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.

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BackgroundTracking recent transmission is a vital part of controlling widespread pathogens such as Mycobacterium tuberculosis. Multiple methods with specific performance characteristics exist for detecting recent transmission chains, usually by clustering strains based on genotype similarities. With such a large variety of methods available, informed selection of an appropriate approach for determining transmissions within a given setting/time period is difficult.MethodsThis study combines whole genome sequence (WGS) data derived from 324 isolates collected 2005–2010 in Kinshasa, Democratic Republic of Congo (DRC), a high endemic setting, with phylodynamics to unveil the timing of transmission events posited by a variety of standard genotyping methods. Clustering data based on Spoligotyping, 24-loci MIRU-VNTR typing, WGS based SNP (Single Nucleotide Polymorphism) and core genome multi locus sequence typing (cgMLST) typing were evaluated.FindingsOur results suggest that clusters based on Spoligotyping could encompass transmission events that occurred almost 200 years prior to sampling while 24-loci-MIRU-VNTR often represented three decades of transmission. Instead, WGS based genotyping applying low SNP or cgMLST allele thresholds allows for determination of recent transmission events, e.g. in timespans of up to 10 years for a 5 SNP/allele cut-off.InterpretationWith the rapid uptake of WGS methods in surveillance and outbreak tracking, the findings obtained in this study can guide the selection of appropriate clustering methods for uncovering relevant transmission chains within a given time-period. For high resolution cluster analyses, WGS-SNP and cgMLST based analyses have similar clustering/timing characteristics even for data obtained from a high incidence setting.

Bacterial factors favoring the unprecedented multidrug-resistant tuberculosis (MDR-TB) epidemic in the former Soviet Union remain unclear. We utilized whole genome sequencing and Bayesian statistics to analyze the evolutionary history, temporal emergence of resistance and transmission networks of MDR Mycobacterium tuberculosis complex isolates from Karakalpakstan, Uzbekistan (2001–2006). One clade (termed Central Asian outbreak, CAO) dating back to 1974 (95% HPD 1969–1982) subsequently acquired resistance mediating mutations to eight anti-TB drugs. Introduction of standardized WHO-endorsed directly observed treatment, short-course in Karakalpakstan in 1998 likely selected for CAO-strains, comprising 75% of sampled MDR-TB isolates in 2005/2006. CAO-isolates were also identified in a published cohort from Russia (2008–2010). Similarly, the presence of mutations supposed to compensate bacterial fitness deficits was associated with transmission success and higher drug resistance rates. The genetic make-up of these MDR-strains threatens the success of both empirical and standardized MDR-TB therapies, including the newly WHO-endorsed short MDR-TB regimen in Uzbekistan.