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.
Bibliographical noteFunding Information: This work was supported by the Wellcome Trust/Newton Fund-Medical Research Council Collaborative Award (200205/Z/15/Z), Bill & Melinda Gates Foundation Trust (OPP1133541), and supported by a grant from Unitaid. This research was funded, in part, by the Wellcome Trust (200205/Z/15/Z). A complete funding statement can be found in appendix 1.
CUK is a consultant for Becton Dickinson, FIND, and the TB Alliance. CUK is collaborating with Janssen, PZA Innovation, and Thermo Fisher Scientific; worked as a consultant for QuantuMDx, the Stop TB Partnership, the WHO Global TB Programme, and the WHO Regional Office for Europe; and gave a paid educational talk for Oxford Immunotec. Hain Lifescience covered CUK's travel and accommodation to present at a meeting. CUK is an unpaid adviser to BioVersys and GenoScreen. ERR is employed by the UK Health Security Agency (UKHSA) and holds an honorary contract with Imperial College London. IFL is director of the Scottish Mycobacteria Reference Laboratory. SN receives funding from German Center for Infection Research, Excellenz Cluster Precision Medicine in Chronic Inflammation, and Leibniz Science Campus Evolutionary Medicine of the LUNG (EvoLUNG)tion EXC 2167. PS is a consultant at Genoscreen. TR is funded by the National Institutes for Health and US Department of Defence and receives salary support from the non-profit organisation FIND. TR is a cofounder, board member, and shareholder of Verus Diagnostics, a company that was founded with the intent of developing diagnostic assays. Verus Diagnostics was not involved in any way with data collection, analysis, or publication of the results, and TR has not received any financial support from Verus Diagnostics. University of California, San Diego (UCSD) Conflict of Interest office has reviewed and approved TR's role in Verus Diagnostics. TR is a coinventor of a provisional patent for a TB diagnostic assay (provisional patent 63/048.989). TR is a coinventor on a patent associated with the processing of tuberculosis sequencing data (European Patent Application 14840432.0 & USSN 14/912,918). TR has agreed to “donate all present and future interest in and rights to royalties from this patent” to UCSD to ensure that he does not receive any financial benefits from this patent. SS works and holds stock options at HaystackAnalytics (Product: Using whole genome sequencing for drug susceptibility testing for Mycobacterium tuberculosis). GFG is listed as an inventor on patent applications for RBD-dimer-based coronavirus vaccines. The patents for these RBD dimers as protein subunit vaccines for SARS-CoV-2 have been licensed to Anhui Zhifei Longcom Biopharmaceutical. IC is a consultant for FIND. DAC reports funding from GlaxoSmithKline and consultancy fees from Biobeats, Oxford University Innovation, and Sensyne Health. CC reports funding from FIND to his institution (Pathology Queensland, Queensland Department of Health) for his laboratory to perform molecular analytic studies (limits of detection) for new molecular platforms manufactured by Cepheid and Bioneer. The other authors declare no competing interests.
Open Access: This is an Open Access article under the CC BY 4.0 license.
Publisher Copyright: © 2022 The Author(s). Published by Elsevier Ltd.
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