Whole-genome sequencing of Mycobacterium tuberculosis as an epidemiological marker
Authors: Dick van Soolingen
Journal: The Lancet Respiratory Medicine (2014)

DNA fingerprinting of Mycobacterium tuberculosis has revolutionised the study of the transmission of tuberculosis. Since the early 1990s, IS6110 restriction fragment length polymorphism (RFLP) typing and variable number of tandem repeat (VNTR) typing have progressed and are being used to answer longstanding epidemiological questions.1Borgdorff MW van Soolingen D The re-emergence of tuberculosis: what have we learnt from molecular epidemiology?.Clin Microbiol Infect. 2013; 19: 889-901Crossref PubMed Scopus (72) Google Scholar DNA fingerprinting of M tuberculosis isolates enables the visualisation of transmission and allows a much more sensitive investigation of this important aspect of tuberculosis control. Although having these possibilities is an advantage to tuberculosis control in daily practice, the findings of possible epidemiological links as shown with DNA fingerprinting are only partly confirmed through the standard epidemiological investigation by patient interviews. As a result, DNA fingerprinting is used to indicate possible epidemiological links, but the verification of these links is dependent on whether we can understand how transmission occurred; if contact between the patients is not thought to be possible, transmission is ruled out. This verification, however, does not include incidental contacts in the public domain and might lead to an underestimation of active transmission. Nevertheless, with RFLP or VNTR typing, transmissions can often be excluded and possible links are at least indicated much more sensitively than through interviews alone.2de Vries G Baars HW Sebek MM van Hest NA Richardus JH Transmission classification model to determine place and time of infection of tuberculosis cases in an urban area.J Clin Microbiol. 2008; 46: 3924-3930Crossref PubMed Scopus (17) Google Scholar Some of the uncertainties, however, about the usefulness of RFLP or VNTR typing are also caused by the slow turnover of these markers; RFLP typing has a half-life of 3–4 years3de Boer AS Borgdorff MW de Haas PE Nagelkerke NJ van Embden JD van Soolingen D Analysis of rate of change of IS6110 RFLP patterns of Mycobacterium tuberculosis based on serial patient isolates.J Infect Dis. 1999; 180: 1238-1244Crossref PubMed Scopus (133) Google Scholar and the change in patterns for VNTR typing has not yet been established accurately. However, most M tuberculosis strains retain their RFLP or VNTR profiles during transmission and the sources—eg, primary, secondary, or tertiary—cannot be distinguished within DNA fingerprint clusters. In 2010 and 2011, the first results of M tuberculosis DNA fingerprinting with whole-genome sequencing (WGS) were reported and it became apparent that this new approach has a higher resolution than conventional typing.4Gardy JL Johnston JC Ho Sui SJ et al.Whole-genome sequencing and social-network analysis of a tuberculosis outbreak.N Engl J Med. 2011; 364: 730-739Crossref PubMed Scopus (571) Google Scholar, 5Schurch AC Kremer K Daviena O et al.High-resolution typing by integration of genome sequencing data in a large tuberculosis cluster.J Clin Microbiol. 2010; 48: 3403-3406Crossref PubMed Scopus (67) Google Scholar Clusters of cases according to RFLP or VNTR typing were subdivided into actual transmission chains and this new information was more in agreement with the contact tracing information. Previous studies of the usefulness of WGS have been retrospective, but in Timothy Walker and colleagues' study in Oxfordshire, UK,6Walker TM Lalor MK Broda A et al.Assessment of Mycobacterium tuberculosis transmission in Oxfordshire, UK, 2007–12, with whole pathogen genome sequences: an observational study.Lancet Respir Med. 2014; (published online March 4.)http://dx.doi.org/10.1016/S2213-2600(14)70027-XGoogle Scholar WGS was applied in the first line to ascertain clustering of tuberculosis cases and to quantify active transmission. This WGS was done with a pairwise determination of the number of single nucleotide polymorphisms (SNPs) separating the isolates; a difference of a maximum of 12 SNPs was applied to rule in a possible transmission. Although this approach is elegant and valid there are still further considerations in the validation process of WGS. As for RFLP and VNTR typing, the usefulness of WGS depends on the population structure of M tuberculosis in the setting in which it is used.7Sloot R Borgdorff MW de Beer JL van Ingen J Supply P van Soolingen D Clustering of tuberculosis cases based on variable-number tandem-repeat typing in relation to the population structure of Mycobacterium tuberculosis in the Netherlands.J Clin Microbiol. 2013; 51: 2427-2431Crossref PubMed Scopus (21) Google Scholar In Walker and colleagues, only in a subset of the pairs of cases with fewer than 12 SNPs difference was an epidemiological link confirmed with interviews and transmission judged likely; these findings are in agreement with those of an earlier study in Amsterdam.8Bryant JM Schurch AC van Deutekom H et al.Inferring patient to patient transmission of Mycobacterium tuberculosis from whole genome sequencing data.BMC Infect Dis. 2013; 13: 110Crossref PubMed Scopus (149) Google Scholar Does this indicate that strains with little or no difference are circulating in these areas without any epidemiological link between the respective cases? Or are we again, and this time with a more accurate and higher resolution marker, not able to understand how transmission of tuberculosis occurred? Is it simply impossible to confirm transmission through interviews with patients? This apparent lack of resolution in typing is also related to the remarkable genomic stability of M tuberculosis. In a study in the UK, a turnover of 0·5 SNPs per genome per year was noted, whereas in the Netherlands a highly similar rate of change of 0·36 SNPs was found.8Bryant JM Schurch AC van Deutekom H et al.Inferring patient to patient transmission of Mycobacterium tuberculosis from whole genome sequencing data.BMC Infect Dis. 2013; 13: 110Crossref PubMed Scopus (149) Google Scholar, 9Walker TM Ip CL Harrell RH et al.Whole-genome sequencing to delineate Mycobacterium tuberculosis outbreaks: a retrospective observational study.Lancet Infectious Dis. 2013; 13: 137-146Summary Full Text Full Text PDF PubMed Scopus (641) Google Scholar Another major concern that has not been sufficiently addressed and investigated generally is that M tuberculosis isolates are not at all homogeneous populations. They comprise several populations with different SNP profiles.10Schurch AC Kremer K Kiers A et al.The tempo and mode of molecular evolution of Mycobacterium tuberculosis at patient-to-patient scale.Infect Genet Evol. 2010; 10: 108-114Crossref PubMed Scopus (63) Google Scholar With the current WGS techniques only the SNP profile in the predominating population is visualised; however, this predominance might shift when the strain is passed on to another person. In view of the concerns described above, WGS is also not an ideal epidemiological marker, but still undoubtedly a major step forward in DNA fingerprinting compared with the conventional RFLP and VNTR typing. In the molecular epidemiology of tuberculosis it would be ideal if at each transmission a minor, but recognisable, difference occurred in the DNA profile of M tuberculosis. Such a difference would enable the deduction of all transmission forks and the separation of transmission for subsequent sources in a chain of transmission in time. Unfortunately, the current WGS is also not a perfect method. I declare that I have no competing interests. Assessment of Mycobacterium tuberculosis transmission in Oxfordshire, UK, 2007–12, with whole pathogen genome sequences: an observational studyAlthough 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. Full-Text PDF Open Access