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

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

3. 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

4. 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 year olds (inc HIV)
>95% burden in poorest countries

5. Estimated TB Incidence Rates, 2001
per pop
< 10
300 or more
No estimate
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© WHO 2003

7. 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

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

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

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

11. 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

13. 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)

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

15. 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)

16. 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

17. 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.

18. 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

19. VNTR (variable number tandem repeats) – The Principle
TAAGGGCCA (X3)
TAAGGGCCA (X2)
TAAGGGCCA (X1)
Strain 1:
Strain 2:

20. ETR–A ETR-B ETR-C ETR-D ETR-E
Digital Code for Strains generated by ETR – VNTR
Strain
Strain

21. Typical MIRU VNTR results

22. 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

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

25. 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”

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

27. 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

28. 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.

29. 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

30. 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