Whole genome sequencing (WGS) of Mycobacterium tuberculosis has rapidly evolved from a research tool to a clinical application for the diagnosis and management of tuberculosis and in public health surveillance. This evolution has been facilitated by the dramatic drop in costs, advances in technology, and concerted efforts to translate sequencing data into actionable information. There is however a risk that, in the absence of a consensus and international standards, the widespread use of WGS technology may result in data and processes that lack harmonisation, comparability and validation. In this review, we outline the current landscape of WGS pipelines and applications and set out best practices for M. tuberculosis WGS, including standards for bioinformatics pipelines, curated repository of resistance-causing variants, phylogenetic analyses, quality control processes, and standardised reporting. 1. Introduction Mycobacterium tuberculosis complex (Mtbc) pathogens are collectively the top infectious disease killer globally, causing 10 million new tuberculosis (TB) cases annually 1. Increasingly, 95 new TB cases are already resistant to rifampicin and isoniazid (termed multidrug resistance; 96 MDR-TB), the key first line drugs 1. Tackling the spread and drug resistance burden of this pathogen requires concerted global effort in prevention, diagnosis, treatment and surveillance.

BackgroundThe World Health Organization recommends universal drug susceptibility testing for Mycobacterium tuberculosis complex to guide treatment decisions and improve outcomes. We assessed whether DNA sequencing can accurately predict antibiotic susceptibility profiles for first-line anti-tuberculosis drugs. MethodsWhole-genome sequences and associated phenotypes to isoniazid, rifampicin, ethambutol and pyrazinamide were obtained for isolates from 16 countries across six continents. For each isolate, mutations associated with drug-resistance and drug-susceptibility were identified across nine genes, and individual phenotypes were predicted unless mutations of unknown association were also present. To identify how whole-genome sequencing might direct first-line drug therapy, complete susceptibility profiles were predicted. These were predicted to be pan-susceptible if predicted susceptible to isoniazid and to other drugs, or contained mutations of unknown association in genes affecting these other drugs. We simulated how negative predictive value changed with drug-resistance prevalence.Results10,209 isolates were analysed. The greatest proportion of phenotypes were predicted for rifampicin (9,660/10,130; (95.4%)) and the lowest for ethambutol (8,794/9,794; (89.8%)). Isoniazid, rifampicin, ethambutol and pyrazinamide resistance was correctly predicted with 97.1%, 97.5% 94.6% and 91.3% sensitivity, and susceptibility with 99.0%, 98.8%, 93.6% and 96.8% specificity, respectively. 5,250 (89.5%) drug profiles were correctly predicted for 5,865/7,516 (78.0%) isolates with complete phenotypic profiles. Among these, 3,952/4,037 (97.9%) predictions of pan-susceptibility were correct. The negative predictive value for 97.5% of simulated drug profiles exceeded 95% where the prevalence of drug-resistance was below 47.0%. ConclusionsPhenotypic testing for first-line drugs can be phased down in favour of DNA sequencing to guide anti- tuberculosis drug therapy.

Antibiotic-resistant tuberculosis poses a global threat, causing the deaths of hundreds of thousands of people annually. While whole-genome sequencing (WGS), with its unprecedented level of detail, promises to play an increasingly important role in diagnosis, data analysis is a daunting challenge. Here, we present a simple-to-use web service (free for academic use at http://phyresse .org). Delineating both lineage and resistance, it provides state-of-the-art methodology to life scientists and physicians untrained in bioinformatics. It combines elaborate data processing and quality control, as befits human diagnostics, with a treasure trove of validated resistance data collected from well-characterized samples in-house and worldwide.