Use and Quality of Blood Cultures for the Diagnosis of Bloodstream Infections: A Cross-Sectional Study in the Ho Teaching Hospital, Ghana, 2019-2021.

Blood Culture and Drug Susceptibility Testing (CDST) remains vital for the diagnosis and management of bloodstream infections (BSIs). While the Ghana National Standard Treatment Guidelines require CDST to be performed in each case of suspected or clinically diagnosed BSI, these are poorly adhered to in the Ho Teaching Hospital (HTH). This study used secondary medical and laboratory records to describe blood CDST requests by clinicians and the quality of CDST processes for the diagnosis of BSI among patients admitted to HTH from 2019 to 2021. Of 4278 patients, 33% were infants. Pneumonia and neonatal sepsis cases were 40% and 22%, respectively. Only 8% (351/4278) had blood CDST requested. Of 94% (329/351) blood CDST processed and reported, only 7% (22/329) were culture-positive, with likely contaminants being recovered from 16% (52/329) of the specimens. The duration from admission to request was 2 days (IQR: 0-5), and Further qualitative studies must be conducted to understand the reasons for low blood CDST utilisation among clinicians and the patient outcomes. Targeted interventions are required to enhance the utilisation of blood CDST by clinicians and the quality of laboratory processes.

Phenotypic and genotypic drug susceptibility patterns of Mycobacterium tuberculosis isolates from pulmonary tuberculosis patients in Central and Southern Ethiopia.

INTRODUCTION: The persistence of tuberculosis (TB) infection in some patients after treatment has highlighted the importance of drug susceptibility testing (DST). This study aimed to determine the drug susceptibility patterns of Mycobacterium tuberculosis (M. tuberculosis) isolates from pulmonary TB (PTB) patients in Central and Southern Ethiopia. METHODS: A health institution-based cross-sectional study was conducted between July 2021 and April 2022. Sputum samples were collected from newly diagnosed smear microscopy and/or Xpert MTB/RIF-positive PTB patients. The samples were processed and cultivated in Lowenstein-Jensen (LJ) pyruvate and glycerol medium. M. tuberculosis isolates were identified using polymerase chain reaction (PCR) based region of difference 9 (RD9) deletion typing. Phenotypic DST patterns of the isolates were characterized using the BACTEC MGIT™ 960 instrument with SIRE kit. Isoniazid (INH) and Rifampicin (RIF) resistant M. tuberculosis isolates were identified using the GenoType® MTBDRplus assay. RESULTS: Sputum samples were collected from 350 PTB patients, 315 (90%) of which were culture-positive, and phenotypic and genotypic DST were determined for 266 and 261 isolates, respectively. Due to invalid results and missing data, 6% (16/266) of the isolates were excluded, while 94% (250/266) were included in the paired analysis. According to the findings, 14.4% (36/250) of the isolates tested positive for resistance to at least one anti-TB drug. Gene mutations were observed only in the rpoB and katG gene loci, indicating RIF and high-level INH resistance. The GenoType® MTBDRplus assay has a sensitivity of 42% and a specificity of 100% in detecting INH-resistant M. tuberculosis isolates, with a kappa value of 0.56 (95%CI: 0.36-0.76) compared to the BACTEC MGIT™ DST. The overall discordance between the two methods was 5.6% (14/250) for INH alone and 0% for RIF resistance and MDR-TB (resistance to both INH and RIF) detection. CONCLUSION: This study reveals a higher prevalence of phenotypic and genotypic discordant INH-resistant M. tuberculosis isolates in the study area. The use of whole-genome sequencing (WGS) is essential for gaining a comprehensive understanding of these discrepancies within INH-resistant M. tuberculosis strains.

Molecular Markers of Early Immune Response in Tuberculosis: Prospects of Application in Predictive Medicine.

Tuberculosis (TB) remains an important public health problem and one of the leading causes of death. Individuals with latent tuberculosis infection (LTBI) have an increased risk of developing active TB. The problem of the diagnosis of the various stages of TB and the identification of infected patients in the early stages has not yet been solved. The existing tests (the tuberculin skin test and the interferon-gamma release assay) are useful to distinguish between active and latent infections. But these tests cannot be used to predict the development of active TB in individuals with LTBI. The purpose of this review was to analyze the extant data of the interaction of M. tuberculosis with immune cells and identify molecular predictive markers and markers of the early stages of TB. An analysis of more than 90 sources from the literature allowed us to determine various subpopulations of immune cells involved in the pathogenesis of TB, namely, macrophages, dendritic cells, B lymphocytes, T helper cells, cytotoxic T lymphocytes, and NK cells. The key molecular markers of the immune response to M. tuberculosis are cytokines (IL-1ß, IL-6, IL-8, IL-10, IL-12, IL-17, IL-22b, IFNÉ£, TNFa, and TGFß), matrix metalloproteinases (MMP-1, MMP-3, and MMP-9), and their inhibitors (TIMP-1, TIMP-2, TIMP-3, and TIMP-4). It is supposed that these molecules could be used as biomarkers to characterize different stages of TB infection, to evaluate the effectiveness of its treatment, and as targets of pharmacotherapy.

Revolutionizing control strategies against Mycobacterium tuberculosis infection through selected targeting of lipid metabolism.

Lipid species play a critical role in the growth and virulence expression of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB). During Mtb infection, foamy macrophages accumulate lipids in granulomas, providing metabolic adaptation and survival strategies for Mtb against multiple stresses. Host-derived lipid species, including triacylglycerol and cholesterol, can also contribute to the development of drug-tolerant Mtb, leading to reduced efficacy of antibiotics targeting the bacterial cell wall or transcription. Transcriptional and metabolic analyses indicate that lipid metabolism-associated factors of Mtb are highly regulated by antibiotics and ultimately affect treatment outcomes. Despite the well-known association between major antibiotics and lipid metabolites in TB treatment, a comprehensive understanding of how altered lipid metabolites in both host and Mtb influence treatment outcomes in a drug-specific manner is necessary to overcome drug tolerance. The current review explores the controversies and correlations between lipids and drug efficacy in various Mtb infection models and proposes novel approaches to enhance the efficacy of anti-TB drugs. Moreover, the review provides insights into the efficacious control of Mtb infection by elucidating the impact of lipids on drug efficacy. This review aims to improve the effectiveness of current anti-TB drugs and facilitate the development of innovative therapeutic strategies against Mtb infection by making reverse use of Mtb-favoring lipid species.

Modeling a UVC irradiation standalone system for inactivating Mycobacterium tuberculosis from indoor spaces.

After the outbreak of the COVID-19 pandemic, a rise in demand has occurred for efficient designs of disinfection systems that utilize ultraviolet-C (UVC) radiation to inactivate airborne microorganisms effectively. This paper proposes what we believe to be a novel standalone system for inactivating Mycobacterium tuberculosis (which requires a higher dosage value than SARS-CoV-2) from a medium size room of 12.5f t×12.5f t×9f t. The structure consists of a UVC source at the center and a spiral pathway guiding the air around the UVC source, thus increasing the residence time of the aerosol particle. The top and bottom louvre and a hollow cylindrical cover (comprising four external cover segments) enclose the UVC source and prevent the danger of direct exposure to indoor occupants. The whole system is modeled in SolidWorks, and flux leakage was examined using the RayViz tool in SolidWorks. Optical/radiometric analysis in ray tracing software TracePro provided the UVC flux value at different locations of the standalone system. Flow simulation carried out in SolidWorks helped calculate aerosol particles' residence time at different airflow trajectories. The designed standalone system demonstrated the capability of delivering 1.87 times more dosage than is required to inactivate Mycobacterium tuberculosis from the ambient air. The standalone system achieves a ventilation rate, i.e., air changes per hour value of 10, according to guidelines from the Council of Scientific & Industrial Research, India.

[Effects of rapid drug sensitivity testing for multidrug-resistant bacteria on the prognosis of patients with severe intra-abdominal infection].

Objective: To examine the clinical value of rapid detection of drug-resistant bacteria by immunochromatography and the effects of rapid detection on the prognosis of patients with severe intra-abdominal infection complicated by carbapenem-resistant Enterobacteriaceae (CRE) bloodstream infection. Methods: This was a retrospective cohort study. We analyzed clinical data of 73 patients with severe abdominal infections with sepsis or septic shock complicated by CRE bloodstream infection admitted to the general surgery department of Jinling Hospital between February 2022 and February 2023. Patients were divided into a colloidal gold immunochromatographic assay (GICA) group (17 patients) and conventional testing group (56 patients) based on whether a GICA for CRE had been performed on the patients' first blood culture sample during the diagnosis and treatment process. There were no statistically significant differences between the GICA and conventional testing groups in age ([55.9±17.3] vs. [47.6±16.4] years), sex ([16 men vs. one woman ] vs. [41 men vs. 15 women]), median Charlson comorbidity index (3.0[2.0,4.0] vs. 3.0[2.0, 4.8]), septic shock (10 vs. 39), or acute kidney injury (8 vs. 40) (all P>0.05). Both groups routinely underwent traditional bacterial identification and drug susceptibility testing. Additionally, patients in the GICA group were tested directly for positive blood cultures using a GICA carbapenemase test kit. The main outcomes were mortality rates on Days 28 and 90 after the first identification of CRE bloodstream infection in both groups. We also compared the microbial clearance rate, duration of hospitalization and intensive care unit stay, and time from onset of CRE bloodstream infection to initiation of targeted and appropriate antibiotics between the two groups. Results: The rate of microbial clearance of bloodstream infection was significantly greater in the GICA group than in the conventional testing group (15/17 vs. 34/56 [60.7%], χ2=4.476, P=0.034), whereas the 28-day mortality tended to be lower in the GICA than conventional testing group [5/17 vs. 44.6% [25/56], χ2=1.250, P=0.264). The 90-day mortality (8/17 vs. 53.6% [30/56], χ2=0.222, P=0.638), median duration of hospitalization (37.0 [18.0, 46.5] days vs. 45.5 [32.2, 64.8] days, Z=-1.867, P=0.062), and median duration of intensive care unit stay (18.0 [6.5, 35.0] days vs. 32.0 [5.0, 51.8] days, Z=-1.251, P=0.209). The median time between the onset of bloodstream infection and administration of antibiotics was 49.0 (38.0, 69.0) hours in the GICA group, which is significantly shorter than the 163.0 (111.8, 190.0) hours in the conventional testing group (Z=-5.731, P<0.001). The median time between the onset of bloodstream infection and administration of appropriate antibiotics was 40.0 (34.0, 80.0) hours in the GICA group, which is shorter than in the conventional testing group (68.0 [38.2, 118.8]) hours; however, this difference is not statistically significant (Z=-1.686, P=0.093). Conclusions: GICA can provide information on carbapenemase- producing pathogens faster than traditional drug sensitivity testing, enabling early administration of the optimal antibiotics. The strategy of 'carbapenemase detection first' for managing bacterial infection has the potential to improve prognosis of patients and reduce mortality rate.

Mycobacterium tuberculosis PE/PPE proteins enhance the production of reactive oxygen species and formation of neutrophil extracellular traps.

Introduction: Neutrophil granulocytes predominate in the lungs of patients infected with Mycobacterium tuberculosis (Mtb) in earlier stages of the disease. During infection, neutrophils release neutrophil extracellular traps (NETs), an antimicrobial mechanism by which a DNA-backbone spiked with antimicrobial components traps the mycobacteria. However, the specific mycobacterial factors driving NET formation remain unclear. Proteins from the proline-glutamic acid (PE)/proline-proline-glutamic acid (PPE) family are critical to Mtb pathophysiology and virulence. Methods: Here, we investigated NET induction by PE18, PPE26, and PE31 in primary human blood-derived neutrophils. Neutrophils were stimulated with the respective proteins for 3h, and NET formation was subsequently assessed using confocal fluorescence microscopy. Intracellular ROS levels and cell necrosis were estimated by flow cytometry. Additionally, the influence of phorbol-12-myristate-13-acetate (PMA), a known NADPH oxidase enhancer, on NET formation was examined. Neutrophil integrity following incubation with the PE/PPE proteins was evaluated using transmission electron microscopy. Results: For the first time, we report that stimulation of primary human blood-derived neutrophils with Mtb proteins PE18, PPE26, and PE31 resulted in the formation of NETs, which correlated with an increase in intracellular ROS levels. Notably, the presence of PMA further amplified this effect. Following incubation with the PE/PPE proteins, neutrophils were found to remain viable and structurally intact, as verified through transmission electron microscopy, indicating the occurrence of vital NET formation. Discussion: These findings offer valuable insights that contribute to a better understanding of host-pathogen interactions during Mtb infection. Moreover, they underscore the significance of these particular Mtb antigens in triggering NET formation, representing a distinctive and previously unrecognized function of PE/PPE antigens.

Pyrrole-thiazolidinone hybrids as a new structural class of broad-spectrum anti-infectives.

A series of pyrrole-thiazolidinone hybrids was designed, synthesized and evaluated for activities against ESKAP bacteria panel and mycobacterial pathogens. From the series, compound 9d showed prominent activity against S. aureus (MIC = 0.5 µg/mL) and compound 9k showed the most promising activity against M. tuberculosis H37Rv (MIC = 0.5 µg/mL). Potent derivatives were found to be non-toxic when tested against Vero cells. Compound 9d upon evaluation in vitro against several MRSA and VRSA strains produced activity comparable or better than standard drugs. In the anti-biofilm assay, 9d reduced S. aureus biofilm by >11% at 10x MIC. The dual inhibitory effect exhibited by pyrrole-thiazolidinone hybrids confirms their potential as new class of promising anti-infective agents.

Exploring the role of the protein tyrosine kinase a (PtkA) in mycobacterial intracellular survival.

Mycobacterium tuberculosis (Mtb) continues to define new paradigms of host-pathogen interaction. There are several host proteins known which are regulated by Mtb infection. The proteins which regulate host biological processes like apoptosis, cell processes, stress proteins, metabolic enzymes, etc. are targeted by the pathogens. Mtb proteins interact directly or indirectly with host proteins and play an important role in their persistence and intracellular growth. Mtb is an intracellular pathogen. It remains dormant for years within the host without activating its immune system. Mtb Protein tyrosine kinase (PtkA) regulates host anti-apoptotic protein, metabolic enzymes, and several other proteins that are involved in stress regulation, cell proliferation, protein folding, DNA repair, etc. PtkA regulates other mycobacterial proteins and plays an important role in its growth and survival. Here we summarized the current knowledge of PtkA and reviewed its role in mycobacterial intracellular survival as it regulates several other mycobacterial proteins and host proteins. PtkA regulates PtpA secretion which is essential for mycobacterial virulence and could be used as an attractive drug target.

Leprosy: The age-old companion of humans - Re-evaluation and comparative analysis of Avar-period cases with Hansen's disease from the Danube-Tisza Interfluve, Hungary.

In recent years, our knowledge of leprosy in the past has substantially been enriched. Nonetheless, much still remains to be discovered, especially in regions and periods from where no written sources are available. To fill in some research gaps, we provide the comparative analysis of eight Avar-period leprosy cases from the Danube-Tisza Interfluve (Hungary). In every case, to reconstruct the biological consequences of leprosy, the detected bony changes were linked with palaeopathological and modern medical information. To reconstruct the social consequences of being affected by leprosy, conceptualisation of the examined individuals' treatment in death was conducted. In every case, the disease resulted in deformation and disfigurement of the involved anatomical areas (rhinomaxillary region, feet, and/or hands) with difficulties in conducting certain physical activities. These would have been disadvantageous for the examined individuals and limited or changed their possibilities to participate in social situations. The most severe cases would have required continuous support from others to survive. Our findings indicate that, despite their very visible disease and associated debility, the examined communities did not segregate leprosy sufferers but provided and cared for them, and maintained a strong enough social network that made their survival possible even after becoming incapable of self-sufficiency.

Co-infection of mice with SARS-CoV-2 and Mycobacterium tuberculosis limits early viral replication but does not affect mycobacterial loads.

Viral co-infections have been implicated in worsening tuberculosis (TB) and during the COVID-19 pandemic, the global rate of TB-related deaths has increased for the first time in over a decade. We and others have previously shown that a resolved prior or concurrent influenza A virus infection in Mycobacterium tuberculosis (Mtb)-infected mice resulted in increased pulmonary bacterial burden, partly through type I interferon (IFN-I)-dependent mechanisms. Here we investigated whether SARS-CoV-2 (SCV2) co-infection could also negatively affect bacterial control of Mtb. Importantly, we found that K18-hACE2 transgenic mice infected with SCV2 one month before, or months after aerosol Mtb exposure did not display exacerbated Mtb infection-associated pathology, weight loss, nor did they have increased pulmonary bacterial loads. However, pre-existing Mtb infection at the time of exposure to the ancestral SCV2 strain in infected K18-hACE2 transgenic mice or the beta variant (B.1.351) in WT C57Bl/6 mice significantly limited early SCV2 replication in the lung. Mtb-driven protection against SCV2 increased with higher bacterial doses and did not require IFN-I, TLR2 or TLR9 signaling. These data suggest that SCV2 co-infection does not exacerbate Mtb infection in mice, but rather the inflammatory response generated by Mtb infection in the lungs at the time of SCV2 exposure restricts viral replication.

Acute Presentation of Tuberculosis Empyema in a Healthy Adolescent.

BACKGROUND Tuberculosis (TB) was the leading cause of infectious death worldwide until the COVID-19 pandemic, which reduced case reporting and disrupted TB diagnosis and services. While Mycobacterium tuberculosis remains a leading cause of morbidity and mortality globally, the disease burden within developed nations remains relatively rare. Although the many complications of TB are well known, no current data exists on those infected with TB who subsequently developed recurrent TB empyema, as it is such a rare complication, especially in pediatric and adolescent populations. CASE REPORT A previously healthy 15-year-old male patient presented with 5-day duration of cough, congestion, intermittent fever, and post-tussive emesis. Although born in the United States, 3 months before presentation, he returned from Senegal, where he had lived for 4 years. Imaging demonstrated consolidation with loculated effusion. Patient underwent video-assisted thoracoscopy and chest tube placement, draining 750 mL of purulent fluid testing positive for rare acid-fast bacilli. Rifampin, isoniazid, pyrazinamide, and ethambutol were administered, with discharge medication compliance ensured by daily videos surveillance through the Department of Health. Although compliant with medications, patient presented to the Emergency Department 2 months later with a multi-loculated fluid recollection and fistula formation requiring chest tube placement. After this discharge, patient experienced resolution of disease following completion of therapy. CONCLUSIONS TB complication should be considered as a differential diagnosis for pleural effusion in the appropriate clinical setting. Providers should not only consider the diagnosis but pursue appropriate testing and management early, particularly in those with risk factors, including travel to an endemic location.

[Management of drug-resistant tuberculosis]. / Management der antibiotikaresistenten Tuberkulose.

The spread of multidrug-resistant Mycobacterium tuberculosis bacteria jeopardizes tuberculosis control, especially in the WHO Europe region. Following the availability of novel drugs and treatment regimens the World Health Organization has updated management recommendations for patients affected by drug-resistant tuberculosis. These novel recommendations include a significant reduction in the duration of therapy. This review presents the epidemiology and diagnostics of antibiotic-resistant tuberculosis as well as up-to-date treatment recommendations.

Mycobacterium tuberculosis ß-lactamase variant reduces sensitivity to ampicillin/avibactam in a zebrafish-Mycobacterium marinum model of tuberculosis.

The ß-lactamase of Mycobacterium tuberculosis, BlaC, hydrolyzes ß-lactam antibiotics, hindering the use of these antibiotics for the treatment of tuberculosis. Inhibitors, such as avibactam, can reversibly inhibit the enzyme, allowing for the development of combination therapies using both antibiotic and inhibitor. However, laboratory evolution studies using Escherichia coli resulted in the discovery of single amino acid variants of BlaC that reduce the sensitivity for inhibitors or show higher catalytic efficiency against antibiotics. Here, we tested these BlaC variants under more physiological conditions using the M. marinum infection model of zebrafish, which recapitulates hallmark features of tuberculosis, including the intracellular persistence of mycobacteria in macrophages and the induction of granuloma formation. To this end, the M. tuberculosis blaC gene was integrated into the chromosome of a blaC frameshift mutant of M. marinum. Subsequently, the resulting strains were used to infect zebrafish embryos in order to test the combinatorial effect of ampicillin and avibactam. The results show that embryos infected with an M. marinum strain producing BlaC show lower infection levels after treatment than untreated embryos. Additionally, BlaC K234R showed higher infection levels after treatment than those infected with bacteria producing the wild-type enzyme, demonstrating that the zebrafish host is less sensitive to the combinatorial therapy of ß-lactam antibiotic and inhibitor. These findings are of interest for future development of combination therapies to treat tuberculosis.

EBUS-GS with the GeneXpert MTB/RIF assay for diagnosis of Mycobacterium tuberculosis infection of isolated pulmonary nodules.

OBJECTIVE: Investigate the use of endobronchial ultrasonography with a guide sheath (EBUS-GS) combined with Gene Xpert MTB/RIF (Xpert) for diagnosis of Mycobacterium tuberculosis (MTB) infection in isolated pulmonary nodules. METHODS: Patients who had isolated pulmonary nodules and unknown diagnoses at our institution from October 2020 to December 2021 were prospectively examined using EBUS-GS and Xpert. The diagnostic values of using EBUS-GS or bronchoalveolar lavage fluid (BALF) with acid-fast staining, MGIT 960 culture, pathological examination, and Xpert for isolated pulmonary nodules caused by MTB infection were compared using receiver operating characteristic (ROC) analysis. RESULTS: There were 135 patients, 64 with isolated pulmonary tuberculomas and 71 with non-tuberculous lesions. The sensitivity of EBUS-GS with Xpert was significantly higher than BALF with Xpert (57.81% vs. 34.78%, P = 0.017). Use of EBUS-GS with Xpert and MGIT 960 culture further increased the sensitivity to 62.50% (95%CI 50.64-74.36) and increased the specificity to 100%. The AUC values of BALF with MGIT 960 culture was 0.663(95%CI 0.543-0.783) and BALF with Xpert was 0.674 (95%CI 0.556-0.792). The AUC values of EBUS-GS with MGIT 960 culture was 0.680 (95%CI 0.554-0.743), with pathological examination was 0.713 (95%CI 0.573-0.760), and with Xpert was 0.789 (95%CI 0.655-0.829). CONCLUSION: Use of EBUS-GS with Xpert had high sensitivity and specificity in the diagnosis of isolated pulmonary tuberculoma. This method has significant potential for use in clinical practice.

Heterogeneity in M. tuberculosis ß-lactamase inhibition by Sulbactam.

For decades, researchers have elucidated essential enzymatic functions on the atomic length scale by tracing atomic positions in real-time. Our work builds on possibilities unleashed by mix-and-inject serial crystallography (MISC) at X-ray free electron laser facilities. In this approach, enzymatic reactions are triggered by mixing substrate or ligand solutions with enzyme microcrystals. Here, we report in atomic detail (between 2.2 and 2.7 Å resolution) by room-temperature, time-resolved crystallography with millisecond time-resolution (with timepoints between 3 ms and 700 ms) how the Mycobacterium tuberculosis enzyme BlaC is inhibited by sulbactam (SUB). Our results reveal ligand binding heterogeneity, ligand gating, cooperativity, induced fit, and conformational selection all from the same set of MISC data, detailing how SUB approaches the catalytic clefts and binds to the enzyme noncovalently before reacting to a trans-enamine. This was made possible in part by the application of singular value decomposition to the MISC data using a program that remains functional even if unit cell parameters change up to 3 Å during the reaction.

Molecular diversity of Mycobacterium tuberculosis isolates in Treatment-experienced Patients from Andhra Pradesh, India.

INTRODUCTION: To get a comprehensive idea about the transmission and epidemiology of TB globally and locally, the use of molecular typing methods has become imperative not only for understanding genetic diversity but also the population structure of Mycobacterium tuberculosis complex (MTBC). We aimed to investigate the drug resistance pattern and genetic diversity of MTBC among previously treated patients with sputum smear-positive pulmonary tuberculosis in a South Indian population. METHODOLOGY: 104 patients with sputum smear positivity and who had previously undergone treatment were selected. Drug susceptibility testing, Spoligotyping, MIRU-VNTR, and SNP typing were performed. RESULTS: Mono-resistance to isoniazid 16 (15.38%) was the highest among all drugs. Out of 104 isolates, 24 (23%) isolates were classified as MDR strains. The distributions of most common lineages were: EAI3-Ind-20 (19.23%), EAI5-13 (12.50%), Beijing-12 (11.54%), CAS1-Delhi- 9 (8.65%), and 7 (6.73%) each of T-H37rv, Unknown and Orphan types. MIRU-VNTR-based analysis revealed that there are two major groups: CAS1-Delhi and Beijing groups. Out of 104 isolates, 82 belonged to well-defined lineages and 6 clusters, and the remaining 22 were singletons. SNP analysis showed no mutations associated with five sets of genes in 33 strains. CONCLUSIONS: The occurrence of 11.54% Beijing strains in South India is an important finding. High frequency of Isoniazid mono resistance noticed. Spoligotyping along with MIRU-VNTR and SNP typing is the best approach to the identification of strain lineages. No mutation with Antigen85C gene represents, can be used for vaccine candidates.

Identification of Hub Genes and Typing of Tuberculosis Infections Based on Autophagy-Related Genes.

Tuberculosis (TB) caused by Mycobacterium tuberculosis is one of the leading causes of morbidity and death in humans worldwide. Some autophagy genes associated with TB and some miRNAs regulating TB have been found, but the identification of autophagy-related genes in M. tuberculosis remains to be explored. Forty-seven autophagy-related genes differentially expressed in TB were identified in this study by analysis of TB-related datasets in the Gene Expression Omnibus (GEO) and autophagy-related genes in the Human Autophagy Database. The potential crucial genes affecting TB were found through the protein-protein interaction (PPI) network, and the possible pathways affected by these genes were verified. Analysis of the PPI network of miRNAs associated with M. tuberculosis infection and their target genes revealed that hsa-let-7, hsa-mir-155, hsa-mir-206, hsa-mir-26a, hsa-mir-30a, and hsa-mir-32 may regulate the expression of multiple autophagy-related genes (MAPK8, UVRAG, UKL2, and GABARAPL1) alone or in combination. Subsequently, Cytoscape was utilized to screen the differentially expressed genes related to autophagy. The hub genes (GABARAPL1 and ULK2) affecting TB were identified. Combined with Gene Set Enrichment Analysis (GSEA), the signaling pathways affected by the hub genes were verified. Finally, we divided TB patients into two subgroups based on autophagy-related genes, and the immune microenvironment of patients in different subgroups was significantly different. Our study found two autophagy-related hub genes that could affect TB and divide TB samples into two subgroups. This finding is of great significance for TB treatment and provides new ideas for exploring the pathogenesis of M. tuberculosis.

Exploring recombinant secretory proteins from Mycobacterium tuberculosis to develop a serological platform for tuberculosis diagnosis.

The lack of a sensitive diagnostic tool for tuberculosis (TB) is the main reason for increasing cause of death in many developing countries. The routine diagnostic tests are either time-consuming or equivocal in terms of results. Hence, there is a need for quicker and accurate diagnostic tests. Certain studies have documented the usage of proteins secreted by Mycobacterium tuberculosis (MTB) in developing a sensitive tool for diagnosing TB. The study aimed to employ PPE41, MPT53, LPQH, CFP10, ESAT6 and TB18.5 proteins and analyze their usage as early diagnostic markers. The proteins were cloned, expressed, purified and applied in ELISA platforms in separate as well as combined systems to assess their early diagnostic features. The results of our study revealed that a cocktail of all six antigen combinations was identified in the maximum number of TB cases. Thus, proteins such as PPE41, MPT53, LPQH, CFP10, ESAT6, and TB18.5 incorporated detection tools could be optimized for an improvised early detection of MTB infections. Moreover, the results suggested that 95.7 % of the MTB-positive serum samples reacted with all the selected antigens of Mycobacterium tuberculosis, while the control serum samples did not react with those antigens. The hexavalent antigen system yielded a novel ELISA platform for better diagnosing MTB infections. Our study yielded a novel technology to diagnose TB, which warrants testing in clinical settings.

Borderline rpoB mutations transmit at the same rate as common rpoB mutations in a tuberculosis cohort in Bangladesh.

The spread of multidrug-resistant tuberculosis (MDR-TB) is a growing problem in many countries worldwide. Resistance to one of the primary first-line drugs, rifampicin, is caused by mutations in the Mycobacterium tuberculosis rpoB gene. So-called borderline rpoB mutations confer low-level resistance, in contrast to more common rpoB mutations which confer high-level resistance. While some borderline mutations show lower fitness in vitro than common mutations, their in vivo fitness is currently unknown. We used a dataset of 394 whole genome sequenced MDR-TB isolates from Bangladesh, representing around 44 % of notified MDR-TB cases over 6 years, to look at differences in transmission clustering between isolates with borderline rpoB mutations and those with common rpoB mutations. We found a relatively low percentage of transmission clustering in the dataset (34.8 %) but no difference in clustering between different types of rpoB mutations. Compensatory mutations in rpoA, rpoB, and rpoC were associated with higher levels of transmission clustering as were lineages two, three, and four relative to lineage one. Young people as well as patients with high sputum smear positive TB were more likely to be in a transmission cluster. Our findings show that although borderline rpoB mutations have lower in vitro growth potential this does not translate into lower transmission potential or in vivo fitness. Proper detection of these mutations is crucial to ensure they do not go unnoticed and spread MDR-TB within communities.