Expression of TNF-alpha-dependent apoptosis-related genes in the peripheral blood of Malagasy subjects with tuberculosis.

The majority of Mycobacterium tuberculosis (Mtb) infections remain asymptomatic with only up to 10% progressing to clinical tuberculosis. However, the constituents of the effective "protective immunity" against tuberculosis responsible for containing most infections remain unknown. Evaluating gene transcriptional profiles in tuberculosis clinical cohorts is one approach to understanding the spectrum of tuberculosis progression. It is clear that apoptosis plays a role in the control of tuberculosis but the utility of apoptosis-related genes as surrogate markers of protection against tuberculosis has not been well investigated. To characterize potential surrogate markers that could discriminate different phases of the clinical tuberculosis spectrum, we investigated gene expression of several TNF-alpha dependent apoptotic genes (TNFR1, TNFR2, FLICE, FLIPs) by real-time RT-PCR of peripheral blood cells from cohorts of individuals with active tuberculosis or potential exposure to tuberculosis. Newly diagnosed tuberculosis patients (n = 23), their close household contacts (n = 80), and community controls (n = 46) were tested at intervals over a period of up to two years. Latent infection or previous Mtb contact was assessed by ELISPOT and TST and complete blood counts were performed during the follow up. Results showed significant upregulation of FLIPs expression by infected individuals regardless of clinical status at entry to the study. A higher percentage of lymphocytes was found in the infected household contacts that remained healthy. In contrast, in individuals with active TB, a significant upregulation of TNFR2 expression, a significantly higher percentage of monocytes and a significantly decreased lymphocyte count were seen, compared to subjects that remained healthy. Moreover, the household contacts who subsequently developed signs of TB also had a significantly high number of monocytes. These data suggest tuberculosis may be associated with decreased T-cell survival (perhaps due to apoptosis) while inhibition of apoptosis in monocytes could lead to a relative increase in these cells: a situation predicted to favour Mtb

Evaluation of nanopore sequencing for Mycobacterium tuberculosis drug susceptibility testing and outbreak investigation: a genomic analysis

BACKGROUND: Mycobacterium tuberculosis whole-genome sequencing (WGS) has been widely used for genotypic drug susceptibility testing (DST) and outbreak investigation. For both applications, Illumina technology is used by most public health laboratories; however, Nanopore technology developed by Oxford Nanopore Technologies has not been thoroughly evaluated. The aim of this study was to determine whether Nanopore sequencing data can provide equivalent information to Illumina for transmission clustering and genotypic DST for M tuberculosis. METHODS: In this genomic analysis, we analysed 151 M tuberculosis isolates from Madagascar, South Africa, and England, which were collected between 2011 and 2018, using phenotypic DST and matched Illumina and Nanopore data. Illumina sequencing was done with the MiSeq, HiSeq 2500, or NextSeq500 platforms and Nanopore sequencing was done on the MinION or GridION platforms. Using highly reliable PacBio sequencing assemblies and pairwise distance correlation between Nanopore and Illumina data, we optimise Nanopore variant filters for detecting single-nucleotide polymorphisms (SNPs; using BCFtools software). We then used those SNPs to compare transmission clusters identified by Nanopore with the currently used UK Health Security Agency Illumina pipeline (COMPASS). We compared Illumina and Nanopore WGS-based DST predictions using the Mykrobe software and mutation catalogue. FINDINGS: The Nanopore BCFtools pipeline identified SNPs with a median precision of 99.3% (IQR 99.1-99.6) and recall of 90.2% (88.1-94.2) compared with a precision of 99.6% (99.4-99.7) and recall of 91.9% (87.6-98.6) using the Illumina COMPASS pipeline. Using a threshold of 12 SNPs for putative transmission clusters, Illumina identified 98 isolates as unrelated and 53 as belonging to 19 distinct clusters (size range 2-7). Nanopore reproduced 15 out of 19 clusters perfectly; two clusters were merged into one cluster, one cluster had a single sample missing, and one cluster had an additional sample adjoined. Illumina-based clusters were also closely replicated using a five SNP threshold and clustering accuracy was maintained using mixed Illumina and Nanopore datasets. Genotyping resistance variants with Nanopore was highly concordant with Illumina, having zero discordant SNPs across more than 3000 SNPs and four insertions or deletions (indels), across 60 000 indels. INTERPRETATION: Illumina and Nanopore technologies can be used independently or together by public health laboratories performing M tuberculosis genotypic DST and outbreak investigations. As a result, clinical and public health institutions making decisions on which sequencing technology to adopt for tuberculosis can base the choice on cost (which varies by country), batching, and turnaround time. FUNDING: Academy for Medical Sciences, Oxford Wellcome Institutional Strategic Support Fund, and the Swiss South Africa Joint Research Award (Swiss National Science Foundation and South African National Research Foundation)

Bi-directional drones to strengthen healthcare provision : experiences and lessons from Madagascar, Malawi and Senegal

Drones are increasingly being used globally for the support of healthcare programmes. Madagascar, Malawi and Senegal are among a group of early adopters piloting the use of bi-directional transport drones for health systems in sub-Saharan Africa. This article presents the experiences as well as the strengths, weaknesses, opportunities and threats (SWOT analysis) of these country projects. Methods for addressing regulatory, feasibility, acceptability, and monitoring and evaluation issues are presented to guide future implementations. Main recommendations for governments, implementers, drone providers and funders include (1) developing more reliable technologies, (2) thorough vetting of drone providers' capabilities during the selection process, (3) using and strengthening local capacity, (4) building in-country markets and businesses to maintain drone operations locally, (5) coordinating efforts among all stakeholders under government leadership, (6) implementing and identifying funding for long-term projects beyond pilots, and (7) evaluating impacts via standardised indicators. Sharing experiences and evidence from ongoing projects is needed to advance the use of drones for healthcare

Local adaptation in populations of Mycobacterium tuberculosis endemic to the Indian Ocean Rim

Background: Lineage 1 (L1) and 3 (L3) are two lineages of the Mycobacterium tuberculosis complex (MTBC) causing tuberculosis (TB) in humans. L1 and L3 are prevalent around the rim of the Indian Ocean, the region that accounts for most of the world's new TB cases. Despite their relevance for this region, L1 and L3 remain understudied. Methods: We analyzed 2,938 L1 and 2,030 L3 whole genome sequences originating from 69 countries. We reconstructed the evolutionary history of these two lineages and identified genes under positive selection. Results: We found a strongly asymmetric pattern of migration from South Asia toward neighboring regions, highlighting the historical role of South Asia in the dispersion of L1 and L3. Moreover, we found that several genes were under positive selection, including genes involved in virulence and resistance to antibiotics . For L1 we identified signatures of local adaptation at the esxH locus, a gene coding for a secreted effector that targets the human endosomal sorting complex, and is included in several vaccine candidates. Conclusions: Our study highlights the importance of genetic diversity in the MTBC, and sheds new light on two of the most important MTBC lineages affecting humans

Host-Directed Therapies for tackling Multi-Drug Resistant TB – learning from the Pasteur-Bechamp debates

Tuberculosis (TB) remains a global emergency causing an estimated 1.5 million deaths annually. For several decades the major focus of TB treatment has been on antibiotic development targeting Mycobacterium tuberculosis (M.tb). The lengthy TB treatment duration and poor treatment outcomes associated with multi-drug resistant TB (MDR-TB) are of major concern. The sparse new TB drug pipeline and widespread emergence of MDR-TB signal an urgent need for more innovative interventions to improve treatment outcomes. Building on the historical Pasteur-Bechamp debates on the role of the ‘microbe’ versus the ‘host internal milieu’ in disease causation, we make the case for parallel investments into host-directed therapies (HDTs). A range of potential HDTs are now available which require evaluation in randomized controlled clinical trials as adjunct therapies for shortening the duration of TB therapy and improving treatment outcomes for drug-susceptible TB and MDR-TB. Funder initiatives that may enable further research into HDTs are described

Towards host-directed therapies for tuberculosis

The treatment of tuberculosis is based on combinations of drugs that directly target Mycobacterium tuberculosis. A new global initiative is now focusing on a complementary approach of developing adjunct host-directed therapies. Despite the availability of effective antibiotics for tuberculosis (TB) for the past half century, it remains an important global health problem; there are ~9 million active TB cases and ~1.5 million TB-induced deaths per year (see the World Health Organization (WHO) Global Tuberculosis Report in Further information). Health services around the world face major barriers to achieving optimal outcomes from current TB treatment regimens. These barriers include: the spread of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB); complex and toxic treatment regimens for MDR-TB; HIV co-infection; pharmacokinetic interactions between TB drugs and antiretroviral drugs; relapse; permanent damage to lung and other tissues; long-term functional disability; immune reconstitution inflammatory syndrome (IRIS); and co-morbidity with non-communicable diseases such as diabetes and chronic obstructive airway diseases. Another fundamental problem is the long duration of TB drug treatment (6 months for drug-sensitive TB and at least 18 months for drug-resistant TB) to achieve a cure, owing to the presence of dormant Mycobacterium tuberculosis bacilli that are phenotypically resistant to current classes of anti-TB drugs, which can only target bacterial replication. There is therefore an urgent need for new TB treatments. However, the TB drug pipeline is thin1, 2. For the past 60 years, efforts to develop new treatments have focused on compounds and regimens that target M. tuberculosis directly. Recently, however, attention has focused on investigating a range of adjunct treatment interventions known as host-directed therapies (HDTs) that instead target the host response to infection. Here, we highlight the rationale for HDTs, the current portfolio of HDTs and their mechanisms of action, and a consortium-based approach to drive forward their evaluation in clinical trials

The 2021 WHO catalogue of Mycobacterium tuberculosis complex mutations associated with drug resistance: a genotypic analysis.

Background: Molecular diagnostics are considered the most promising route to achievement of rapid, universal drug susceptibility testing for Mycobacterium tuberculosis complex (MTBC). We aimed to generate a WHO-endorsed catalogue of mutations to serve as a global standard for interpreting molecular information for drug resistance prediction. Methods: In this systematic analysis, we used a candidate gene approach to identify mutations associated with resistance or consistent with susceptibility for 13 WHO-endorsed antituberculosis drugs. We collected existing worldwide MTBC whole-genome sequencing data and phenotypic data from academic groups and consortia, reference laboratories, public health organisations, and published literature. We categorised phenotypes as follows: methods and critical concentrations currently endorsed by WHO (category 1); critical concentrations previously endorsed by WHO for those methods (category 2); methods or critical concentrations not currently endorsed by WHO (category 3). For each mutation, we used a contingency table of binary phenotypes and presence or absence of the mutation to compute positive predictive value, and we used Fisher's exact tests to generate odds ratios and Benjamini-Hochberg corrected p values. Mutations were graded as associated with resistance if present in at least five isolates, if the odds ratio was more than 1 with a statistically significant corrected p value, and if the lower bound of the 95% CI on the positive predictive value for phenotypic resistance was greater than 25%. A series of expert rules were applied for final confidence grading of each mutation. Findings: We analysed 41 137 MTBC isolates with phenotypic and whole-genome sequencing data from 45 countries. 38 215 MTBC isolates passed quality control steps and were included in the final analysis. 15 667 associations were computed for 13 211 unique mutations linked to one or more drugs. 1149 (7·3%) of 15 667 mutations were classified as associated with phenotypic resistance and 107 (0·7%) were deemed consistent with susceptibility. For rifampicin, isoniazid, ethambutol, fluoroquinolones, and streptomycin, the mutations' pooled sensitivity was more than 80%. Specificity was over 95% for all drugs except ethionamide (91·4%), moxifloxacin (91·6%) and ethambutol (93·3%). Only two resistance mutations were identified for bedaquiline, delamanid, clofazimine, and linezolid as prevalence of phenotypic resistance was low for these drugs. Interpretation: We present the first WHO-endorsed catalogue of molecular targets for MTBC drug susceptibility testing, which is intended to provide a global standard for resistance interpretation. The existence of this catalogue should encourage the implementation of molecular diagnostics by national tuberculosis programmes. Funding: Unitaid, Wellcome Trust, UK Medical Research Council, and Bill and Melinda Gates Foundation

Detection of multidrug-resistant tuberculosis from stored DNA samples : a multicenter study

Background: In low-income countries, rapid detection of tuberculosis (TB) drug resistance is often restricted by the difficulties of transporting and storing sputum samples from remote health centers to the reference laboratories where molecular tests are available. The aim of this study was to evaluate the performance of four transport and storage systems for molecular detection of rifampicin (RIF) and isoniazid (INH) resistance. Methods: This was a multicenter study. Molecular detection of RIF and INH resistance was performed directly from smear-positive TB sputa spotted on a slide, FTA card, GenoCard, and ethanol using the Genotype MTBDRplus assay. The performance of the DNA extraction method from each storage support to detect drug resistance was assessed by calculating their sensitivity and specificity compared to the phenotypic method. Results: From all sites, the overall sensitivity and specificity for RIF-resistance detection was 88% and 85%, respectively, for slides, 86% and 92%, respectively, for GenoCard, 87% and 89%, respectively, for FTA card, and 88% and 92%, respectively, for ethanol. For INH-resistance detection, the overall sensitivity and specificity was 82% and 90%, respectively, for slides, 85% and 96%, respectively, for GenoCard, 86% and 92%, respectively, for FTA card, and 86% and 94%, respectively, for ethanol. Conclusion: Smear slides and filter cards showed to be very useful tools to facilitate DNA extraction from sputum samples with the potential to accelerate the detection of drug resistance in remote areas