Advantages of long- and short-reads sequencing for the hybrid investigation of the Mycobacterium tuberculosis genome

Introduction: In the fight to limit the global spread of antibiotic resistance, computational challenges associated with sequencing technology can impact the accuracy of downstream analysis, including drug resistance identification, transmission, and genome resolution. About 10% of Mycobacterium tuberculosis (MTB) genome is constituted by the PE/PPE family, a GC-rich repetitive genome region. Although sequencing using short read technology is widely used, it is well recognized its limit in the PE/PPE regions due to the unambiguously mapping process onto the reference genome. The aim of this study was to compare the performances of short-reads (SRS), long-reads (LRS) and hybrid-reads (HYBR) based analysis over different common investigative tasks: genome coverage estimation, variant calling and cluster analysis, drug resistance detection and de novo assembly. Methods: For the study 13 model MTB clinical isolates were sequenced with both SRS and LRS. HYBR were produced correcting the long reads with the short reads. The fastq from the three approaches were then processed using a customized version of MTBseq for genome coverage estimation and variant calling and using two different assemblers for de novo assembly evaluation. Results: Estimation of genome coverage performances showed lower 8X breadth coverage for SRS respect to LRS and HYBR: considering the PE/PPE genes, SRS showed low results for the PE_PGRS family, while obtained acceptable coverage in PE and PPE genes; LRS and HYBR reached optimal coverages in PE/PPE genes. For variant calling HYBR showed the highest resolution, detecting the highest percentage of uniquely identified mutations compared to LRS and SRS. All three approaches agreed on the identification of two major clusters, with HYBR identifying an higher number of SNPs between the two clusters. Comparing the quality of the assemblies, HYBR and LRS obtained better results than SRS. Discussion: In conclusion, depending on the aim of the investigation, both SRS and LRS present complementary advantages and limitations implying that for a full resolution of MTB genomes, where all the mentioned analyses and both technologies are needed, the use of the HYBR approach represents a valid option and a well-rounded strategy

Genetic sequencing for surveillance of drug resistance in tuberculosis in highly endemic countries: A multi-country population-based surveillance study

Background: In many countries, regular monitoring of the emergence of resistance to anti-tuberculosis drugs is hampered by the limitations of phenotypic testing for drug susceptibility. We therefore evaluated the use of genetic sequencing for surveillance of drugresistance in tuberculosis.Methods: Population-level surveys were done in hospitals and clinics in seven countries (Azerbaijan, Bangladesh, Belarus, Pakistan, Philippines, South Africa, and Ukraine) to evaluate the use of genetic sequencing to estimate the resistance of Mycobacterium tuberculosisisolates to rifampicin, isoniazid, ofloxacin, moxifloxacin, pyrazinamide, kanamycin, amikacin, and capreomycin. For each drug, we assessed the accuracy of genetic sequencing by a comparison of the adjusted prevalence of resistance, measured by genetic sequencing, with the true prevalence of resistance, determined by phenotypic testing.Findings: Isolates were taken from 7094 patients with tuberculosis who were enrolled in the study between November, 2009, and May, 2014. In all tuberculosis cases, the overall pooled sensitivity values for predicting resistance by genetic sequencing were 91% (95% CI 87-94) for rpoB (rifampicin resistance), 86% (74-93) for katG, inhA, and fabG promoter combined (isoniazid resistance), 54% (39-68) for pncA (pyrazinamide resistance), 85% (77-91) for gyrA and gyrB combined (ofloxacin resistance), and 88% (81-92) for gyrA and gyrB combined (moxifloxacin resistance). For nearly all drugs and in most settings, there was a large overlap in the estimated prevalence of drug resistanceby genetic sequencing and the estimated prevalence by phenotypic testing.Interpretation: Genetic sequencing can be a valuable tool for surveillance of drug resistance, providing new opportunities to monitor drug resistance in tuberculosis in resource-poor countries. Before its widespread adoption for surveillance purposes, there is a need to standardise DNA extraction methods, recording and reporting nomenclature, and data interpretation.Findings: Bill & Melinda Gates Foundation, United States Agency for International Development, Global Alliance for Tuberculosis DrugDevelopment

Anti-tuberculosis drug resistance in Slovakia, 2018–2019: The first whole-genome epidemiological study

Objective: The resistance of Mycobacterium (M.) tuberculosis to antituberculosis drugs poses a major threat to global public health. Whole genome sequencing (WGS) is an increasingly preferred method in the diagnostics and monitoring of the transmission dynamics of resistant forms of tuberculosis (TB). The aim of the study was to, for the first time, use the sequencing-based analysis to study the transmission and resistance patterns of a systematic and recent collection of extensively drug resistant (XDR) and multidrug resistant tuberculosis (MDR-TB) isolates and to expand our knowledge about drug resistant (DR) TB epidemiological dynamics in Slovakia.Design: A total of 495 patients with pulmonary TB, who were referred to National Reference Laboratory for Mycobacteriology (Vysne Hagy, Slovakia) in the years 2018-2019, were studied. Out of the total of 495 patients, 4 XDR-TB (0.8%) and 8 (1.6%) MDR-TB isolates were identified by conventional drug susceptibility testing on Lowenstein-Jensen solid medium and subjected to whole genome sequencing. Sequencing data were evaluated for molecular-epidemiological analysis and identification of resistance patterns.Results: Phylogenetic and cluster analysis showed extensive recent transmission events and the predominance of Euro-American lineage 4.7 in Slovakia. However, phylogenetic analysis revealed the circulation of several lineages that originally occurred in Eastern European countries. Resistance patterns for first- and second-line antituberculosis drugs characterized by whole genome sequencing were in high concordance with the results of phenotypic drug susceptibility testing.Conclusion: Forty percent of at least MDR-TB isolates were not genetically linked, indicating that appropriate measures should be taken to monitor and prevent the spread of drug-resistant tuberculosis within the country as well as in other regions

Countrywide implementation of whole genome sequencing: an opportunity to improve tuberculosis management, surveillance and contact tracing in low incidence countries

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Update on the diagnosis of tuberculosis

Background Tuberculosis remains a global public health threat, and the development of rapid and precise diagnostic tools is the key to enabling the early start of treatment, monitoring response to treatment, and preventing the spread of the disease. Objective An overview of recent progress in host- and pathogen-based tuberculosis diagnostics. Sources We conducted a PubMed search of recent relevant articles and guidelines on tuberculosis screening and diagnosis. Content An overview of currently used methods and perspectives in the following areas of tuberculosis diagnostics is provided: immune-based diagnostics, X-ray, clinical symptoms and scores, cough detection, culture of Mycobacterium tuberculosis and identifying its resistance profile using phenotypic and genotypic methods, including next generation sequencing, sputum- and non-sputum-based molecular diagnosis of tuberculosis and monitoring of response to treatment. Implications A brief overview of the most relevant advances and changes in international guidelines regarding screening and diagnosing tuberculosis is provided in this review. It aims at reviewing all relevant areas of diagnostics, including both pathogen- and host-based methods.PostprintPeer reviewe

Characterization of Genomic Variants Associated with Resistance to Bedaquiline and Delamanid in Naive Mycobacterium tuberculosis Clinical Strains

The role of mutations in genes associated with phenotypic resistance to bedaquiline (BDQ) and delamanid (DLM) in Mycobacterium tuberculosis complex (MTBc) strains is poorly characterized. A clear understanding of the genetic variants' role is crucial to guide the development of molecular-based drug susceptibility testing (DST). In this work, we analyzed all mutations in candidate genomic regions associated with BDQ- and DLM-resistant phenotypes using a whole-genome sequencing (WGS) data set from a collection of 4,795 MTBc clinical isolates from six countries with a high burden of tuberculosis (TB). From WGS analysis, we identified 61 and 163 unique mutations in genomic regions potentially involved in BDQ- and DLM-resistant phenotypes, respectively. Importantly, all strains were isolated from patients who likely have never been exposed to these medicines. To characterize the role of mutations, we calculated the free energy variation upon mutations in the available protein structures of Ddn (DLM), Fgd1 (DLM), and Rv0678 (BDQ) and performed MIC assays on a subset of MTBc strains carrying mutations to assess their phenotypic effect. The combination of structural and phenotypic data allowed for cataloguing the mutations clearly associated with resistance to BDQ (n = 4) and DLM (a = 35), only two of which were previously described, as well as about a hundred genetic variants without any correlation with resistance. Significantly, these results show that both BDQ and DLM resistance related mutations are diverse and distributed across the entire region of each gene target, which is of critical importance for the development of comprehensive molecular diagnostic tools

Exportation of MDR TB to europe from setting with actively transmitted persistent strains in peru

We performed a cross-border molecular epidemiology analysis of multidrug-resistant tuberculosis in Peru, Spain, and Italy. This analysis revealed frequent transmission in Peru and exportation of a strain that recreated similar levels of transmission in Europe during 2007–2017. Transnational efforts are needed to control transmission of multidrug-resistant tuberculosis globally

Prevalence and genetic profiles of isoniazid resistance in tuberculosis patients: A multicountry analysis of cross-sectional data.

BACKGROUND: The surveillance of drug resistance among tuberculosis (TB) patients is central to combatting the global TB epidemic and preventing the spread of antimicrobial resistance. Isoniazid and rifampicin are two of the most powerful first-line anti-TB medicines, and resistance to either of them increases the risk of treatment failure, relapse, or acquisition of resistance to other drugs. The global prevalence of rifampicin resistance is well documented, occurring in 3.4% (95% CI 2.5%-4.4%) of new TB patients and 18% (95% CI 7.6%-31%) of previously treated TB patients in 2018, whereas the prevalence of isoniazid resistance at global and regional levels is less understood. In 2018, the World Health Organization (WHO) recommended a modified 6-month treatment regimen for people with isoniazid-resistant, rifampicin-susceptible TB (Hr-TB), which includes rifampicin, pyrazinamide, ethambutol, and levofloxacin. We estimated the global prevalence of Hr-TB among TB patients and investigated associated phenotypic and genotypic drug resistance patterns. METHODS AND FINDINGS: Aggregated drug resistance data reported to WHO from either routine continuous surveillance or nationally representative periodic surveys of TB patients for the period 2003-2017 were reviewed. Isoniazid data were available from 156 countries or territories for 211,753 patients. Among these, the global prevalence of Hr-TB was 7.4% (95% CI 6.5%-8.4%) among new TB patients and 11.4% (95% CI 9.4%-13.4%) among previously treated TB patients. Additional data on pyrazinamide and levofloxacin resistance were available from 6 countries (Azerbaijan, Bangladesh, Belarus, Pakistan, the Philippines, and South Africa). There were no cases of resistance to both pyrazinamide and levofloxacin among Hr-TB patients, except for the Philippines (1.8%, 95% CI 0.2-6.4) and Belarus (5.3%, 95% CI 0.1-26.0). Sequencing data for all genomic regions involved in isoniazid resistance were available for 4,563 patients. Among the 1,174 isolates that were resistant by either phenotypic testing or sequencing, 78.6% (95% CI 76.1%-80.9%) had resistance-conferring mutations in the katG gene and 14.6% (95% CI 12.7%-16.8%) in both katG and the inhA promoter region. For 6.8% (95% CI 5.4%-8.4%) of patients, mutations occurred in the inhA promoter alone, for whom an increased dose of isoniazid may be considered. The main limitations of this study are that most analyses were performed at the national rather than individual patient level and that the quality of laboratory testing may vary between countries. CONCLUSIONS: In this study, the prevalence of Hr-TB among TB patients was higher than the prevalence of rifampicin resistance globally. Many patients with Hr-TB would be missed by current diagnostic algorithms driven by rifampicin testing, highlighting the need for new rapid molecular technologies to ensure access to appropriate treatment and care. The low prevalence of resistance to pyrazinamide and fluoroquinolones among patients with Hr-TB provides further justification for the recommended modified treatment regimen

Triage test for all-oral drug-resistant tuberculosis (DR-TB) regimen : a phase IV study to assess effectiveness, feasibility, acceptability and cost-effectiveness of the Xpert MTB/XDR assay for rapid triage and treatment of DR-TB

This publication was produced by the TRiAD study, which is part of the EDCTP2 programme supported by the European Union (Grant RIA2019-2888-TRIAD).INTRODUCTION : The TriAD study will assess the Xpert MTB/XDR (Xpert XDR; Cepheid) assay to detect tuberculosis (TB) drug resistance in sputum testing positive for TB to rapidly triage and treat patients with a short all-oral treatment regimen. METHODS AND ANALYSIS : In this study, approximately 4800 Xpert MTB/RIF or Ultra MTB-positive patients (irrespective of rifampicin (RIF) resistance (RR) status) from several clinical sites across South Africa, Nigeria and Ethiopia will be enrolled over 18-24 months and followed-up for approximately 6 months post-TB treatment completion. Participants will be enrolled into one of two cohorts based on Xpert MTB/RIF and Xpert XDR results: Mycobacterium tuberculosis ( M.tb) positive participants with RR in Cohort 1 (n=880) and M.tb positive RIF susceptible TB patients with isoniazid mono-resistance irrespective of presence of resistance to fluoroquinolones, second-line injectable drugs or ethionamide in Cohort 2 (n=400). Cohort 1 will be compared with historical cohorts from each implementing sites. The primary study outcomes include time to initiation of an appropriate treatment regimen by resistance profile and the proportion of patients with favourable treatment outcomes compared with historical cohorts from each of the implementing sites. Secondary outcomes include feasibility, acceptability and cost-effectiveness of this approach to inform policies and guidelines for programmatic implementation of this triage and treat model for drug-resistant tuberculosis management. Utility of the tuberculosis molecular bacterial load assay (TB-MBLA) for real-time treatment response assessment will also be evaluated. ETHICS AND DISSEMINATION : The University of KwaZulu-Natal Biomedical Research Ethics Committee (BREC) and local research committees have provided ethical review and approval (BREC/00002654/2021, HREC 210805, NHREC/01/01/2007 and EPHI-IRB-459-2022). The South African Health Products Regulatory Authority (SAHPRA) have granted regulatory approval for the TRiAD Study (SAHPRA MD20211001). Trial results will be disseminated through conference presentations, peer-reviewed publications and the clinical trial registry. TRIAL REGISTRATION NUMBER : Clinicaltrials.gov; Trial registration number: NCT05175794; South African National Clinical Trials Register (SANCTR DOH-27-012022-4720).Peer reviewe