Prediction of Susceptibility to First-Line Tuberculosis Drugs by DNA Sequencing.

BACKGROUND: The World Health Organization recommends drug-susceptibility testing of Mycobacterium tuberculosis complex for all patients with tuberculosis to guide treatment decisions and improve outcomes. Whether DNA sequencing can be used to accurately predict profiles of susceptibility to first-line antituberculosis drugs has not been clear. METHODS: We obtained whole-genome sequences and associated phenotypes of resistance or susceptibility to the first-line antituberculosis drugs isoniazid, rifampin, ethambutol, and pyrazinamide 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 profiles were predicted to be susceptible to all four drugs (i.e., pansusceptible) if they were predicted to be susceptible to isoniazid and to the other drugs or if they contained mutations of unknown association in genes that affect susceptibility to the other drugs. We simulated the way in which the negative predictive value changed with the prevalence of drug resistance. RESULTS: A total of 10,209 isolates were analyzed. The largest proportion of phenotypes was predicted for rifampin (9660 [95.4%] of 10,130) and the smallest was predicted for ethambutol (8794 [89.8%] of 9794). Resistance to isoniazid, rifampin, ethambutol, and pyrazinamide was correctly predicted with 97.1%, 97.5%, 94.6%, and 91.3% sensitivity, respectively, and susceptibility to these drugs was correctly predicted with 99.0%, 98.8%, 93.6%, and 96.8% specificity. Of the 7516 isolates with complete phenotypic drug-susceptibility profiles, 5865 (78.0%) had complete genotypic predictions, among which 5250 profiles (89.5%) were correctly predicted. Among the 4037 phenotypic profiles that were predicted to be pansusceptible, 3952 (97.9%) were correctly predicted. CONCLUSIONS: Genotypic predictions of the susceptibility of M. tuberculosis to first-line drugs were found to be correlated with phenotypic susceptibility to these drugs. (Funded by the Bill and Melinda Gates Foundation and others.).

Longitudinal Outbreak of Multidrug-Resistant Tuberculosis in a Hospital Setting, Serbia.

A retrospective population-based molecular epidemiologic study of multidrug-resistant Mycobacterium tuberculosis complex strains in Serbia (2008-2014) revealed an outbreak of TUR genotype strains in a psychiatric hospital starting around 1990. Drug unavailability, poor infection control, and schizophrenia likely fueled acquisition of additional resistance and bacterial fitness-related mutations over 2 decades.

Antibiotic resistance prediction for Mycobacterium tuberculosis from genome sequence data with Mykrobe.

Two billion people are infected with Mycobacterium tuberculosis, leading to 10 million new cases of active tuberculosis and 1.5 million deaths annually. Universal access to drug susceptibility testing (DST) has become a World Health Organization priority. We previously developed a software tool, Mykrobe predictor, which provided offline species identification and drug resistance predictions for M. tuberculosis from whole genome sequencing (WGS) data. Performance was insufficient to support the use of WGS as an alternative to conventional phenotype-based DST, due to mutation catalogue limitations.  Here we present a new tool, Mykrobe, which provides the same functionality based on a new software implementation. Improvements include i) an updated mutation catalogue giving greater sensitivity to detect pyrazinamide resistance, ii) support for user-defined resistance catalogues, iii) improved identification of non-tuberculous mycobacterial species, and iv) an updated statistical model for Oxford Nanopore Technologies sequencing data. Mykrobe is released under MIT license at https://github.com/mykrobe-tools/mykrobe. We incorporate mutation catalogues from the CRyPTIC consortium et al. (2018) and from Walker et al. (2015), and make improvements based on performance on an initial set of 3206 and an independent set of 5845 M. tuberculosis Illumina sequences. To give estimates of error rates, we use a prospectively collected dataset of 4362 M. tuberculosis isolates. Using culture based DST as the reference, we estimate Mykrobe to be 100%, 95%, 82%, 99% sensitive and 99%, 100%, 99%, 99% specific for rifampicin, isoniazid, pyrazinamide and ethambutol resistance prediction respectively. We benchmark against four other tools on 10207 (=5845+4362) samples, and also show that Mykrobe gives concordant results with nanopore data.  We measure the ability of Mykrobe-based DST to guide personalized therapeutic regimen design in the context of complex drug susceptibility profiles, showing 94% concordance of implied regimen with that driven by phenotypic DST, higher than all other benchmarked tools.

Pulmonary isolation and clinical relevance of nontuberculous mycobacteria during nationwide survey in Serbia, 2010-2015.

The rates of pulmonary colonization and disease due to nontuberculous mycobacteria (NTM) appear to be increasing globally, but diversity of species recovered as well as clinical relevance of NTM isolates differ considerably by geographic region. The first nationwide study of isolation frequency and clinical significance of NTM in Serbia included all patients with respiratory specimens yielding a positive NTM culture over the six-year period, 2010-2015. We analyzed trends in annual NTM isolation and NTM pulmonary disease (PD) incidence rates, with NTM PD cases defined in accordance with microbiological criteria established by the American Thoracic Society/Infectious Diseases Society of America (ATS/IDSA). 777 pulmonary NTM isolates were collected from 565 patients, of whom 126 (22.3%) met the ATS/IDSA criteria. The annual NTM isolation and NTM PD incidence rates per 100,000 changed over 2010-2015 from 0.9 to 1.6 (p = 0.1746) and from 0.18 to 0.48 (p = f0.0040), respectively. Both isolation and disease rates increased considerably with age, while higher NTM PD rates were also associated with residence in urbanized areas. Diversity of NTM species isolated was shown to be region-specific, with M. xenopi as the most prevalent species (17.3%), and increasing isolation rates of rapid growing mycobacteria (RGM) (p = 0.0072). M. xenopi was also the most common cause of NTM PD (28.6%), followed by RGM (27.8%). With 73% clinically relevant isolates, M. abscessus was identified as the most clinically relevant NTM species. While NTM PD obviously remains a rare disease in Serbia, the overall results justify recognition of NTM as pathogens of rising importance, and require further characterization of their epidemiology in the country.