A CRISPR-Cas12a-based platform for ultrasensitive rapid highly specific detection of Mycobacterium tuberculosis in clinical application.

Tuberculosis, caused by Mycobacterium tuberculosis (MTB), is the second leading cause of death after COVID-19 pandemic. Here, we coupled multiple cross displacement amplification (MCDA) technique with CRISPR-Cas12a-based biosensing system to design a novel detection platform for tuberculosis diagnosis, termed MTB-MCDA-CRISPR. MTB-MCDA-CRISPR pre-amplified the specific sdaA gene of MTB by MCDA, and the MCDA results were then decoded by CRISPR-Cas12a-based detection, resulting in simple visual fluorescent signal readouts. A set of standard MCDA primers, an engineered CP1 primer, a quenched fluorescent ssDNA reporter, and a gRNA were designed targeting the sdaA gene of MTB. The optimal temperature for MCDA pre-amplification is 67°C. The whole experiment process can be completed within one hour, including sputum rapid genomic DNA extraction (15 minutes), MCDA reaction (40 minutes), and CRISPR-Cas12a-gRNA biosensing process (5 minutes). The limit of detection (LoD) of the MTB-MCDA-CRISPR assay is 40 fg per reaction. The MTB-MCDA-CRISPR assay does not cross reaction with non-tuberculosis mycobacterium (NTM) strains and other species, validating its specificity. The clinical performance of MTB-MCDA-CRISPR assay was higher than that of the sputum smear microscopy test and comparable to that of Xpert method. In summary, the MTB-MCDA-CRISPR assay is a promising and effective tool for tuberculosis infection diagnosis, surveillance and prevention, especially for point-of-care (POC) test and field deployment in source-limited regions.

Role of non-coding RNAs in tuberculosis and their potential for clinical applications.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains the leading cause of mortality due to infectious diseases, only surpassed in 2020 by COVID-19. Despite the development in diagnostics, therapeutics, and evaluation of new vaccines for TB, this infectious disease remains uncontrollable due to the emergence of multidrug-resistant (MDR) and extremely drug-resistant (XDR) TB, among other factors. The development in transcriptomics (RNomics) has enabled the study of gene expression in TB. It is considered that non-coding RNAs (ncRNAs) from host [microRNAs (miRNAs)] and Mtb [small RNAs (sRNAs)] are important elements in TB pathogenesis, immune resistance, and susceptibility. Many studies have shown the importance of host miRNAs in regulating immune response against Mtb via in vitro and in vivo mice models. The bacterial sRNAs play a major role in survival, adaptation, and virulence. Here, we review the characterization and function of host and bacteria ncRNAs in TB and their potential use in clinical applications as diagnostic, prognostic, and therapeutic biomarkers.

[The problem of formation of new risks of medicinal resistance of patients with tuberculosis in condition of active use of antibiotics by population under COVID-19].

In pandemic conditions, situation of active and uncontrolled use by population of antimicrobial preparations treating COVID-19 occurs. So, new risks of development of medication resistance among patients with various infectious diseases, tuberculosis included, appear. The purpose of the study is to characterize prevalence of antimicrobial preparations use by population in relationship with development of medication resistance in patients with tuberculosis during COVID-19 pandemic. Material and methods. The analysis of sales of antimicrobial medicines was implemented on the basis of published official data from the joint-stock company DSM Group presenting monthly audit of the Russian pharmaceutical market. The determination of primary antibiotic resistance was carried out in 2018-2020 on 3312 patients with tuberculosis. The modified method of proportions on liquid nutrient medium in system with automated accounting of microorganisms growth, the method of absolute concentrations and the method of polymerase chain reaction with real-time detection were applied. The results of the study. It was established that the most demanding antimicrobial medications among population were ceftriaxone, azithromycin, levofloxacin, moxifloxacin, azithromycin. At the same time, the maximum increase in sales in 2020 up to 150% as compared with of 2019 was determined in medications derived from quinolone moxifloxacin, levofloxacin, which began to be used in treatment of coronavirus infection. At the same time, these medications are traditionally used in tuberculosis treatment. But in 2020, alarming trend was established that limits treatment of tuberculosis patients. The primary resistance of mycobacteria was also established in newly diagnosed tuberculosis patients, also for the same antimicrobial medications of quinolone derivatives, and increasing in proportion of patients with primary medication resistance to levofloxacin, moxifloxacin in 2020 as compared to 2018 was 189-480%. At the same time, increasing of resistance to other antibiotics made up to 60.8% on average. Conclusion. The study results imply alarming scenario of medication resistance shifts towards very virulent and highly medication-resistant genotypes. This trend can result in conditions of successful transmission of deadly medication-resistant mutants that can seriously undermine effectiveness of implemented programs of struggle with tuberculosis worldwide.

Activation of transcription factor CREB in human macrophages by Mycobacterium tuberculosis promotes bacterial survival, reduces NF-kB nuclear transit and limits phagolysosome fusion by reduced necroptotic signaling.

Macrophages are a first line of defense against pathogens. However, certain invading microbes modify macrophage responses to promote their own survival and growth. Mycobacterium tuberculosis (M.tb) is a human-adapted intracellular pathogen that exploits macrophages as an intracellular niche. It was previously reported that M.tb rapidly activates cAMP Response Element Binding Protein (CREB), a transcription factor that regulates diverse cellular responses in macrophages. However, the mechanism(s) underlying CREB activation and its downstream roles in human macrophage responses to M.tb are largely unknown. Herein we determined that M.tb-induced CREB activation is dependent on signaling through MAPK p38 in human monocyte-derived macrophages (MDMs). Using a CREB-specific inhibitor, we determined that M.tb-induced CREB activation leads to expression of immediate early genes including COX2, MCL-1, CCL8 and c-FOS, as well as inhibition of NF-kB p65 nuclear localization. These early CREB-mediated signaling events predicted that CREB inhibition would lead to enhanced macrophage control of M.tb growth, which we observed over days in culture. CREB inhibition also led to phosphorylation of RIPK3 and MLKL, hallmarks of necroptosis. However, this was unaccompanied by cell death at the time points tested. Instead, bacterial control corresponded with increased colocalization of M.tb with the late endosome/lysosome marker LAMP-1. Increased phagolysosomal fusion detected during CREB inhibition was dependent on RIPK3-induced pMLKL, indicating that M.tb-induced CREB signaling limits phagolysosomal fusion through inhibition of the necroptotic signaling pathway. Altogether, our data show that M.tb induces CREB activation in human macrophages early post-infection to create an environment conducive to bacterial growth. Targeting certain aspects of the CREB-induced signaling pathway may represent an innovative approach for development of host-directed therapeutics to combat TB.

Comparison of two diagnostic intervention packages for community-based active case finding for tuberculosis: an open-label randomized controlled trial.

Two in every five patients with active tuberculosis (TB) remain undiagnosed or unreported. Therefore community-based, active case-finding strategies require urgent implementation. However, whether point-of-care (POC), portable battery-operated, molecular diagnostic tools deployed at a community level, compared with conventionally used POC smear microscopy, can shorten time-to-treatment initiation, thus potentially curtailing transmission, remains unclear. To clarify this issue, we performed an open-label, randomized controlled trial in periurban informal settlements of Cape Town, South Africa, where we TB symptom screened 5,274 individuals using a community-based scalable mobile clinic. Some 584 individuals with HIV infection or symptoms of TB underwent targeted diagnostic screening and were randomized (1:1) to same-day smear microscopy (n = 296) or on-site DNA-based molecular diagnosis (n = 288; GeneXpert). The primary aim was to compare time to TB treatment initiation between the arms. Secondary aims included feasibility and detection of probably infectious people. Of participants who underwent targeted screening, 9.9% (58 of 584) had culture-confirmed TB. Time-to-treatment initiation occurred significantly earlier in the Xpert versus the smear-microscopy arm (8 versus 41 d, P = 0.002). However, overall, Xpert detected only 52% of individuals with culture-positive TB. Notably, Xpert detected almost all of the probably infectious patients compared with smear microscopy (94.1% versus 23.5%, P = <0.001). Xpert was associated with a shorter median time to treatment of probably infectious patients (7 versus 24 d, P = 0.02) and a greater proportion of infectious patients were on treatment at 60 d compared with the probably noninfectious patients (76.5% versus 38.2%, P < 0.01). Overall, a greater proportion of POC Xpert-positive participants were on treatment at 60 d compared with all culture-positive participants (100% versus 46.5%, P < 0.01). These findings challenge the traditional paradigm of a passive case-finding, public health strategy and argues for the implementation of portable DNA-based diagnosis with linkage to care as a community-oriented, transmission-interruption strategy. The study was registered with the South African National Clinical Trials Registry (application ID 4367; DOH-27-0317-5367) and ClinicalTrials.gov (NCT03168945).

Evolution of tuberculosis diagnostics: From molecular strategies to nanodiagnostics.

Tuberculosis has remained a global concern for public health affecting the lives of people for ages. Approximately 10 million people are affected by the disease and 1.5 million succumb to the disease worldwide annually. The COVID-19 pandemic has highlighted the role of early diagnosis to win the battle against such infectious diseases. Thus, advancement in the diagnostic approaches to provide early detection forms the foundation to eradicate and manage contagious diseases like tuberculosis. The conventional diagnostic strategies include microscopic examination, chest X-ray and tuberculin skin test. The limitations associated with sensitivity and specificity of these tests demands for exploring new techniques like probe-based assays, CRISPR-Cas and microRNA detection. The aim of the current review is to envisage the correlation between both the conventional and the newer approaches to enhance the specificity and sensitivity. A significant emphasis has been placed upon nanodiagnostic approaches manipulating quantum dots, magnetic nanoparticles, and biosensors for accurate diagnosis of latent, active and drug-resistant TB. Additionally, we would like to ponder upon a reliable method that is cost-effective, reproducible, require minimal infrastructure and provide point-of-care to the patients.

Integrating tuberculosis and COVID-19 molecular testing in Lima, Peru: a cross-sectional, diagnostic accuracy study.

BACKGROUND: Integrated molecular testing could be an opportunity to detect and provide care for both tuberculosis and COVID-19. Many high tuberculosis burden countries, such as Peru, have existing GeneXpert systems for tuberculosis testing with GeneXpert Xpert MTB/RIF Ultra (Xpert Ultra), and a GeneXpert SARS-CoV-2 assay, GeneXpert Xpert Xpress SARS-CoV-2 (Xpert Xpress), is also available. We aimed to assess the feasibility of integrating tuberculosis and COVID-19 testing using one sputum specimen with Xpert Ultra and Xpert Xpress in Lima, Peru. METHODS: In this cross-sectional, diagnostic accuracy study, we recruited adults presenting with clinical symptoms or suggestive history of tuberculosis or COVID-19, or both. Participants were recruited from a total of 35 primary health facilities in Lima, Peru. Participants provided one nasopharyngeal swab and one sputum sample. For COVID-19, we tested nasopharyngeal swabs and sputum using Xpert Xpress; for tuberculosis, we tested sputum using culture and Xpert Ultra. We compared diagnostic accuracy of sputum testing using Xpert Xpress with nasopharyngeal swab testing using Xpert Xpress. Individuals with positive Xpert Xpress nasopharyngeal swab results were considered COVID-19 positive, and a positive culture indicated tuberculosis. To assess testing integration, the proportion of cases identified in sputum by Xpert Xpress was compared with Xpert Xpress on nasopharyngeal swabs, and sputum by Xpert Ultra was compared with culture. FINDINGS: Between Jan 11, 2021, and April 26, 2022, we recruited 600 participants (312 [52%] women and 288 [48%] men). In-study prevalence of tuberculosis was 13% (80 participants, 95% CI 11-16) and of SARS-CoV-2 was 35% (212 participants, 32-39). Among tuberculosis cases, 13 (2·2%, 1·2-3·7) participants were concurrently positive for SARS-CoV-2. Regarding the diagnostic yield of integrated testing, Xpert Ultra detected 96% (89-99) of culture-confirmed tuberculosis cases (n=77), and Xpert Xpress-sputum detected 67% (60-73) of COVID-19 cases (n=134). All five study staff reported that integrated molecular testing was easy and acceptable. INTERPRETATION: The diagnostic yield of Xpert Xpress on sputum was moderate, but integrated testing for tuberculosis and COVID-19 with GeneXpert was feasible. However, systematic testing for both diseases might not be the ideal approach for everyone presenting with presumptive tuberculosis or COVID-19, as concurrent positive cases were rare during the study period. Further research might help to identify when integrated testing is most worthwhile and its optimal implementation. FUNDING: Canadian Institutes of Health Research and International Development Research Centre. TRANSLATION: For the Spanish translation of the abstract see Supplementary Materials section.

Tuberculosis vaccines update: Is an RNA-based vaccine feasible for tuberculosis?

OBJECTIVES: Despite concerted efforts, Mycobacterium tuberculosis (M.tb), the pathogen that causes tuberculosis (TB), continues to be a burden on global health, regaining its dubious distinction in 2022 as the world's biggest infectious killer with global COVID-19 deaths steadily declining. The complex nature of M.tb, coupled with different pathogenic stages, has highlighted the need for the development of novel immunization approaches to combat this ancient infectious agent. Intensive efforts over the last couple of decades have identified alternative approaches to improve upon traditional vaccines that are based on killed pathogens, live attenuated agents, or subunit recombinant antigens formulated with adjuvants. Massive funding and rapid advances in RNA-based vaccines for immunization have recently transformed the possibility of protecting global populations from viral pathogens, such as SARS-CoV-2. Similar efforts to combat bacterial pathogens such as M.tb have been significantly slower to implement. METHODS: In this review, we discuss the application of a novel replicating RNA (repRNA)-based vaccine formulated and delivered in nanostructured lipids. RESULTS: Our preclinical data are the first to report that RNA platforms are a viable system for TB vaccines and should be pursued with high-priority M.tb antigens containing cluster of differentiation (CD4+) and CD8+ T-cell epitopes. CONCLUSION: This RNA vaccine shows promise for use against intracellular bacteria such as M.tb as demonstrated by the feasibility of construction, enhanced induction of cell-mediated and humoral immune responses, and improved bacterial burden outcomes in in vivo aerosol-challenged preclinical TB models.

Trends in pediatric tuberculosis diagnosis utilizing xpert Mycobacterium tuberculosis/Rifampicin in a poor-resource, high-burden region: A retrospective, multicenter study.

Background: The burden of tuberculosis (TB) in Nigeria remains high, and diagnosis in children, a challenge. We aimed to document yield from Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) as a mode of diagnosis for children and the variables associated with a positive result. Methods: This was a retrospective review of TB treatment cards of children aged 0-15 years managed from January 2017 to December 2021 across six public tertiary institutions in Nigeria. The data obtained were analyzed using the descriptive and inferential statistics. Statistical significance was set at P < 0.05. Results: Of 1489 children commenced on TB treatment, 1463 (97.9%) had sufficient data for analysis the median age of study participants was 60 months (interquartile range [IQR]: 24, 120), and 814 (55.6%) were males. Xpert MTB/RIF test was performed in 862 (59%) participants and MTB was detected in 171 (19.8%) participants, of which 6.4% (11/171) had RIF resistance reported. The use of Xpert MTB/RIF rose from 56.5% in 2017 to 64% in 2020 but fell to 60.9% in 2021. We found that older age (> 10 years), the presence of pulmonary TB (PTB), and a negative human immunodeficiency virus (HIV) status were associated with positive Xpert MTB/RIF tests (P = 0.002, 0.001, and 0.012, respectively). Conclusion: The utilization of Xpert MTB/RIF in children increased in the years before the COVID-19 pandemic. Factors associated with MTB detection by Xpert MTB/RIF include older age, the presence of PTB, and a negative HIV status. Clinical and radiological evaluation continues to play vital roles in the diagnosis of childhood TB in Nigeria.

A tale of three in symbiosis: TB-COVID-19-Bordetella coinfection.

Coinfections/mixed infections are common in the respiratory tract. Many times existing organisms have similar risk factors and clinical features that make the diagnosis difficult. Coronavirus diagnosed in 2019 (COVID-19) and tuberculosis (TB) are two such diseases. Patients with TB have lower cellular immunity and impaired pulmonary function. In such environment, atypical organisms, can infect and make the outcome unfavorable. A 21-year-old malnourished (body mass index- 15 kg/m2) girl presented with fever and cough for 10 days. Sputum for Cartridge Based Nucleic Acid Amplification Test demonstrated Mycobacterium tuberculosis with no rifampin resistance. Fever persisted (100-101°F) and saturation was dropping even after 10 days of antitubercular treatment. A repeat reverse transcription-polymerase chain reaction was done and was positive. In view of persistent symptoms after 20 days, bronchoscopy was done, and cultures showed Bordetella bronchiseptica. Fever and symptoms resolved completely after initiation of the sensitive drug. Diagnostic delay in coinfections can lead to increased morbidity and mortality.

Knowledge domain and emerging trends in HIV-MTB co-infection from 2017 to 2022: A scientometric analysis based on VOSviewer and CiteSpace.

Co-infection with Mycobacterium tuberculosis (MTB) in human immunodeficiency virus (HIV)-infected individuals is one of the leading causes of death. Also, research on HIV and MTB (HIV-MTB) co-infection was found to have a downward trend. In this work, we performed the knowledge domain analysis and visualized the current research progress and emerging trends in HIV-MTB co-infection between 2017 and 2022 by using VOSviewer and CiteSpace. The relevant literatures in this article were collected in the Web of Science (WoS) database. VOSviewer and CiteSpace bibliometric software were applied to perform the analysis and visualization of scientific productivity and frontier. Among all the countries, USA was dominant in the field, followed by South Africa, and England. Among all the institutions, the University of Cape Town (South Africa) had more extensive collaborations with other research institutions. The Int J Tuberc Lung Dis was regarded as the foremost productive journal. Survival and mortality analysis, pathogenesis, epidemiological studies, diagnostic methods, prognosis improvement of quality of life, clinical studies and multiple infections (especially co-infection with COVID-19) resulted in the knowledge bases for HIV-MTB co-infection. The clinical research on HIV-MTB co-infection has gradually shifted from randomized controlled trials to open-label trials, while the cognition of HIV-TB has gradually shifted from cytokines to genetic polymorphisms. This scientometric study used quantitative and qualitative methods to conduct a comprehensive review of research on HIV-MTB co-infection published over the past 5 years, providing some useful references to further the study of HIV-MTB co-infection.

Non-amplification nucleic acid detection with thio-NAD cycling.

The PCR technique is indispensable in biology and medicine, but some difficulties are associated with its use, including false positive or false negative amplifications. To avoid these issues, a non-amplification nucleic acid detection protocol is needed. In the present study, we propose a method in which nucleic-acid probe hybridization is combined with thio-NAD cycling to detect nucleic acids without amplification. We report our application of this method for the detection of the gene of MPT64 in Mycobacterium tuberculosis. Two different cDNA probes targeted the mpt64 gene: the first probe was used to immobilize the mpt64 gene, and the second probe, linked with alkaline phosphatase (ALP), was hybridized to a target sequence in the mpt64 gene. A substrate was then hydrolyzed by ALP, and a cycling reaction was conducted by a dehydrogenase with its co-factors (thio-NAD and NADH). The single-stranded DNA, double-stranded DNA, plasmid DNA for the mpt64 gene, and whole genome of M. tuberculosis var. BCG were detected at the level of 105-106 copies/assay, whereas the non-tuberculosis mycobacteria (e.g., M. avium, M. intracellulare, M. kansasii, and M. abscessus) were below the limits of detection. The present method enables us to avoid the errors inherent in nucleic acid amplification methods.

Detection of Mycobacterium tuberculosis Complex Using the Xpert MTB/RIF Ultra Assay on the Stool of Pediatric Patients in Dushanbe, Tajikistan.

We report the findings of a prospective laboratory diagnostic accuracy study to evaluate the sensitivity, specificity, and predictive values of the Xpert MTB/RIF Ultra assay for Mycobacterium tuberculosis detection in fresh stool specimens from children under 15 years of age with confirmed tuberculosis (TB) disease from Dushanbe, Tajikistan. Six hundred eighty-eight (688) participants were enrolled from April 2019 to October 2021. We identified 16 participants (2.3%) with confirmed TB disease, defined as ≥1 TB sign/symptom plus microbiologic confirmation. With the Xpert MTB/RIF Ultra assay for stool, we found a sensitivity of 68.8% (95% CI, 46.0 to 91.5) and a specificity of 98.7% (95% CI, 97.8 to 99.5) in confirmed TB disease. Our results are comparable to other published studies; however, our cohort was larger and our confirmed TB disease rate lower than most. We also demonstrated that this assay was feasible to implement in a centralized hospital laboratory in a low-middle-income Central Asian country. However, we encountered obstacles such as lack of staffing, material ruptures, outdated government protocols, and decreased case presentation due to COVID-19. We found eight patients whose only positive test was an Xpert Ultra stool assay. None needed treatment during the study; however, three were treated later, suggesting such cases require close observation. Our report is the first from Central Asia and one of a few from a low-middle-income country. We believe our study demonstrates the generalizability of the Xpert MTB/RIF Ultra assay on fresh stool specimens from children and provides further evidence supporting WHO's approval of this diagnostic strategy. IMPORTANCE The importance of this report is that it provides further support for WHO's recent recommendation that fresh stool is an acceptable sample for GeneXpert TB testing in children, especially small children who often cannot produce an adequate sputum sample. Diagnosing TB in this age group is difficult, and many cases are missed, leading to unacceptable rates of TB illness and death. In our large cohort of children from Dushanbe, Tajikistan, the GeneXpert stool test was positive in 69% of proven cases of TB, and there were very few false-positive tests. We also showed that this diagnostic strategy was feasible to implement in a low-middle-income country with an inefficient health care delivery system. We hope that many more programs will adopt this form of diagnosing TB in children.

Antibiotic resistance acquisition versus primary transmission in the presentation of extensively drug-resistant tuberculosis.

Mycobacterium tuberculosis is the leading cause of mortality worldwide due to a single bacterial pathogen. Of concern is the negative impact that the COVID-19 pandemic has had on the control of tuberculosis (TB) including drug-resistant forms of the disease. Antimicrobial resistance increases the likelihood of worsened outcomes in TB patients including treatment failure and death. Multidrug-resistant (MDR) strains, resistant to first-line drugs isoniazid and rifampin, and extensively drug-resistant (XDR) strains with further resistance to second-line drugs (SLD), threaten control programs designed to lower TB incidence and end the disease as a public health challenge by 2030, in accordance with UN Sustainable Development Goals. Tackling TB requires an understanding of the pathways through which drug resistance emerges. Here, the roles of acquired resistance mutation, and primary transmission, are examined with regard to XDR-TB. It is apparent that XDR-TB can emerge from MDR-TB through a small number of additional resistance mutations that occur in patients undergoing drug treatment. Rapid detection of resistance, to first-line drugs and SLD, at the initiation of and during treatment, and prompt adjustment of regimens are required to ensure treatment success in these patients. Primary transmission is predicted to make an increasing contribution to the XDR-TB caseload in the future. Much work is required to improve the implementation of the World Health Organization-recommended infection control practices and block onward transmission of XDR-TB patients to contacts including health-care workers. Finally, limiting background resistance to fluoroquinolones in pre-XDR strains of M. tuberculosis will necessitate better antimicrobial stewardship in the broader use of this drug class.

Drug resistant tuberculosis: Implications for transmission, diagnosis, and disease management.

Drug resistant tuberculosis contributes significantly to the global burden of antimicrobial resistance, often consuming a large proportion of the healthcare budget and associated resources in many endemic countries. The rapid emergence of resistance to newer tuberculosis therapies signals the need to ensure appropriate antibiotic stewardship, together with a concerted drive to develop new regimens that are active against currently circulating drug resistant strains. Herein, we highlight that the current burden of drug resistant tuberculosis is driven by a combination of ongoing transmission and the intra-patient evolution of resistance through several mechanisms. Global control of tuberculosis will require interventions that effectively address these and related aspects. Interrupting tuberculosis transmission is dependent on the availability of novel rapid diagnostics which provide accurate results, as near-patient as is possible, together with appropriate linkage to care. Contact tracing, longitudinal follow-up for symptoms and active mapping of social contacts are essential elements to curb further community-wide spread of drug resistant strains. Appropriate prophylaxis for contacts of drug resistant index cases is imperative to limit disease progression and subsequent transmission. Preventing the evolution of drug resistant strains will require the development of shorter regimens that rapidly eliminate all populations of mycobacteria, whilst concurrently limiting bacterial metabolic processes that drive drug tolerance, mutagenesis and the ultimate emergence of resistance. Drug discovery programs that specifically target bacterial genetic determinants associated with these processes will be paramount to tuberculosis eradication. In addition, the development of appropriate clinical endpoints that quantify drug tolerant organisms in sputum, such as differentially culturable/detectable tubercle bacteria is necessary to accurately assess the potential of new therapies to effectively shorten treatment duration. When combined, this holistic approach to addressing the critical problems associated with drug resistance will support delivery of quality care to patients suffering from tuberculosis and bolster efforts to eradicate this disease.

Failing upwards: Genetics-based strategies to improve antibiotic discovery and efficacy in Mycobacterium tuberculosis.

Therapeutic advances in the 20th century significantly reduced tuberculosis (TB) mortality. Nonetheless, TB still poses a massive global health challenge with significant annual morbidity and mortality that has been amplified during the COVID-19 pandemic. Unlike most common bacterial infectious diseases, successful TB treatment requires months-long regimens, which complicates the ability to treat all cases quickly and effectively. Improving TB chemotherapy by reducing treatment duration and optimizing combinations of drugs is an important step to reducing relapse. In this review, we outline the limitations of current multidrug regimens against TB and have reviewed the genetic tools available to improve the identification of drug targets. The rational design of regimens that sterilize diverse phenotypic subpopulations will maximize bacterial killing while minimizing both treatment duration and infection relapse. Importantly, the TB field currently has all the necessary genetic and analytical tools to screen for and prioritize drug targets in vitro based on the vulnerability of essential and non-essential genes in the Mtb genome and to translate these findings in in vivo models. Combining genetic methods with chemical screens offers a formidable strategy to redefine the preclinical design of TB therapy by identifying powerful new targets altogether, as well as targets that lend new efficacy to existing drugs.

K-mer applied in Mycobacterium tuberculosis genome cluster analysis.

According to studies carried out, approximately 10 million people developed tuberculosis in 2018. Of this total, 1.5 million people died from the disease. To study the behavior of the genome sequences of Mycobacterium tuberculosis (MTB), the bacterium responsible for the development of tuberculosis (TB), an analysis was performed using k-mers (DNA word frequency). The k values ranged from 1 to 10, because the analysis was performed on the full length of the sequences, where each sequence is composed of approximately 4 million base pairs, k values above 10, the analysis is interrupted, as consequence of the program's capacity. The aim of this work was to verify the formation of the phylogenetic tree in each k-mer analyzed. The results showed the formation of distinct groups in some k-mers analyzed, taking into account the threshold line. However, in all groups, the multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains remained together and separated from the other strains.

Efficient elimination of airborne pathogens: a study on aerosolized Mycobacterium tuberculosis and SARS-CoV-2 using ZeBox technology.

BACKGROUND: Despite multifactorial evidence, the safe and effective elimination of free-floating micro-organisms remains a significant scientific challenge. ZeBox Technology exploits microbial Zeta Potential, to extract and eliminate them from free-flowing air, using a non-ionizing electric field, in combination with a microbicidal surface. AIM: Evaluation of ZeBox Technology against aerosolized SARS-CoV-2 and Mycobacterium tuberculosis under controlled conditions. METHODS: SARS-CoV-2 and M. tuberculosis H37Ra were used in this study. Individual micro-organisms were aerosolized using a Collison nebulizer inside an air-sealed test chamber. Air samples were collected from the chamber on to a Mixed Cellulose Ester membrane, at various time points, and used for enumeration. SARS-CoV-2 was enumerated using qRT-PCR, while M. tuberculosis H37Ra was quantified using standard microbiological procedures. FINDINGS: We established a viable aerosolized microbial load of ∼10E9 and ∼10E6 for SARS-CoV-2 and M. tuberculosis H37Ra, respectively, inside the test chamber. Under ideal conditions, the floating microbial load was at a steady-state level of 10E9 for SARS-CoV-2 and 10E6 for M. tuberculosis. When the ZeBox-Technology-enabled device was operated, the microbial load reduced significantly. A reduction of ∼10E4.7 was observed for M. tuberculosis, while a reduction of ∼10E7 for SARS-CoV-2 was observed within a short duration. The reduction in airborne SARS-CoV-2 load was qualitatively and quantitatively measured using fluorescence analysis and qRT-PCR methods, respectively. CONCLUSION: This validation demonstrates the efficacy of the developed technology against two of the deadliest micro-organisms that claim millions of lives worldwide. In conjunction with the existing reports, the present validation proved the true broad-spectrum elimination capability of ZeBox technology.