E. Grace Smith

Whole-genome sequencing to delineate Mycobacterium tuberculosis outbreaks: a retrospective observational study

Summary Background Tuberculosis incidence in the UK has risen in the past decade. Disease control depends on epidemiological data, which can be difficult to obtain. Whole-genome sequencing can detect microevolution within Mycobacterium tuberculosis strains. We aimed to estimate the genetic diversity of related M tuberculosis strains in the UK Midlands and to investigate how this measurement might be used to investigate community outbreaks. Methods In a retrospective observational study, we used Illumina technology to sequence M tuberculosis genomes from an archive of frozen cultures. We characterised isolates into four groups: cross-sectional, longitudinal, household, and community. We measured pairwise nucleotide differences within hosts and between hosts in household outbreaks and estimated the rate of change in DNA sequences. We used the findings to interpret network diagrams constructed from 11 community clusters derived from mycobacterial interspersed repetitive-unit–variable-number tandem-repeat data. Findings We sequenced 390 separate isolates from 254 patients, including representatives from all five major lineages of M tuberculosis. The estimated rate of change in DNA sequences was 0·5 single nucleotide polymorphisms (SNPs) per genome per year (95% CI 0·3–0·7) in longitudinal isolates from 30 individuals and 25 families. Divergence is rarely higher than five SNPs in 3 years. 109 (96%) of 114 paired isolates from individuals and households differed by five or fewer SNPs. More than five SNPs separated isolates from none of 69 epidemiologically linked patients, two (15%) of 13 possibly linked patients, and 13 (17%) of 75 epidemiologically unlinked patients (three-way comparison exact p<0·0001). Genetic trees and clinical and epidemiological data suggest that super-spreaders were present in two community clusters. Interpretation Whole-genome sequencing can delineate outbreaks of tuberculosis and allows inference about direction of transmission between cases. The technique could identify super-spreaders and predict the existence of undiagnosed cases, potentially leading to early treatment of infectious patients and their contacts. Funding Medical Research Council, Wellcome Trust, National Institute for Health Research, and the Health Protection Agency.

Whole-genome sequencing for prediction of Mycobacterium tuberculosis drug susceptibility and resistance: a retrospective cohort study.

Summary Background Diagnosing drug-resistance remains an obstacle to the elimination of tuberculosis. Phenotypic drug-susceptibility testing is slow and expensive, and commercial genotypic assays screen only common resistance-determining mutations. We used whole-genome sequencing to characterise common and rare mutations predicting drug resistance, or consistency with susceptibility, for all first-line and second-line drugs for tuberculosis. Methods Between Sept 1, 2010, and Dec 1, 2013, we sequenced a training set of 2099 Mycobacterium tuberculosis genomes. For 23 candidate genes identified from the drug-resistance scientific literature, we algorithmically characterised genetic mutations as not conferring resistance (benign), resistance determinants, or uncharacterised. We then assessed the ability of these characterisations to predict phenotypic drug-susceptibility testing for an independent validation set of 1552 genomes. We sought mutations under similar selection pressure to those characterised as resistance determinants outside candidate genes to account for residual phenotypic resistance. Findings We characterised 120 training-set mutations as resistance determining, and 772 as benign. With these mutations, we could predict 89·2% of the validation-set phenotypes with a mean 92·3% sensitivity (95% CI 90·7–93·7) and 98·4% specificity (98·1–98·7). 10·8% of validation-set phenotypes could not be predicted because uncharacterised mutations were present. With an in-silico comparison, characterised resistance determinants had higher sensitivity than the mutations from three line-probe assays (85·1% vs 81·6%). No additional resistance determinants were identified among mutations under selection pressure in non-candidate genes. Interpretation A broad catalogue of genetic mutations enable data from whole-genome sequencing to be used clinically to predict drug resistance, drug susceptibility, or to identify drug phenotypes that cannot yet be genetically predicted. This approach could be integrated into routine diagnostic workflows, phasing out phenotypic drug-susceptibility testing while reporting drug resistance early. Funding Wellcome Trust, National Institute of Health Research, Medical Research Council, and the European Union.

Cluster of human tuberculosis caused by Mycobacterium bovis: evidence for person-to-person transmission in the UK

BACKGROUND Despite a recent resurgence in the incidence of bovine tuberculosis in UK cattle herds, no associated rise in the number of cases in man has been noted. Disease due to human Mycobacterium bovis infection usually occurs in older patients, in whom drinking unpasteurised milk in the past is the probable source of infection. Person-to-person transmission is very rare. METHODS After identification of two epidemiologically-linked cases of human M bovis infection through routine laboratory and surveillance activities, all patients identified with M bovis infection in the Midlands from 2001-05 (n=20) were assessed by DNA fingerprinting (MIRU-VNTR and spoligotyping), with additional interviews for patients with a clustered strain. FINDINGS A cluster of six cases was identified. All clustered cases were young and UK-born; five patients had pulmonary disease, and one patient died due to M bovis meningitis, with four patients possessing factors predisposing to tuberculosis. All patients had common social links through visits to bars in two different areas. With the exception of the first case, there was an absence of zoonotic links or consumption of unpasteurised dairy products, suggesting that person-to-person transmission had occurred. INTERPRETATION This report of several instances of M bovis transmission between people in a modern urban setting emphasises the need to maintain control measures for human and bovine tuberculosis. Transmission and subsequent disease was probably due to a combination of host and environmental factors. Prospective surveillance and DNA fingerprinting identified the cluster, enabling health protection teams to set up control measures and prevent further transmission.

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

Summary 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.

Assessment of Mycobacterium tuberculosis transmission in Oxfordshire, UK, 2007-12, with whole pathogen genome sequences: an observational study.

Summary Background Patients born outside the UK have contributed to a 20% rise in the UK’s tuberculosis incidence since 2000, but their effect on domestic transmission is not known. Here we use whole-genome sequencing to investigate the epidemiology of tuberculosis transmission in an unselected population over 6 years. Methods We identified all residents with Oxfordshire postcodes with a Mycobacterium tuberculosis culture or a clinical diagnosis of tuberculosis between Jan 1, 2007, and Dec 31, 2012, using local databases and checking against the national Enhanced Tuberculosis Surveillance database. We used Illumina technology to sequence all available M tuberculosis cultures from identified cases. Sequences were clustered by genetic relatedness and compared retrospectively with contact investigations. The first patient diagnosed in each cluster was defined as the index case, with links to subsequent cases assigned first by use of any epidemiological linkage, then by genetic distance, and then by timing of diagnosis. Findings Although we identified 384 patients with a diagnosis of tuberculosis, country of birth was known for 380 and we sequenced isolates from 247 of 269 cases with culture-confirmed disease. 39 cases were genomically linked within 13 clusters, implying 26 local transmission events. Only 11 of 26 possible transmissions had been previously identified through contact tracing. Of seven genomically confirmed household clusters, five contained additional genomic links to epidemiologically unidentified non-household members. 255 (67%) patients were born in a country with high tuberculosis incidence, conferring a local incidence of 109 cases per 100 000 population per year in Oxfordshire, compared with 3·5 cases per 100 000 per year for those born in low-incidence countries. However, patients born in the low-incidence countries, predominantly UK, were more likely to have pulmonary disease (adjusted odds ratio 1·8 [95% CI 1·2–2·9]; p=0·009), social risk factors (4·4 [2·0–9·4]; p<0·0001), and be part of a local transmission cluster (4·8 [1·6–14·8]; p=0·006). Interpretation Although inward migration has contributed to the overall tuberculosis incidence, our findings suggest that most patients born in high-incidence countries reactivate latent infection acquired abroad and are not involved in local onward transmission. Systematic screening of new entrants could further improve tuberculosis control, but it is important that health care remains accessible to all individuals, especially high-risk groups, if tuberculosis control is not to be jeopardised.

Mycobacterial Interspersed Repetitive Unit Typing of Mycobacterium tuberculosis Compared to IS6110-Based Restriction Fragment Length Polymorphism Analysis for Investigation of Apparently Clustered Cases of Tuberculosis

ABSTRACT An evaluation of the utility of IS6110-based restriction fragment length polymorphism (RFLP) typing compared to a combination of variable number tandem repeat (VNTR) typing and mycobacterial interspersed repetitive unit (MIRU) typing was undertaken. A total of 53 patient isolates of Mycobacterium tuberculosis from four presumed episodes of cross-infection were examined. Genomic DNA was extracted from the isolates by a cetyl trimethylammonium bromide method. The number of copies of tandem repeats of the five loci ETRA to ETRE and 12 MIRU loci was determined by PCR amplification and agarose gel electrophoresis of the amplicons. VNTR typing identified the major clusters of strains in the three investigations in which they occurred (each representing a different evolutionary clade: 32333, 42235, and 32433). The majority of unrelated isolates (by epidemiology and RFLP typing) were also identified by VNTR typing. The concordance between the RFLP and MIRU typing was complete, with the exception of two isolates with RFLP patterns that differed by one band each from the rest of the major epidemiologically linked groups of isolates in investigation A. All of these isolates had identical MIRU and VNTR types. A further pair of isolates differed in the number of tandem repeat copies at two MIRU alleles but had identical RFLP patterns. The speed of the combined VNTR and MIRU typing approach enabled results for some of the investigations to be supplied in “real time,” influencing choices in contact tracing. The ease of comparison of results of MIRU and VNTR typing, which are recorded as single multidigit numbers, was also found to greatly facilitate investigation management and the communication of results to health care professionals.

Same-day diagnostic and surveillance data for tuberculosis via whole genome sequencing of direct respiratory samples.

ABSTRACT Routine full characterization of Mycobacterium tuberculosis is culture based, taking many weeks. Whole-genome sequencing (WGS) can generate antibiotic susceptibility profiles to inform treatment, augmented with strain information for global surveillance; such data could be transformative if provided at or near the point of care. We demonstrate a low-cost method of DNA extraction directly from patient samples for M. tuberculosis WGS. We initially evaluated the method by using the Illumina MiSeq sequencer (40 smear-positive respiratory samples obtained after routine clinical testing and 27 matched liquid cultures). M. tuberculosis was identified in all 39 samples from which DNA was successfully extracted. Sufficient data for antibiotic susceptibility prediction were obtained from 24 (62%) samples; all results were concordant with reference laboratory phenotypes. Phylogenetic placement was concordant between direct and cultured samples. With Illumina MiSeq/MiniSeq, the workflow from patient sample to results can be completed in 44/16 h at a reagent cost of £96/£198 per sample. We then employed a nonspecific PCR-based library preparation method for sequencing on an Oxford Nanopore Technologies MinION sequencer. We applied this to cultured Mycobacterium bovis strain BCG DNA and to combined culture-negative sputum DNA and BCG DNA. For flow cell version R9.4, the estimated turnaround time from patient to identification of BCG, detection of pyrazinamide resistance, and phylogenetic placement was 7.5 h, with full susceptibility results 5 h later. Antibiotic susceptibility predictions were fully concordant. A critical advantage of MinION is the ability to continue sequencing until sufficient coverage is obtained, providing a potential solution to the problem of variable amounts of M. tuberculosis DNA in direct samples.

Insidious risk of severe mycobacterium chimaera infection in cardiac surgery patients

Background. An urgent UK investigation was launched to assess risk of invasive Mycobacterium chimaera infection in cardiothoracic surgery and a possible association with cardiopulmonary bypass heater-cooler units following alerts in Switzerland and The Netherlands. Methods. Parallel investigations were pursued: (1) identification of cardiopulmonary bypass–associated M. chimaera infection through national laboratory and hospital admissions data linkage; (2) cohort study to assess patient risk; (3) microbiological and aerobiological investigations of heater-coolers in situ and under controlled laboratory conditions; and (4) whole-genome sequencing of clinical and environmental isolates. Results. Eighteen probable cases of cardiopulmonary bypass–associated M. chimaera infection were identified; all except one occurred in adults. Patients had undergone valve replacement in 11 hospitals between 2007 and 2015, a median of 19 months prior to onset (range, 3 months to 5 years). Risk to patients increased after 2010 from <0.2 to 1.65 per 10000 person-years in 2013, a 9-fold rise for infections within 2 years of surgery (rate ratio, 9.08 [95% CI, 1.81–87.76]). Endocarditis was the most common presentation (n = 11). To date, 9 patients have died. Investigations identified aerosol release through breaches in heater-cooler tanks. Mycobacterium chimaera and other pathogens were recovered from water and air samples. Phylogenetic analysis found close clustering of strains from probable cases. Conclusions. We identified low but escalating risk of severe M. chimaera infection associated with heater-coolers with cases in a quarter of cardiothoracic centers. Our investigations strengthen etiological evidence for the role of heater-coolers in transmission and raise the possibility of an ongoing, international point-source outbreak. Active management of heater-coolers and heightened clinical awareness are imperative given the consequences of infection.

Identifying lineage effects when controlling for population structure improves power in bacterial association studies.

Bacteria pose unique challenges for genome-wide association studies because of strong structuring into distinct strains and substantial linkage disequilibrium across the genome1,2. Although methods developed for human studies can correct for strain structure3,4, this risks considerable loss-of-power because genetic differences between strains often contribute substantial phenotypic variability5. Here, we propose a new method that captures lineage-level associations even when locus-specific associations cannot be fine-mapped. We demonstrate its ability to detect genes and genetic variants underlying resistance to 17 antimicrobials in 3,144 isolates from four taxonomically diverse clonal and recombining bacteria: Mycobacterium tuberculosis, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae. Strong selection, recombination and penetrance confer high power to recover known antimicrobial resistance mechanisms and reveal a candidate association between the outer membrane porin nmpC and cefazolin resistance in E. coli. Hence, our method pinpoints locus-specific effects where possible and boosts power by detecting lineage-level differences when fine-mapping is intractable.

Contact investigations for outbreaks of Mycobacterium tuberculosis: advances through whole genome sequencing

The control of tuberculosis depends on the identification and treatment of infectious patients and their contacts, who are currently identified through a combined approach of genotyping and epidemiological investigation. However, epidemiological data are often challenging to obtain, and genotyping data are difficult to interpret without them. Whole genome sequencing (WGS) technology is increasingly affordable, and offers the prospect of identifying plausible transmission events between patients without prior recourse to epidemiological data. We discuss the current approaches to tuberculosis control, and how WGS might advance public health efforts in the future.