Chasing TB Clusters – the role of molecular strain typing NPHS Staff Conference 10th October 2007 Michael Ruddy Gwen Lowe Lika Nehaul

Introduction Background Epidemiology & laboratory role Overview of molecular strain typing M tb Outbreak control Summary

TB Clinical disease caused by Mycobacterium tuberculosis (M tb) “consumption” “Captain of the men of death” “Great white plague” Killed 1/4 Europeans 19th Century killed 1 billion worldwide in last 200 years

TB One third world infected (~2 billion) ~10% immune competent develop disease ~8 million new cases pa 2-3 million deaths pa major cause death 15-49 year olds (inc HIV) >95% burden in poorest countries

Estimated TB Incidence Rates, 2001 per 100 000 pop < 10 10 - 24 25 - 49 50 - 99 100 - 299 300 or more No estimate The designations employed and the presentation of material on this map do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. White lines on maps represent approximate border lines for which there may not yet be full agreement. © WHO 2003

Percentage per tested cases Multi drug resistance (MDR), EU 2005 Percentage per tested cases Within the EU (27 countries), the Baltic states account for most of the MDR cases reported. In 2005, around 18% of all cases tested were MDR, while in other EU countries the percentage ranged between 0-6% XDR-TB cases have been also reported in the EU, and most of them were again reported from the Baltic States * Countries with incomplete data Source: EuroTB

TB Notifications England & Wales 1913-2005 Source: HPA ETS Sept 07

TB Notifications England & Wales 1982 to 2005 Source: HPA ETS Sept 07

TB Notifications UK By Region 2005 Source: HPA ETS Sept 07

Role of Laboratory Microscopy (ZN stain for AFB) Culture (solid & liquid) Identification (conventional & molecular) Drug sensitivity testing (conventional & molecular) “Serology” (gamma interferon testing) Molecular strain typing

Pink / red acid-fast bacilli seen in Ziehl-Neelsen stain of sputum. (M Pink / red acid-fast bacilli seen in Ziehl-Neelsen stain of sputum. (M.Tb)

M.kansasii (left) and M.tuberculosis (right) growing on LJ media Phenotypic tests M.kansasii (left) and M.tuberculosis (right) growing on LJ media

Mtb Typing: Why perform? Local outbreak investigation (confirm suspected epidemiological links) Detect unknown links – targeted interventions Exclude lab cross contamination Detect emergence of new strains Detect globally prevalent strains (?inform vaccine strategies/treatment protocols)

Which molecular method? Reproducibility No. of types RFLP 100% 84 Mixed-linker PCR 81 Spoligotyping 94% 61 DRE – PCR 58% 63 ETR – VNTR 97% 56 MIRU – VNTR 78 Mostrom et al 2002

RFLP (restriction fragment length polymorphism) The Reference Method RFLP DNA genotyping profiles of a series of M. tuberculosis isolates for direct visual and computer-assisted assessment of position and number of bands on the gel.

MIRU-VNTR Mycobacterial Interspersed Repetitive Units Variable Number Tandem Repeats Mini satellite regions of repeating sequences of nucleotides ?function, highly conserved Number of times the sequence repeats at each region varies >40 such regions (MIRU) useful to type strains 15 specific loci adopted across UK

VNTR (variable number tandem repeats) – The Principle TAAGGGCCA (X3) TAAGGGCCA (X2) TAAGGGCCA (X1) Strain 1: 2 4 1 8 3 Strain 2: 3 3 1 4 4

ETR–A ETR-B ETR-C ETR-D ETR-E Digital Code for Strains generated by ETR – VNTR Strain 1 1 3 2 2 3 Strain 2 3 3 2 3 3

Typical MIRU VNTR results

Combined results ETR MIRU Isolate Patient A B C D E 2 4 10 16 20 23 24   ETR MIRU Isolate Patient A B C D E 2 4 10 16 20 23 24 26 27 31 39 40 3 1 5 6 7 F G - H

VNTR-MIRU Generate a 15 digit strain designation number Geographical incidence of TB isolates with 15-VNTR profile ‘422225435173533’, which corresponds to the Leicester outbreak strain. Generate a 15 digit strain designation number Collated in UK National database

Summary 1st April 2005 prospective MIRU-VNTR all M tb isolates WCM 15 digit strain type to local database (1000 isolates) Local – national – international datasets Beware false clusters! Lab – HPT teamwork vital Secondary typing (?additional 7 loci ?RFLP) “epidemiologically confirmed genotype cluster”

Acknowledgements Alan Paull Daniel Thomas Eleri Davies Gwen Lowe Gwyneth Samuel Kay Parry Lewis White Lika Nehaul Rhian Williams Robin Howe Sally Corden

The workplace cluster Large office- approx 2000 employees Case A- Smear positive –late shift Case B- Friend- late shift same floor Case C- Late shift different floor- family member with TB A/B/C been to same school- where large outbreak several years previously Case D –Late shift same floor

The workplace cluster-2 A +B +D same high prevalence back ground. A+D had BCG C – different high prevalence background- had BCG Office had recruit a friend policy- so clusters of individuals with links.

Hypotheses Cluster is coincidence (cases from high prevalence background where TB is much more common) First three cases (A/B/C) associated with school outbreak (infected from another case? Reactivation?) Fourth coincidence First two cases friends (A/B), so close contact, third infected from family member, fourth different strain Outbreak of TB in workplace

How typing helped Typing from A/C/D indistinguishable (no isolate from B) Family member of C had different typing Conclusion: workplace outbreak Typing influenced management Late shift on floor where A/B/D worked offered screening. No new cases detected. Subsequent work links identified for A/C/D