Universal genotyping reveals province-level differences in the molecular epidemiology of tuberculosis.

OBJECTIVES: Compare the molecular epidemiology of tuberculosis (TB) between two large Canadian provinces-Ontario and British Columbia (BC)-to identify genotypic clusters within and across both provinces, allowing for an improved understanding of genotype data and providing context to more accurately identify clusters representing local transmission. DESIGN: We compared 24-locus Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats (MIRU-VNTR) genotyping for 3,314 Ontario and 1,602 BC clinical Mycobacterium tuberculosis isolates collected from 2008 through 2014. Laboratory data for each isolate was linked to case-level records to obtain clinical and demographic data. RESULTS: The demographic characteristics of persons with TB varied between provinces, most notably in the proportion of persons born outside Canada, which was reflected in the large number of unique genotypes (n = 3,461). The proportion of clustered isolates was significantly higher in BC. Substantial clustering amongst non-Lineage 4 TB strains was observed within and across the provinces. Only two large clusters (≥10 cases/cluster) representing within province transmission had interprovincial genotype matches. CONCLUSION: We recommend expanding analysis of shared genotypes to include neighbouring jurisdictions, and implementing whole genome sequencing to improve identification of TB transmission, recognize outbreaks, and monitor changing trends in TB epidemiology.

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.).

Genotyping and Whole-Genome Sequencing to Identify Tuberculosis Transmission to Pediatric Patients in British Columbia, Canada, 2005-2014.

Background: Tuberculosis (TB) in children is often an indicator of recent transmission. Genotyping and whole-genome sequencing (WGS) can enhance pediatric TB investigations by confirming or refuting transmission events. Methods: Mycobacterium tuberculosis isolates from all pediatric patients <18 years with culture-confirmed TB in British Columbia (BC) from 2005 to 2014 (n = 49) were genotyped by Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeat (MIRU-VNTR) and compared with adult isolates. Genotypically clustered cases underwent WGS. Clinical, demographic, and contact data were reviewed for each case. Results: Twenty-three children were Canadian-born, 7 to Canadian-born parents (CBP) and 16 to foreign-born parents (FBP). Of the 26 foreign-born children, all were born in Asia (81%) or Africa (19%). Using molecular and epidemiological data, we determined that 15 children had acquired their infection within BC, and household transmission explained all 7 Canadian-born (FBP) children that acquired TB locally. In contrast, 6 of 7 Canadian-born (CBP) children were exposed via a non-household community source. Eight Canadian-born (FBP) children acquired their infections through travel to their parents' place of birth. All but 1 of the foreign-born children acquired their infection outside of BC. Conclusions: Genotyping and genomic data reveal that drivers of pediatric transmission vary according to a child's age, birthplace, and their parents' place of birth.

Universal Genotyping for Tuberculosis Prevention Programs: a 5-Year Comparison with On-Request Genotyping.

Prospective universal genotyping of tuberculosis (TB) isolates is used by many laboratories to detect clusters of cases and inform contact investigations. Prior to universal genotyping, most TB prevention programs genotyped isolates on request only, relying on requests from public health professionals whose knowledge of a patient's clinical, demographic, and epidemiological characteristics suggested potential transmission. To justify the switch from on-request to universal genotyping-particularly in the public health domain, with its limited resources and competing priorities-it is important to demonstrate the additional benefit provided by a universal genotyping program. We compared the clustering patterns revealed by retrospective 24-locus mycobacterial interspersed repetitive unit-variable-number tandem repeat genotyping of all culture-positive isolates over a 5-year period to the patterns previously established by our genotyping-on-request program in the low-incidence setting of British Columbia, Canada. We found that 23.8% of isolates were requested during the study period, and while requested isolates had increased odds of belonging to a genotype cluster (adjusted odds ratio, 2.3; 95% confidence interval, 1.5 to 3.3), only 54.6% clustered with the requested comparator strain. Universal genotyping revealed 94 clusters ranging in size from 2 to 53 isolates (mean = 5) and involving 432 individuals. On-request genotyping missed 54 (57.4%) of these clusters and 130 (30.1%) clustered individuals. Our results underscore that TB patient networks are complex, with unrecognized linkages between patients, and a prospective province-wide universal genotyping program provides an informative, bias-free tool to explore transmission to a degree not possible with on-request genotyping.

Molecular Epidemiology of Tuberculosis in British Columbia, Canada: A 10-Year Retrospective Study.

Background: Understanding regional molecular epidemiology allows for the development of more efficient tuberculosis prevention strategies in low-incidence settings. Methods: We analyzed 24-locus mycobacterial interspersed repetitive-unit-variable-number tandem repeat (MIRU-VNTR) genotyping for 2290 Mycobacterium tuberculosis clinical isolates collected in the province of British Columbia (BC), Canada, in 2005-2014. Laboratory data for each isolate were linked to case-level clinical and demographic data. These data were used to describe the molecular epidemiology of tuberculosis across the province. Results: We detected >1500 distinct genotypes across the 4 major M. tuberculosis lineages, reflecting BC's diverse population. Disease site and clustering rates varied across lineages, and MIRU-VNTR was used to group the 2290 isolates into 189 clusters (2-70 isolates per cluster), with an overall clustering rate of 42.4% and an estimated local transmission rate of 34.1%. Risk factors for clustering varied between Canadian-born and foreign-born individuals; the former had increased odds (odds ratio, 7.8; 95% confidence interval [CI], 6.2-9.6) of belonging to a genotypic cluster, although nearly one-quarter of clusters included both Canadian- and foreign-born persons. Large clusters (≥10 cases) occurred more frequently within the M. tuberculosis Euro-American lineage, and individual-level risk factors associated with belonging to a large cluster included being Canadian born (adjusted odds ratio, 3.3; 95% CI, 2.3-4.8), residing in a rural area (2.3; 1.2-4.5), and illicit drug use (2.0; 1.2-3.4). Conclusions: Although tuberculosis in BC largely arises through reactivation of latent tuberculosis in foreign-born persons, locally transmitted infections occur in discrete populations with distinct disease and risk factor profiles, representing groups for targeted interventions.

Rapid, comprehensive, and affordable mycobacterial diagnosis with whole-genome sequencing: a prospective study.

BACKGROUND: Slow and cumbersome laboratory diagnostics for Mycobacterium tuberculosis complex (MTBC) risk delayed treatment and poor patient outcomes. Whole-genome sequencing (WGS) could potentially provide a rapid and comprehensive diagnostic solution. In this prospective study, we compare real-time WGS with routine MTBC diagnostic workflows. METHODS: We compared sequencing mycobacteria from all newly positive liquid cultures with routine laboratory diagnostic workflows across eight laboratories in Europe and North America for diagnostic accuracy, processing times, and cost between Sept 6, 2013, and April 14, 2014. We sequenced specimens once using local Illumina MiSeq platforms and processed data centrally using a semi-automated bioinformatics pipeline. We identified species or complex using gene presence or absence, predicted drug susceptibilities from resistance-conferring mutations identified from reference-mapped MTBC genomes, and calculated genetic distance to previously sequenced UK MTBC isolates to detect outbreaks. WGS data processing and analysis was done by staff masked to routine reference laboratory and clinical results. We also did a microcosting analysis to assess the financial viability of WGS-based diagnostics. FINDINGS: Compared with routine results, WGS predicted species with 93% (95% CI 90-96; 322 of 345 specimens; 356 mycobacteria specimens submitted) accuracy and drug susceptibility also with 93% (91-95; 628 of 672 specimens; 168 MTBC specimens identified) accuracy, with one sequencing attempt. WGS linked 15 (16% [95% CI 10-26]) of 91 UK patients to an outbreak. WGS diagnosed a case of multidrug-resistant tuberculosis before routine diagnosis was completed and discovered a new multidrug-resistant tuberculosis cluster. Full WGS diagnostics could be generated in a median of 9 days (IQR 6-10), a median of 21 days (IQR 14-32) faster than final reference laboratory reports were produced (median of 31 days [IQR 21-44]), at a cost of £481 per culture-positive specimen, whereas routine diagnosis costs £518, equating to a WGS-based diagnosis cost that is 7% cheaper annually than are present diagnostic workflows. INTERPRETATION: We have shown that WGS has a scalable, rapid turnaround, and is a financially feasible method for full MTBC diagnostics. Continued improvements to mycobacterial processing, bioinformatics, and analysis will improve the accuracy, speed, and scope of WGS-based diagnosis. FUNDING: National Institute for Health Research, Department of Health, Wellcome Trust, British Colombia Centre for Disease Control Foundation for Population and Public Health, Department of Clinical Microbiology, Trinity College Dublin.