Applications of whole genome sequencing to TB epidemiology

Next generation molecular epidemiology Lecture 17: Applications of whole genome sequencing to TB epidemiology National Institute of Infectious Disease January 18, 2017 Scope of investigations covered by next generation molecular epidemiology Identifying …  risk factors that could not be identified by conventional or early-generation molecular biology laboratory methods  new or hidden transmission pathways  direction of transmission of an infectious agent  endogenous reactivation vs exogenous reinfection  ecological niche from which clonal pathogenic strains are selected and disseminate  pathogen microbial population structures associated with a syndrome  host commensal microbial population structures that determine noncommunicable disease outcomes Methods used to genotypeM tuberculosis        IS6110 RFLP Spoligotyping MIRU-VNTR “Deligotyping” Repetitive element PCR SNP analysis Whole genome sequencing Endonucleases used to cut DNA 5’ overhand Blunt end PvuII cutting site PvuII site: Restriction fragment length polymorphism (RFLP) analysis of M tuberculosis DNA (Ferrazoli et al) IS6110 Southern blot hybridization of RFLP analysis (Ferrazoli et al) Spoligotyping with M tuberculosis (Kamerbeek et al, J Clin Microbiol, 1997) Based on polymorphism within clustered regularlyinterspaced short palindromic repeats (CRISPR) Analysis of spoligotypes • SPOTCLUST website for analysis of spoligofamilies (http://cgi2.cs.rpi.edu/vitoli/SPOTCLUST.html) • SpolDB4 database (43) and SITVIT database (http://www.pasteurguadeloupe.fr:8081/SITVITDemo/) Variation in the Number of Tandem Repeats (VNTR) -A variation in the number of short, repeated segments found in a specific locus -Amplification of these loci (using primers specific to flanking regions) will produce DNA fragments whose lengths vary between strains Mycobacterial Interspersed Repetitive Units MIRU-VNTR for M tuberculosis rationale • The Mycobacterium tuberculosis genome contains 41 loci with direct tandem repeats of 50 – 70 bp • The number of repeats per locus varies between strains • Strains can be typed based on the number of repeats per locus 12-loci MIRU VNTR 24-loci MIRU VNTR as discriminating as IS6110-RFLP MIRU 02 04 10 16 20 23 24 26 27 31 39 40 Size (bp) 283 332 201 168 365 287 414 303 226 264 226 220 No of copies 2 MIRU pattern: 232234253322 http://www.miru-vntrplus.org/ 2 SNP analysis of M. tuberculosis strains Beijing family strains (Gutacker et al, JID, 2006) Role of Beijing clade in the transmission of MDRTB • China (Heilongjiang Province: Wang J et al, J Clin Microbiol, 2011): – Overall: 8% – Beijing clade: 27% • Russian Federation and former Soviet Union countries (Niemann S et al, J Clin Microbiol, 2010; Afanas’ev MV et al, Eur J Clin Microbiol Infect Dis, 2011): – Overall (retreatment cases): ~40% – Beijing clade: 70-78% Epidemiologic studies using IS6110-based strain-typing analysis • • • • • • • • • • Differentiating recent from past transmission Confirming an outbreak in institutional or community settings Identifying an outbreak in what appears to be sporadic cases of TB Identifying risk factors in community-based settings Identifying virulence factors associated with M tuberculosis Tracking geographic spread of clones of M tuberculosis Managing a clinical case of TB Detecting drug-resistant strains of M tuberculosis Evaluating laboratory contamination Evaluating effect of an intervention (e.g., BCG, INH prophylaxis) Differentiating new cases of TB arising from recent infection vs remote past infection • Recent transmission TB: – Rapidly progressive or primary TB (CDC: within yrs of new transmission) • Remote past transmission TB: – Reactivation TB • Assumptions: – Identical IS6110 pattern observed in or more persons (called cluster) with newly-diagnosed TB represent cases due to recent exogenous infection – Isolates with unique RFLP pattern (noncluster pattern) in a community represent isolates from a case of reactivation of endogenous infection Caveats: • May not apply in rural areas with highly stable populations (Braden et al, JID, 1997) • Low-risk countries: IS6110-based test may underestimate the proportion of recent infection in young people and overestimate in older people with TB (Vinnycky et al, EID, 2003) • High-risk countries: IS6110-based test may underestimate the proportion of disease due to recent infection (Vinnycky et al, EID, 2003) Proportion (%) of M tuberculosis isolates belonging to cluster IS6110 RFLP groups (low-incidence regions) 100 90 80 70 60 50 40 30 20 10 New York City, 198990 San Los Angeles, Texas, 1994- Colorado, Francisco, 1994-96 95 1992-94 1991-92 Arkansas, Amsterdam, 1992-93 1992-95 Proportion (%) of M tuberculosis isolates belonging to cluster IS6110 RFLP groups (high-incidence regions) 100 90 80 70 60 50 40 30 20 10 Tunisa, 1992-93 Ethiopia, Tanzania, Sao Paulo, Taiwan, 1992-93 1992-93 Brazil, 1993-94 1995-97 Vitόria, Brazil, 2000-10 Rio de Janeiro, 1996 Campinas, 1996-99 Methods used to genotype M tuberculosis  IS6110 RFLP  Spoligotyping  MIRU-VNTR  “Deligotyping”  Repetitive element PCR  SNP analysis  Whole genome sequencing Are these genotyping tests able to truly differentiate recent vs past transmission? Problems with assumption based on genotyping tests  More recent studies show the same genotypes of M tuberculosis in the same geographic sites (>20 yrs!) Beijing clade C-strain in NYC  What is the definition of “recent transmission”  What is the threshold of SNPs needed to claim recent transmission vs past transmission? Discussion for presentation TB  Walker, TB et al Whole-genome sequencing to delineate Mycobacteriumtuberculosis outbreaks: a retrospective observational study Lancet ID, 2013  Casali, N et al Whole Genome Sequence Analysis of a Large Isoniazid-Resistant Tuberculosis Outbreak in London: A Retrospective Observational Study PLoS Medicine, 2016 Discussion  What are the advantages of WGS over other genotyping tests to study epidemiology of TB?  What epidemiologic problems can be addressed by WGS that cannot be addressed by early-generation genotyping tests?  What type of public health intervention can be devised based on WGS data?  What are the disadvantages of WGS over other genotyping tests to study epidemiology of TB?  What criteria can be used to infer recent transmission TB using WGS data?  What other types of information are needed to demonstrate recent transmission TB in addition to WGS data? ... presentation TB  Walker, TB et al Whole- genome sequencing to delineate Mycobacteriumtuberculosis outbreaks: a retrospective observational study Lancet ID, 2013  Casali, N et al Whole Genome Sequence... disadvantages of WGS over other genotyping tests to study epidemiology of TB?  What criteria can be used to infer recent transmission TB using WGS data?  What other types of information are needed to. .. factors associated with M tuberculosis Tracking geographic spread of clones of M tuberculosis Managing a clinical case of TB Detecting drug-resistant strains of M tuberculosis Evaluating laboratory