elevated production of polyfunctional T cells producing signature Th1 cytokines, long lasting CD4+ and CD8+ memory T cell response and balanced Th17/Treg cells ratio. Interestingly, post-infection, and not post-vaccination immunotherapy, was able to significantly reduce mycobacterial loads in spleen and lungs of experimentally infected mice. Conclusions: Small molecule mediated transient disruption of anti-inflammatory effectors, such as IL-10/STAT3, during vaccination or post infection with Mtb, may serve to enhance the efficacy of existing and forthcoming TB vaccines. The strategy can also be tested concomitantly with standard chemotherapy (either in vaccinated or unvaccinated host) to enhance the efficacy of therapeutic regimen and to reduce the treatment length.

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Aims and objectives: In the present study, we report on the cross-border clusters involving Italian MDR-TB strains collected in the framework of the ECDC supported EUSeqMyTB project. This pilot study was conducted as retrospective/prospectic WGS-based surveillance project to monitor possible cross-border TB transmission involving strains isolated in Europe. Methods: Within the EUSeqMyTB study period (approximately 24 months) 2218 RR/MDR-TB strains from 25 EU countries were collected, including a total of 127 strains from Italy. Additional RR/MDR-TB strains were collected and sequenced in Italy as part of the national WGS-based surveillance. All strains were subjected to WGS according to standard protocol for the Ion torrent or Illumina platform. Sequencing of Italian strains was performed by Illumina. WGS data were subjected to quality check (See Tagliani et al ERJ 2020) and analysis was performed by SNP based pipeline and Core Genome MSLT (SeqSphere+ Redom GmbH, Germany) Results: The Euroamerican superlineage was the most represented in Italy (64% of cases). The Italian strains were included in the 3 major cross-border clusters identified in the study. The largest cluster comprised a total of 30 patients from 3 different countries, including 22 individuals born in the same European country. Italy contributed to this cluster with 12 cases isolated during the study period and with additional two cases after the end of the study in 2019. The clustered strains isolated in Italy were from both Italian born and foreign born TB cases, the majority of which were from another EU country. For the majority of the clustered cases isolated in Italy the epidemiological link was identified and the cluster was linked to a pub located in Milan, Italy. A subgroup of the MDR-TB cases showed additional resistance to 2^nd line anti TB drugs as well as resistance to bedaquiline (linked to a large deletion in the rv0678 gene). One clustered case showed also a minimum inhibitory concentration for delamanid close to the drug critical concentration. For the additional two large cross-border clusters identified in the EUSeqMyTB pilot study, Italy contributed with respectively 2 and 5 cases. All of them were linked to migration from sub-Saharan Africa countries and no case of transmission to Italian born population was identified neither during the study timeframe nor afterwards. Conclusions: We documented that WGS-based surveillance of RR/MDR-TB strains highly support contact tracing investigation. The implementation of WGS in routine should require the performance of WGS as soon as the strain become available and not in batches with delay. Information exchange with neighboring countries should be facilitate. We report on an ongoing transmission of a pre-XDR strain linked to a larger EU cluster and that the transmitted strain is highly resistant to therapy with the current MDR recommended treatment.

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Aims and objectives: Mycobacterium tuberculosis (M. tuberculosis) is one of the major threatening infectious diseases and public health in the world. Spoligotyping is a method for r genotyping assay and molecular epidemiology of M. tuberculosis strains. The aim of this study was to determine the molecular epidemiology of M. tuberculosis by spoligotype isolated from patients with tuberculosis at Kurdistan province, Iran. Methods: a total of 47 M. tuberculosis isolates were collected from TB patient at Kurdistan province, Iran. All sample cultures on Lowenstein-Jensen medium and were incubated at 37 °C and were kept for 8 weeks. Bacterial isolates were identified as M. tuberculosis using standard biochemical tests. Multi-drug resistance (MDR) were determined by using proportional method. Spoligotyping was performed. Results: Spoligotyping of 47 strains created 8 patterns. Out of 47 samples, 23 (48.94%) were new Spoligotype International Type (SIT). In preexisting shared pattern STI 2669 were highest degree 23.40% that belong to Ural family of M. tuberculosis spoligotypes. Frequency of MDR were 6.38%. Resistance to Isoniazid and Rifampin were 14.89% and 10.64%, respectively. Conclusion: In overall, half of isolated strains from patients showed new patterns that, indicated the dynamic transmission of tuberculosis. Therefore, highlights dominance of differ pattern of tuberculosis distribution in west Iran. The low frequency of resistance is very interested.

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Aims and objectives: Tuberculosis (TB) remain a leading cause of death globally among among infectious diseases that has killed more number of people than any other infectious diseases. It was Robert Koch who recognized the spectrum of pathology of tuberculosis (TB) in different animal species. Methods: The examination of clinical specimens from infected humans and animals confirmed the variable patterns of pathological reactions in different animal species. Guinea pigs are innately susceptible while humans, mice and rabbits show different level of resistance depending upon their genotype. The studies of TB in laboratory animals like mice, rabbits and guinea pigs have significantly increased the understanding of the aetiology,viurulence, and pathogenesis of the disease. By introducing less than five virulence organisms into guinea pigs by the respiratory route can produce lung lesions, bacteraemia and fatal diseases, which has helped the extrapolation of results of such experiments to humans beings. The similarities in the course of course of clinical infection between guinea pigs and humans allow us to model different models of TB and to evaluate the protective efficacy of candidate in such systems. The only limitation of this model is a dearth of immunogical reagents required for the qualitative and quantitative evaluation of the immune responses, special reference to cytokines and cell phenotypes. Further limitation is the higher cost of the guinea pigs as compared with the mice. Results: The rabbit is relatively resistant to M TB infection, however following infection with virulent Mycobacterium bovis, the rabbit produces pulmonary cavities like humans. The rabbit model, however, is also limited by the lack of immunological reagents. Mice are the animal choice of studying the immunology of Mycobacterial infections and contributed much to our current understanding of the roles of various immunological mechanisms of resistance. The resistance of mice to the development of classic TB disease, however, represents a significant disadvantage to the mouse model. Although non-human primates are closely related to humans, owing to high cost and handing difficulties they have not been exploited to a large extent. Conclusions: As all existing animal models fall to mimic the human disease perfectly, efforts should be focused on the development of the non-human primate (s) as the alternative animal model for TB.

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Aims and objectives: Tuberculosis (TB) is a leading infectious cause of mortality worldwide and millions of people continue to develop active TB each year. Mycobacterium bovis (M. bovis) is a member of Mycobacterium tuberculosis (Mtb) complex causing bovine tuberculosis and imposing high zoonotic threat to human health. Kallikreins (KLKs) belong to a subgroup of secreted serine proteases and their role is established in various physiological and pathological processes. Since the KLKs expression has been linked to various cancerous, degenerative and infectious conditions, it is likely that KLKs expression may mediate host immune response against M. bovis infection. Methods: In the current study, we investigated the expression of KLK12 in M. bovis infection in vivo and in vitro by using C57BL/6 mice and two types of murine macrophages, RAW264.7 cells and bone marrow derived macrophages (BMDMs). In this study, we used two strains of M. bovis, M. bovis C68004 strain and M. bovis N strain. To define the role of KLK12 in immune response regulation of murine macrophages, we produced KLK12 knockdown BMDMs by using siRNA transfection. We investigated the expression of various proteins through QRT-PCR, Immunoblot and ELISA. Apoptotic cells were detected by flow cytometry. Furthermore, cell viability and phagocytic ability of macrophages was also determined after transfection and infection. CFU assay was performed to know the intracellular survival of M. bovis. Results: Our results exhibited an increase in the expression of KLK12 in the lung and spleen of M. bovis infected mice. A dose (MOI) and time dependent up-regulation was also noticed in RAW264.7 and BMDMs. Then, we evaluated the role of KLK12 in innate immune response regulation against M. bovis. Interestingly, knockdown of KLK12 resulted into significant down regulation of autophagy and apoptosis in M. bovis infected BMDMs. Furthermore, we demonstrated that this KLK12 mediated regulation of autophagy and apoptosis involves mTOR/AMPK/TSC2 and BAX/Bcl-2/Cytochrome c/Caspase 3 pathways, respectively. Similarly, inflammatory cytokines IL-1β, IL-6, IL-12 and TNF-α were also significantly down regulated in KLK12 knockdown macrophages but difference in IL-10 and IFN-β expression was non-significant. Our finding also depicted that knockdown of KLK12 enhances the intracellular survival of M. bovis. Conclusion: Taken together, these findings suggest that up-regulation of KLK12 in M. bovis infected murine macrophages plays a substantial role in the protective immune response regulation by modulating autophagy, apoptosis and pro-inflammatory pathways. To our knowledge, this is the first report on expression and the role of KLK12 in M. bovis infection and the data may contribute to a new paradigm for diagnosis and treatment of bovine and human TB.

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Aims and objectives: Niger is a challenging environment, with limited resources for TB control. Health staff are scarce, access to care is limited due to long distances between communities and health facilities and security threats. The Short Treatment Regimen (STR) has been implemented nationwide for rifampicin resistant tuberculosis (RR-TB), since 2008. No previous publication has shown the results from countrywide programmatic implementation using few exclusion criteria. The National Tuberculosis Programme and the Damien Foundation conducted a retrospective observational study to evaluate the management of RR-TB from 2008 to 2017. Methods: Sputum specimens were collected before treatment initiation and monthly for smear and culture during treatment. Follow-up after cure continued up to one year after cure with smear and culture performed every 6 months. Individuals with confirmed RR-TB were treated in the three national Units. Initially, the high-dose gatifloxacin-based Bangladesh regimen was used. In October 2013 high-dose gatifloxacin was replaced by normal-dose moxifloxacin when gatifloxacin became unavailable on the market. From October 2015, a modified STR, with linezolid replacing kanamycin during the intensive phase was used in patients with hearing loss on audiometry. Systematic clinical and laboratory monitoring of patients on treatment was introduced in 2008. From 2008 to September 2013, hearing loss was monitored clinically and with audiometry at the end of the intensive phase. Thereafter, bimonthly audiometry has been carried out in all patients given SLIs. Results: Among 1,550 patients tested for rifampicin resistance, mainly previously treated patients, 411 (26.5%) were diagnosed with pulmonary RR-TB. The proportion of retreatment cases tested for RR-TB increased from 11.2% in 2008 to 60.5% in 2017. Of the 411 patients with confirmed RR-TB, 359 (87.3%) were enrolled on treatment and 324 (90.3%) started on STR. 24 patients received a modified STR mainly because of hearing loss at baseline or during treatment. Among 300 patients on standardised STR, 250 (83.3%) (IC95%: 78.7-87.1) were cured relapse-free, ten (3.3%) had failure, 28 (9.3%) died, eight (2.7%) were lost to follow-up and four (1.3%) relapsed. Among the 14 patients with bacteriological unfavourable outcome (failure and relapse), nine had initial resistance to fluoroquinolones (FQ) and five had susceptible strains. Among 251 patients with initial susceptibility to FQ there were 4 (1.6%) acquisition of resistance. Of 24 patients receiving a modified STR, 21 (87.5%) were cured relapse-free. Serious ototoxicity was reported in 6 of 120 patients (5%) with clinical monitoring, audiometry at the end of the intensive phase and switch from kanamycin to capreomycin and in 2 of 74 patients (2.8%) with bimonthly audiometry and reduction of kanamycin dose from daily to thrice a week. In 130 patients with bimonthly audiometry and switch from kanamycin to linezolid, serious hearing loss was detected in 2 (1.5%) patients. Since 2016 onwards no serious hearing loss was recorded in Niger. Conclusions: A comprehensive nationwide approach to multidrug-resistant tuberculosis management using the STR was feasible and successful. Hearing loss was manageable replacing the injectable with linezolid. Our study confirms the effectiveness and safety of the STR.

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Aims and objectives: The standardized short treatment regimen (STR) is implemented in Niger to treat multidrug-resistant tuberculosis (MDR-TB) since 2008 with excellent outcomes. However, ototoxicity secondary to injectable is a concern and data on modified oral STR are limited. The aim of this study is to test if linezolid can replace the injectable. Both drugs have a similar mechanism of action and the use of linezolid in replacement of SLI may preserve bedaquiline as core-drug in a salvage regimen to treat possible failures and relapses. Linezolid is used only in the intensive phase to reduce the risk of peripheral neuropathy and optic neuritis. Methods: A prospective longitudinal study was conducted on a modified STR replacing kanamycin with linezolid (600 mg/d) in case of hearing loss (grade 1) at baseline or during treatment. WHO definitions were used for outcomes and adverse events were assessed according to French National Agency for Research on AIDS. Audiometry was performed bimonthly from 2016 to 2017 and monthly from 2018 onwards. Results: A total of 173 patients were treated with the STR for pulmonary MDR-TB from 2016 to June 2018. Among them, 22 (12.7%) had a modified STR, switching from kanamycin to linezolid during treatment (14) or starting with linezolid (8) because of hearing loss. Fourteen (63.6%) were males, 2 (9.1%) were HIV positive, the median age was 37.5 years (range 21-71) and the median BMI was 16.5 kg/m² (range 13-24.2). No patients had strains resistant to fluoroquinolones and/or injectables. The median time from treatment start to switch kanamycin to linezolid was 2 months (range 0-3) and the median duration of treatment with linezolid was 2 months (range 1-5). All patients had smear and culture conversion with a median and range of 1 month (1-6) and 2 months (2-4) respectively. Nineteen patients (86.4%) were cured and 3 died of respiratory failure (2) and severe immunosuppression (1). Eighteen patients were assessed for follow-up 6 months and 12 were assessed 12 months after cure. All remained smear and culture negative. Severe adverse events were anaemia in 5 cases (22.7%) with a median onset of 1 month (range 1-3) and thrombocytopenia in 1 case (4.5%). Linezolid was temporarily discontinued and these patients received blood transfusions. The drug was reintroduced at reduced dosage (300 mg) with no further problems. Seven patients (31.8%) had a reversible mild to moderate peripheral neuropathy which was reversible at the end of treatment. No patients had optic neuritis. From 2016 onwards no serious hearing loss secondary to SLI use was detected nationwide in Niger. Conclusions: A modified STR with linezolid may achieve high cure rates with manageable adverse events.

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Aims and objectives: The prevalence of pulmonary tuberculosis as well as Non Tuberculous Mycobacteria (NTM), has been on the increase worldwide. In order to improve the control of the disease, genotyping and drug susceptibility of Mycobacterium tuberculosis complex (MTBC) are important. This study sought to determine drug susceptibility and genetic diversity of MTBC as well as NTM in three West/Central African countries. Methods: A collection of 503 isolates (158 from Cameroon, 202 from Nigeria and 143 from Ghana) obtained from culture using either Lowenstein–Jensen medium, Middlebrook 7H9 Broth and/or Xpert MTB/RIF were included in the study and some isolates tested for drug susceptibility using the proportion method, BACTEC MGIT 960 or Line Probe Assay (LPA). Isolates were further subjected to IS6110 typing, large sequence polymorphisms, and spoligotyping. All IS6110-negative strains suspected as NTM were tested using hsp65 gene amplification, DNA sequencing and blast analysis. Results: The Cameroon family was the most prevalent genetic strain [128/202 (63.4%) in Nigeria, 54/150 (36%) in Cameroon and 27/119 (22.7%) in Ghana] and SIT 61 the largest cluster with 111/202 (55%) and 43/150 (27%) isolates in Nigeria and Cameroon respectively. Multidrug resistance (MDR) was observed in 29/202 (14.4%) cases in Nigeria, and HIV positivity [37/202 (18.3%) patients] was associated with rifampicin 9/37 (24.3%) resistance (p=0.012), as well as gender (p=0.009). In Cameroon, 13/147 (8.8 %) were resistant to rifampicin (RIF), with 6 (46.2%) of the 13 RIF resistant isolates associated with UgandaI sublineage (p=0.0007). Other sublineages such as Haarlem, Ghana, West African 1 were recorded in the three countries. NTMs isolates included M. colombiense, M. engbaekii, M. intracellulare strain, M. gordonae, M. fortuitum, M. paraense and Nocardia veteran. Conclusion: Neglected NTM timely diagnosis should be reinforced in the study area. Drug resistance in TB/HIV co-infected patients and its association with a sublineage calls for a surveillance strategy to curb its expansion in the west/central Africa.

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Aims and objectives: Tuberculosis (TB) caused by Mycobacterium tuberculosis complex remains a leading cause of morbidity and mortality worldwide. The zoonotic infectious condition represents a never-ending challenge towards which drug discovery efforts are needed. The current study was designed to evaluate the in vitro antimycobacterial activity of ethanolic extracts from roots, stem bark, leaves and unripe fruits derived from Solanum torvum, a shrub traditionally used against respiratory tract illnesses, including tuberculosis. Methods: The phenotypic colorimetric micro plate alamar blue assay (MABA) was used to study the antimycobacterial activity of the ethanolic extracts against six mycobacterial strains. Each experiment was run in triplicate. Data generated was analyzed using descriptive statistics to obtain mean minimum inhibitory concentration values. Results: The roots, stem bark, leaves and unripe fruits exhibited minimum inhibitory concentration values of 1.250 mg/mL, 0.078 mg/mL, 1.250 mg/mL and 0.625 mg/mL against the pathogenic mycobacterial strain, M. tuberculosis H37Rv (ATCC 27294) respectively. Conclusions: In conclusion, Solanum torvum stem bark has demonstrated moderate activity against the pathogenic Mycobacterium tuberculosis strain. This observation validates the ethno pharmacological use of the plant species against tuberculosis. Further studies are required to isolate, elucidate the structure and characterize the antimycobacterial compounds responsible for the observed activity. These will potentially contribute towards bioprospecting for a new class of ligands with activity against sensitive and drug resistant strains of M. tuberculosis.

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Aims and objectives: Tuberculosis (TB) worldwide re-emergence constitutes a major public health problem with multi-drug resistant (MDR) strains becoming a greater threat. Genotyping and drug susceptibility of Mycobacterium tuberculosis complex (MTBC) are important in order to improve the control of the disease. This study sought to determine drug susceptibility and genetic diversity of MTBC in three West/Central African countries, namely Cameroon, Nigeria and Ghana located in the Gulf of Guinea. Methods: A collection of 498 isolates (150 from Cameroon, 202 from Nigeria and 146 from Ghana) obtained from culture using Lowenstein–Jensen medium, Middlebrook 7H9 Broth and/or Xpert MTB/RIF and Line Probe Assay (LPA) were included in the study and some isolates tested for drug susceptibility using the proportion method. Isolates were further subjected to IS6110 typing, region of difference 1, 4, 9, 12, 702, 711, spoligotyping and hsp65 gene sequencing analysis. Results: The LAM10_CAM was the most prevalent genetic family [128/202 (63.4%) in Nigeria, 46/123 (37.4%) in Ghana and 54/150 (36%) in Cameroon] and SIT 61 the largest cluster with 111/202 (55%) and 43/150 (27%) isolates in Nigeria and Cameroon respectively. Multidrug resistance (MDR) was observed in 29/202 (14.4%) cases in Nigeria, and HIV positivity [37/202 (18.3%) patients] was associated with rifampicin 9/37 (24.3%) resistance (p=0.012), as well as gender (p=0.009). In Cameroon, 13/147 (8.8 %) were resistant to rifampicin (RIF), with 6 (46.2%) of the 13 RIF resistant isolates associated with UgandaI sub-lineage (p=0.0007). Other sublineages such as Haarlem, Ghana, West African 1 were recorded in the three countries. Conclusion: Rifampicin resistance in TB/HIV co-infected patient and its association with a sublineage calls for a reinforced surveillance strategy to curb drug resistance development in the Gulf of Guinea.

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Aims and objectives: In 2016, Guinea had an estimated notification rate of 177 new tuberculosis (TB) cases per 100.000 population, with 360 estimated-number of rifampicin-resistant (RR) TB cases. In 2014, Damien Foundation and the National Tuberculosis Programme (NTP) of Guinea started a biomedical-social-support to people treated by multidrug-resistant TB (MDR-TB) in one-pilot health-facility. The aim of this study is to analysis effectiveness of biomedical-social-support on MDR-TB-care. Methods: All MDR-TB-cases treated during 2016 to 2017 were analysed. Treatment-outcomes were compared according to the provision of biomedical-social-support in one pilot-health-facility to two-health-facilities without it. In biomedical-social-support, all biological-tests, ancillary drugs were provided free of charge and a nutritional-kit and transport-refunds were monthly provided during the whole treatment. Treatment regimen included 20-month treatment regimen with Kanamycin (Km), Levofloxacin (Lfx), Cycloserine (Cs), Pyrazinamide (Z) and Prothionamide (Pto) during 6-month in the intensive-phase, followed by 12-18-month of same drugs but Km. Results: We included 75 MDR-TB cases, 7(9%) HIV-positive. Mean-age was 26years (IQR 15-49). All cases were pulmonary-TB, from which 10(13%) were new-cases. There were 27 MDR-TB cases with biomedical-social support and 48 without it. Mean delay of treatment-start in days was 20(IQR 9-110) in the pilot health-facility compared to 34(IQR 9-111). Treatment outcomes in the group with biomedical-social support were: cured 22(82%), treatment-completion 0(0%), death 2(7%), failure 1(4%) and 2(7%) lost-to-follow-up compared to those without biomedical-social support 23(48%), 2(4%), 9(19%), 2(4%) and 12(25%) respectively. Treatment success to unfavourable- outcomes (failure, death and lost-to-follow-up) in the pilot health-facility was 82% and 18% respectively compared to 52% and 48% respectively in those health-facilities without biomedical-social support (p<0.01). Conclusions: The introduction of biomedical-social support to people affected by MDR-TB was successful in Guinea. People who benefited from this strategy had more favourable treatment-outcomes. The biomedical-social support could improve treatment-success if extended to all MDR-TB people under treatment.

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Aims and objectives: The purpose of our study was to examine the efficacy and safety of intravenous chemotherapy during intensive treatment phase in patients with newly diagnosed pulmonary tuberculosis (pulmonary TB). Methods: The study involved 92 patients with newly diagnosed pulmonary TB aged between 20 and 68. All patients with newly diagnosed pulmonary TB and chemosensitive tuberculosis were enrolled in the study. The patients were allocated to two groups. The first (control) group of 46 patients received standard chemotherapy orally. The second (main) group consisted of 46 patients who were prescribed isoniazid, rifampin, ethambutol by i.v. infusion, and pyrazinamide orally as a part of the standard treatment. Results: Symptoms of intoxication and chest manifestations in pulmonary TB patients from the second group were eliminated faster than the same symptoms in the group 1. In the group 2, the mycobacterial clearance in sputum smears was achieved more rapidly, and up to 2 months it was reached in 37 patients (80.43%), while in the control group in 25 patients (54.35%),p = 0.0066. Destruction healing and inflitrative change alleviation after 4 months was reached in 38 patients (82.61%) (in control group — 28 (60.87%), (p = 0.0192). No additional negative effects were detected when compared with the control group at any time. Conclusions: Thanks to i.v. chemotherapy, clinical manifestations of the in-patients with pulmonary TB were eliminated faster, severe side effects of anti-TB drugs were not noticed, time of bacterial clearance and healing of destruction was shorter, healing frequency of destructions increased and the of residual changes decreased.

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Aims and objectives: In the present study, we report on the first seven cases of acquired bedaquiline and clofazimine resistance identified in Pakistan. The study was conducted within the framework of a retrospective surveillance project to monitor the acquisition of resistance to bedaquiline implemented by the National TB Reference Laboratory in Pakistan and TB Supranational Reference Laboratory. Methods: Seventy three sequential isolates from 31 drug-resistant-tuberculosis patients on bedaquiline-containing regimens were retrospectively tested for bedaquiline resistance by MIC testing in 7H11 medium, and for all isolates showing an increased MIC compared to the baseline isolate, further MIC testing for bedaquiline and clofazimine was conducted using the Bactec MGIT960 (BD, Franklin Lakes, NJ, USA). The H37Rv (ATCC 27294) strain was used as a susceptible control in MIC testing. Bedaquiline dry powder was supplied by Janssen-Pharmaceutica (Beerse, Belgium). WGS was carried out with the Illumina Nextera-XT DNA sample preparation kit to prepare paired-end libraries of 150-bp read length to sequence on an Illumina NextSeq platform. Data analysis and single nucleotide polymorphism (SNP) calling were performed using the MTBseq-Pipeline on low-frequency detection mode. Genes associated with resistance to bedaquiline and/or clofazimine (atpE, Rv0678, pepQ, and Rv1979c) were screened for mutations. Results: We studied 73 isolates from patients (n=31; 8 MDR, 16 pre-XDR, and 7 XDR) enrolled in bedaquiline-containing regimens. The sequential isolates tested included two strains from 22 patients, three from 7 patients, and four from 2 patients. All baseline strains included in the study were sensitive to bedaquiline. Seven patients developed an increase in bedaquiline MICs in 7H11 medium (range, 0.125 to >0.5mg·liter⁻¹) during therapy, and six of them became resistant to bedaquiline according to the current critical concentration proposed for the drug. In all seven patients we detected genetic variants in Rv0678 that were not present at baseline isolates. Moreover in five out of seven patients the treatment was ultimately failed. Conclusions: We documented cases failing therapy that developed specific mutations in Rv0678 and had increased MICs associated with cross-resistance to clofazimine during treatment. This study underlines the relevance of surveillance programs following the introduction of new drugs.

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Aims and objectives: of our study was to investigate the effectiveness of intravenously (i/v) isoniazid (H) and ethambutol (E) administration in patients with new sputum positive drug-susceptible pulmonary tuberculosis (TB) with tuberculous meningoencephalitis (TM) and human immunodeficiency virus (HIV) co-infection in the intensive phase of treatment. Methods: Fifty-four patients with TB / TM and HIV co-infection were enrolled in this study. Group 1 included 23 patients treated with E and H i/v, while rifampicin and pyrazinamide were prescribed orally. Group 2 consisted of 31 patients treated with the first-line anti-TB drugs orally. Concentrations of H and E in blood serum were detected using a chromatographic method. Results: A significant improvement in clinical symptoms and X-ray signs in patients treated i/v with H and E was observed compared with group 2. Sputum Mycobacterium tuberculosis positivity was observed during the second month of treatment in 25.0 % of patients from group 1 and 76.1% of patients from the control group (p<0.01). In addition, 9 (39.1%) patients died up to six months when H and E were prescribed i/v compared with 22 (70.9%) in group 2 (p=0.02). Conclusions: In TB / TM with HIV, i/v H and E treatment was more effective than oral H and E treatment at 2 months of intensive treatment in sputum conversion as well as in clinical improvement, accompanied by significantly higher mean serum concentrations. In addition, the mortality rate was lower in i/v H and E treatment than oral.

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Aims & Objective: Post TB sequelae like hemoptysis and recurrent chest infections are a source of lot of morbidity around the globe. Many of these patients present to thoracic surgeons and surgical intervention is able to offer significant relief in many of these patients. Methods: Retrospective analysis was carried out for cases operated upon for post TB seqalae in the period from March 1997 to December 2017 in the Thoracic Surgery Department of the National Institute of Tuberculosis and Respiratory Diseases, New Delhi, India. A total of 1325 cases of post TB sequelae were taken up for surgery Out of these, those operated upon primarily for troublesome hemoptysis were 1154 cases. 171 cases were taken up for surgery because they had for recurrent chest infections. Out of these, 558 had tubercular cavities or destroyed lungs, 213 had aspergilloma and post tubercular bronchiectasis was present in 554 cases. Procedures performed were pneumonectomy in 417 patients, lobectomy in 732, bilobectomy in 98, segmental and wedge resections in 16 and thoracoplasty in 62 cases. Results: A total of 60 deaths occurred in this series. There were 18 early and 42 late deaths. 7 patients died of hemorrhage, 4 of respiratory failure, 3 sudden deaths in postoperative period (possible cause-Cardiac arrhythmia or cardiac herniation) and 4 of sepsis. Bronchopleural fistula developed postoperatively in 88 cases, which was managed by tube thoracostomy, followed by thoracoplasty, if required. Other complications like wound infection, atelectasis and pneumonitis were quite manageable. Control of hemoptysis was achieved in majority of cases. Minor re-bleeds occurred in 37 cases. Massive or recurrent troublesome re-bleed took place in 8 cases, which required completion pneumonectomy. Relief of symptoms was achieved in all of the remaining cases. Conclusion: Overall quite gratifying results are achieved if surgical intervention is offered in patients suffering from debilitating post TB symptoms in carefully selected and well prepared cases.

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Aims and objectives: Due to the growing burden of coronavirus 2019 and considerable rate of RT-PCR false-negative results, a complementary diagnostic tool is necessary for the detection of active cases. Methods: This study was performed in Masih Daneshvari Hospital, Tehran, Iran, among admitted symptomatic cases clinically and radiologically suspected to COVID-19 and their first RT-PCR assay was negative. Results of second RT-PCR assay and serologic study were compared. Results: In the period of study 137 symptomatic patients with negative RT-PCR assay on the first sample were recruited. Second RT-PCR assay was positive for 45 (32.8%) of them. IgM and IgG antibodies against SARS-CoV-2 were positive in 83 (60.6%) and 86 (62.8%) of patients respectively. Finally, the serologic assay was diagnostic among 73% of patients and the yield of serology was higher after the first week of illness (p-value <0.001). The combination of RT-PCR and serology increased the diagnostic yield to 83.9%. Conclusions: This study showed that the serologic assay in parallel to RT-PCR may be useful as a complementary tool for diagnosis of COVID-19 among admitted cases.

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Aims and objectives: Mycobacterium tuberculosis (Mtb) is a bacterium that causes tuberculosis (TB) in humans. tuberculoprotein are a widely used for detection of primary infection with Mtb. The production of tuberculoprotein is accompanied by some difficulties that require a series of modifications in the production and purification processes. In this study, we aimed to compare two methods for precipitation Mycobacterium tuberculosis tuberculoprotein and investigated its protein profiles. Methods: Mtb C strain grown in Löwenstein-Jensen and Dorset Henley liquid medium. Bacteria were inactivated by subjecting to 68°C for 90 min. After inactivation the culture was filtered and tuberculoprotein were precipitated by ammonium sulfate (AS) and Trichloroacetic acid (TCA). Protein concentrations were determined by using the Lowry protein assay. In finally, the approximate molecular weights in the purified fractions were determined by sodium dodecyl sulfate (SDS)-PAGE. Results: The results showed that concentration of extracted tuberculoprotein by AS precipitation method was lower than TCA method. SDS-PAGE pattern of proteins precipitation with TCA showed proteins with wide range of molecular weight but proteins precipitation with AS showed low molecular weight proteins. Conclusions: Finally the results of this study showed that optimum methods besides a novel approach of AS precipitation in production of tuberculoprotein are suitable and applicable.

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Aims and objectives: There is an emergence need to identifying Mycobacterial proteins of value to diagnostic assays and vaccine formulations. Glycosylation is an important post-translational modification of proteins and affects more than half of all the proteins in a cell. O-linked glycosylation refers to the event in which a carbohydrate is covalently linked to the hydroxyl group of serine or threonine. This modification influences a number of properties of proteins including proteolytic resistance, solubility, immunological properties and ligand binding. Host cells can bind to Mycobacterial cell wall carbohydrates via a class of surface localized or secreted proteins known as lectins, and these interactions strongly contribute to bacterial adhesion and uptake, and are also associated with the capability of Mtb to survive. This study represents the attempt to predict o-glycosylation pathways of immunogenic TB proteins as potential therapeutic strategies against TB. Methods: A Mycobacterium tuberculosis culture filtrate enriched with mannose-containing proteins that were purified by conA-Affinity chromatography. One and two dimensional gel electrophoresis carried out by standard methods. Gel staining procedures are based on the periodic Acid-Schiff reaction. The Protein spots of interest were excised from the gel and then analysis by MALDI Mass Spectrometry. GlycoPP software, Version 1.0, was used for glycosylation prediction and protein sequences. Results: Protein bands or spots were excised from the gel and analyzed by mass spectrometry. These proteins have O-glycosite residue and a glycan attached covalently and enzymatically at amide or hydroxyl group which includes Rv0475, Rv3367, Rv3699, Rv0251c, Rv2558 and Rv1284. Rv0475 has 17 Potential O-Linked Glycosylated Sites but 6 sites of Potential Glycosylated. Rv3367 has 67 Potential O-Linked Glycosylated Sites and 55 sites of Potential Glycosylated. Additionally, Rv3699 has 26 Potential O-Linked Glycosylated Sites and 10 sites Potential Glycosylated. Conclusions: This study may be shown the role of glycoprotein's in Mycobacterium pathogenesis, disease development, and its implications in drug development. All of these proteins have several putative antigenic epitopes and induces an immune response especially production of antibodies. In addition, Rv3367as a member of the Mycobacterium tuberculosis PE family and PGRS subfamily, gly-rich proteins have carbohydrate moieties and also are known to play a role in several pathological processes. Because of the importance of M.tuberculosis immunogenic antigens, the availability of the secretion and glycosylation of MTB proteins is a tool that will allow a deeper understanding of role these proteins play in the pathogenesis of disease and provide the basis for more sensitive and discriminative clinical diagnostic tests and improving to formulate new Mycobacterial vaccines.

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Aims and objectives: Molecular techniques have considerable advantages for rapid detection, a reduction of infectiousness, prevention of further resistance development and surveillance of drug-resistant TB. MTBDRsl VER 2.0 was used to detect resistance to second-line anti-tuberculosis drugs on rifampicin-resistant M. tuberculosis (RR-MTB) isolates compared to the minimum inhibitory concentrations (MICs) and whole genome sequencing (WGS). Methods: The MTBDRsl VER 2.0 (Hain Life Science, Nehren, Germany) and WGS (San Diego, CA, USA) was performed for tracing mutations in resistant-related genes involved in resistance to fluoroquinolone (FLQs) and second-line injectable drugs (SLIDs). The broth microdilution method using 7H9 Middlebrook media supplemented with OADC was used to determine the MICs. Results: The MTBDRsl VER 2.0 correctly detected 5/6 (83.3%) of FLQ-resistant strains having gyrA mutations. The MUT1 A1401G (7 strains) and MUT2 G1484T (1 strain) mutations in rrs gene were detected in 8 AMK/KAN/CAP-resistant strains. Four low-level KAN-resistant strains with the G-10A/C-12T (3 strains) and C-14T (1 strain) mutations in eis gene was diagnosed using MTBDRsl VER 2.0. Conclusions: Five error results were found in detecting resistance to kanamycin and capreomycin compared to the phenotypic drug susceptibility testing and WGS. Failing wild type-bands without improved mutant-bands did not indicate a reliable resistance. WGS could efficiently resolve the discrepancies of the results. MTBDRsl showed better performance in detecting extensively drug-resistant strains.

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Aims and objectives: The current study has evaluated the MICs and MBCs of ZnONPs, MgONPs, and MgONPs-ZnONPs against H₃₇Rv Mtb and MDR-Mtb. Methods: Mixture, magnesium oxide nanoparticles (NPs) and zinc oxide (MgONPs-ZnONPs) were prepared. The microplate alamar blue (MABA) assay and the proportion method were used to evaluate of anti-tubercular activity against MDR-MTB. MTT test was done to MgONPs-ZnONPs against Vero and HepG2 cell lines. Results: The MIC of MgONPs and ZnONPs were 0.195 and 0.468 μg.ml^-1 against 10⁴ of H₃₇Rv Mtb. As well, 0.166 μg.ml^-1 of MgONPs-ZnONPs was able to inhibit 10^-4 H₃₇Rv Mtb. The MIC of MgONPs against 10⁴ concentrations of MDR-Mtb was 12.5 μg.ml^-1. The MIC of MgONPs/ZnONPs against 10⁴ concentrations of MDR-Mtb reached to 0.664 μg.ml^-1. The MBC value of ZnONPs increased to 1.875 μg.ml^-1 against 10^-4 concentrations of MDR-Mtb. Testing showed that the MBCs of MgONPs/ZnONPs reached to 1.328 μg.ml^-1 against 10⁴ concentrations of MDR-Mtb. The IC₅₀ against MDR-TB was 0.779 μg.ml^-1 for ZnONPs and 0.883 μg.ml^-1 for MgONPs-ZnONPs. The MgONPs-ZnONPs was not toxic to Vero cell lines however ZnONPs could inhibit the Vero and HepG₂ cell lines. Conclusion: We found that ZnONPs and mixture MgONPs-ZnONPs not only have higher bactericide behavior but might have also synergistic effects against MDR-TB.

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