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Summary: 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.

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 ( n = 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., (Copyright © 2020 American Society for Microbiology.)

Setting: National Institute of Diseases of the Chest and Hospital, Dhaka; Bangladesh Institute of Research and Rehabilitation in Diabetes, Endocrine and Metabolic Disorders, Dhaka; and Chittagong Chest Disease Hospital, Chittagong, Bangladesh., Objective: To present operational data and discuss the challenges of implementing FAST (Find cases Actively, Separate safely and Treat effectively) as a tuberculosis (TB) transmission control strategy., Design: FAST was implemented sequentially at three hospitals., Results: Using Xpert® MTB/RIF, 733/6028 (12.2%, 95%CI 11.4-13.0) patients were diagnosed with unsuspected TB. Patients with a history of TB who were admitted with other lung diseases had more than twice the odds of being diagnosed with unsuspected TB as those with no history of TB (OR 2.6, 95%CI 2.2-3.0, P < 0.001). Unsuspected multidrug-resistant TB (MDR-TB) was diagnosed in 89/1415 patients (6.3%, 95%CI 5.1-7.7). Patients with unsuspected TB had nearly five times the odds of being diagnosed with MDR-TB than those admitted with a known TB diagnosis (OR 4.9, 95%CI 3.1-7.6, P < 0.001). Implementation challenges include staff shortages, diagnostic failure, supply-chain issues and reliance on external funding., Conclusion: FAST implementation revealed a high frequency of unsuspected TB in hospitalized patients in Bangladesh. Patients with a previous history of TB have an increased risk of being diagnosed with unsuspected TB. Ensuring financial resources, stakeholder engagement and laboratory capacity are important for sustainability and scalability.

The present study was an attempt to establish a suitable method for the effective diagnosis of extra-pulmonary tuberculosis in Bangladesh. In this regard, detection of Mycobacterium tuberculosis from 390 different extra-pulmonary specimens was performed by Bright-Field microscopy, light-emitting diode fluorescence microscopy and Lowenstein-Jensen culture methods, followed by an extensive comparison among these methods. M. tuberculosis was detected in 53 cases through the conventional Lowenstein-Jensen culture method; 49 cases were detected under Bright-Field microscope, whereas the light-emitting diode fluorescence microscopy detected 64 cases. Out of 53 culture-positive isolates, 12 were found to be multi-drug resistant. Light-emitting diode fluorescence microscopy was found to be more sensitive and effective than both the Bright-Field microscopy and the Lowenstein-Jensen culture methods. Incidentally, light-emitting diode fluorescence microscopy appeared imperative to detecting the multi-drug resistant tuberculosis., (Copyright © 2012 Asian-African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved.)