bluuebunny/crossref_metadata_2025_split huggingface dataset:
In-depth analysis of multi- and extensively drug-resistant mycobacterium tuberculosis isolates in the capital of iran
Authors: Vaziri Farzam 
Published: January 2021

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.
Description of a novel selective medium (abscessus selective agar; asa) for the isolation of mycobacterium abscessus complex
Authors: Moore John E , Millar Beverley C 
Published: January 2021

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 30o C. 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.
Tuberculosis and COVID-19 Co-Infection
Authors: Rezaei Mitra , Babamahmoodi Abdolreza , Marjani Majid 
Published: January 2021

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.

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.
Tuberculosis and diabetes
Authors: chema Marouane , sana kammoun , mariem Frikha , salma Hachicha , asma Ghorbel , salma Smaoui , neila Zalila , férièle Messadi Akrout 
Published: January 2021

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.
DNA vaccine against tuberculosis by using five mycobacterium specific genes
Authors: Shahzad Mirza Imran 
Published: January 2021

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.

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.
Treatment Outcomes of Non-tuberculous mycobacteriosis (NTM) in children with Cystic Fibrosis
Authors: Mir F , Nathwani AA , Shakoor S 
Published: January 2021

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
Cloning and Expression of virulent proteins CFP-10 and ESAT-6 of Mycobacterium bovis AN5 for diagnosis of bovine tuberculosis
Authors: Reza Arefpajoohi , Tagi Zahraei Salehi , Nader Mosavari , Ramak Yahya raeyat 
Published: January 2021

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
A Potent Inhibitor of IL-10/STAT3 Axis Signaling Modulates Anti-Inflammatory Responses and Boosts Anti-Tuberculosis Immunity in rBCG30 Immunized Mice
Authors: Ahmad Faraz , Umar Mohd Saad , Khan Nazoora , Gupta Pushpa , Gupta Umesh Datta , Owais Mohammad 
Published: January 2021

Aims and Objectives:
In the present study we tried to improve anti-TB immunity offered by 2nd 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 (H37 Rv).

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.
Use of whole genome sequencing to identify transmission of resistant tuberculosis strains in italy
Authors: Tagliani Elisa , Borroni Emanuele , Cirillo Daniela Maria 
Published: January 2021

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 2nd 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.
Animal Models of Tuberculosis
Authors: Gupta U. D. , Gupta Pushpa 
Published: January 2021

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.