Jason T. Evans

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.

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.

Emergence and spread of a human-transmissible multidrug-resistant nontuberculous mycobacterium

Global spread of aggressive mycobacteria Many mycobacteria, in addition to those causing leprosy and tuberculosis, are capable of infecting humans. Some can be particularly dangerous in patients suffering from immunosuppression or chronic disease, such as cystic fibrosis (CF). Bryant et al. observed clusters of near-identical isolates of drug-resistant Mycobacterium abscessus in patients reporting to CF clinics. The similarity of the isolates suggests transmission between patients, rather than environmental acquisition. Although this bacterium is renowned for its environmental resilience, the mechanism for its long-distance transmission among the global CF patient community remains a puzzle. Science, this issue p. 751 Near-identical isolates of Mycobacterium abscessus indicate recent chains of transmission among cystic fibrosis patients. Lung infections with Mycobacterium abscessus, a species of multidrug-resistant nontuberculous mycobacteria, are emerging as an important global threat to individuals with cystic fibrosis (CF), in whom M. abscessus accelerates inflammatory lung damage, leading to increased morbidity and mortality. Previously, M. abscessus was thought to be independently acquired by susceptible individuals from the environment. However, using whole-genome analysis of a global collection of clinical isolates, we show that the majority of M. abscessus infections are acquired through transmission, potentially via fomites and aerosols, of recently emerged dominant circulating clones that have spread globally. We demonstrate that these clones are associated with worse clinical outcomes, show increased virulence in cell-based and mouse infection models, and thus represent an urgent international infection challenge.

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.

Automated High-Throughput Mycobacterial Interspersed Repetitive Unit Typing of Mycobacterium tuberculosis Strains by a Combination of PCR and Nondenaturing High-Performance Liquid Chromatography

ABSTRACT Mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing of Mycobacterium tuberculosis complex isolates is portable, 100% reproducible, and highly discriminatory. Nondenaturing high-performance liquid chromatography (non-dHPLC) with use of a WAVE microbial analysis system is a promising method of PCR amplicon analysis as it is low cost and requires no preanalysis processing. The aims of this study were to validate the application of WAVE microbial analysis system technology to MIRU-VNTR typing. A collection of 70 strains were cultivated in liquid culture and extracted using the QIAamp DNA minikit. Novel primers were designed to target the 12 MIRU-VNTR loci (P. Supply et al., J. Clin. Microbiol. 39:3563-3571, 2001). After amplification, each PCR product was analyzed on a WAVE microbial analysis system. The fragment size was calculated from the chromatogram, and the number of tandem repeats at each locus was determined. For the collection of 70 strains 100% concordance was achieved when comparing MIRU-VNTR profiles obtained from agarose gel electrophoresis and PCRs analyzed on a WAVE microbial analysis system. The calculated fragment sizes, obtained from the WAVE microbial analysis system, were sufficiently accurate to ensure 100% confidence when assigning the number of tandem repeats to a MIRU-VNTR locus. This study is the first to report the successful use of non-dHPLC for screening for variations in the number of MIRU-VNTRs in mycobacterial DNA. Non-dHPLC analysis was demonstrated to be a rapid, low-labor input method for the detection and analysis of MIRU-VNTR amplicons. The combination with non-dHPLC further enhances the utility of MIRU-VNTR typing.

Ethnicity and mycobacterial lineage as determinants of tuberculosis disease phenotype

Background Emerging evidence suggests that Mycobacterium tuberculosis (Mtb) lineage and host ethnicity can determine tuberculosis (TB) clinical disease patterns but their relative importance and interaction are unknown. Methods We evaluated prospectively collected TB surveillance and Mtb strain typing data in an ethnically heterogeneous UK population. Lineage assignment was denoted using 15-loci mycobacterial interspersed repetitive units containing variable numbers of tandem repeats (MIRU-VNTR) and MIRU-VNTRplus. Geographical and ethnic associations of the six global Mtb lineages were identified and the influence of lineage and demographic factors on clinical phenotype were assessed using multivariate logistic regression. Results Data were available for 1070 individuals with active TB which was pulmonary only, extrapulmonary only and concurrent pulmonary–extrapulmonary in 52.1%, 36.9% and 11.0% respectively. The most prevalent lineages were Euro-American (43.7%), East African Indian (30.2%), Indo-Oceanic (13.6%) and East Asian (12.2%) and were geo-ethnically restricted with, for example, Indian subcontinent ethnicity inversely associated with Euro-American lineage (OR 0.23; 95% CI 0.14 to 0.39) and positively associated with the East African-Indian lineage (OR 4.04; 95% CI 2.19 to 7.45). Disease phenotype was most strongly associated with ethnicity (OR for extrathoracic disease 21.14 (95% CI 6.08 to 73.48) for Indian subcontinent and 14.05 (3.97 to 49.65) for Afro-Caribbean), after adjusting for lineage. With East Asian lineage as the reference category, the Euro-American (OR 0.54; 95% CI 0.32 to 0.91) and East-African Indian (OR 0.50; 95% CI 0.29 to 0.86) lineages were negatively associated with extrathoracic disease, compared with pulmonary disease, after adjusting for ethnicity. Conclusions Ethnicity is a powerful determinant of clinical TB phenotype independently of mycobacterial lineage and the role of ethnicity-associated factors in pathogenesis warrants investigation.

Undetected Multidrug-Resistant Tuberculosis Amplified by First-line Therapy in Mixed Infection

Infections with >1 Mycobacterium tuberculosis strain(s) are underrecognized. We show, in vitro and in vivo, how first-line treatment conferred a competitive growth advantage to amplify a multidrug-resistant M. tuberculosis strain in a patient with mixed infection. Diagnostic techniques that identify mixed tubercle bacilli populations are needed to curb the spread of multidrug resistance.

Investigating Transmission of Mycobacterium bovis in the United Kingdom in 2005 to 2008

ABSTRACT Due to an increase in bovine tuberculosis in cattle in the United Kingdom, we investigated the characteristics of Mycobacterium bovis infection in humans and assessed whether extensive transmission of M. bovis between humans has occurred. A cross-sectional study linking demographic, clinical, and DNA fingerprinting (using 15-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat [MIRU-VNTR] typing) data on cases reported between 2005 and 2008 was undertaken. A total of 129 cases of M. bovis infection in humans were reported over the period, with a decrease in annual incidence from 0.065 to 0.047 cases per 100,000 persons. Most patients were born pre-1960, before widespread pasteurization was introduced (73%), were of white ethnicity (83%), and were born in the United Kingdom (76%). A total of 102 patients (79%) had MIRU-VNTR typing data. A total of 31 of 69 complete MIRU-VNTR profiles formed eight distinct clusters. The overall clustering proportion determined using the n − 1 method was 33%. The largest cluster, comprising 12 cases, was indistinguishable from a previously reported West Midlands outbreak strain cluster and included those cases. This cluster was heterogeneous, having characteristics supporting recent zoonotic and human-to-human transmission as well as reactivation of latent disease. Seven other, smaller clusters identified had demographics supporting recrudescence rather than recent infection. A total of 33 patients had incomplete MIRU-VNTR profiles, of which 11 may have yielded 2 to 6 further small clusters if typed to completion. The incidence of M. bovis in humans in the United Kingdom remains low, and the epidemiology is predominantly that of reactivated disease.

Rapid Genotyping of CTX-M Extended-Spectrum β-Lactamases by Denaturing High-Performance Liquid Chromatography

ABSTRACT Denaturing high-performance liquid chromatography (dHPLC) is a powerful technique which has been used extensively to detect genetic variation. This is the first report of the application of dHPLC for rapid genotyping of bacterial β-lactamase genes. The technique was specifically developed to genotype members of all blaCTX-M DNA homology groups. Thirteen well-defined blaCTX-M extended-spectrum β-lactamase (ESBL)-producing strains were used to develop and optimize the dHPLC genotyping assay. Further evaluation was carried out with a blinded panel of 62 clinical isolates. The results of blaCTX-M genotyping achieved by dHPLC were comparable to the typing results obtained by DNA sequencing. Applying the newly developed dHPLC-based genotyping method, we successfully genotyped all 73 blaCTX-M ESBL-producing strains from the 4-month survey study. Furthermore, we found the first reported cases in the United Kingdom of clinically significant disease caused by CTX-M-14- and CTX-M-1-producing Escherichia coli strains. We conclude that the novel dHPLC assay is highly accurate, rapid, and cost-effective for the genotyping of blaCTX-M-producing ESBLs and has great potential for determining the clinical relevance of different and new blaCTX-M genotypes, as well as for epidemiological studies and surveillance programs.

Global Origin of Mycobacterium tuberculosis in the Midlands, UK

DNA fingerprinting data for 4,207 Mycobacterium tuberculosis isolates were combined with data from a computer program (Origins). Largest population groups were from England (n = 1,031) and India (n = 912), and most prevalent strains were the Euro-American (45%) and East African–Indian (34%) lineages. Combining geographic and molecular data can enhance cluster investigation.