1. TB typing and its impact on Public health in Wales Dr P. Lewis White Public Health Wales Microbiology Cardiff

2. Background – the need M. tuberculosis: –Leading cause of death Second to HIV 1.77 million deaths in 2007 –WHO: 2001: 200 million cases 2020: 1 Billion cases –Population movement –HIV –Drug resistance –Infection can be controlled and treated Kanduma et al., 2003 JAM; Glaziou et al. 2009 Clin Chest Med 2009 –9040 documented UK cases 15-44: 60% Males: 55% 14.6 per 100000 –92% English cases –5% Scottish –2% Welsh –1% Northern Irish HPA annual report on TB in UK 2010

3. Modified from HPA annual report on TB in UK 2010

4. HPA annual report on TB in UK 2010 19 High-risk PCTs

5. TB rates across the UK Modified from HPA annual report on TB in UK 2010

6. 14 7 57 4 5 2 Three year average rate in Wales

7. Cardiff Distribution – 10yr mean IR 0 0 0 0 8.7 <1 80.2 41.1 12.4 23.4 30.0 15.1 9.9 6.8 Wong MSc Thesis 2010

8. Background – Benefits of Typing Confirm or refute possible outbreaks of infection Determine transmission risk factors: –improved controls Determine the origin of infection Laboratory cross contamination Inform and focus contact tracing procedures Determine new infection or relapse Globally, Nationally or regional.

9. Background – Typing Whole Genome analysis –Sequencing – SNPs or large sequence polymorphisms –Genome/Genome Hybridisation –Technically demanding –Time consuming –Cost effective –Evolutionary rather than epidemiological investigations Regions –Strain specific profile = Fingerprint –>2 strains with the same fingerprint = Cluster –Cluster = high probability of linkage –Could = recent transmission

10. Background – the genome Monomorphic bacteria – little sequence diversity TB genome –4.4Mb –4000 protein-coding sequences –High GC content Biased amino acid content –Homogenous throughout the world Very few silent mutations –Transposons Inherently variable Transpositions, deletions, inversions and duplications Insertion sequences –genetic polymorphism –Used for discrimination –IS6110

11. Insertion Sequence 6110 Present in different copy numbers (0-25) Integrated at various sites (hot spots) Epidemiological investigations IS6110 RFLP – Gold standard –PvuII –0.9 – 10kb –Gel electrophoresis –Southern blotting – IS6110 probe Problems –5-10ug of DNA –Cumbersome –Result comparison –Low or no copy strains (25%) –Non-TB species –Not random distribution

12. The alternatives MethodReproducibilityNo. of types RFLP100%84 Mixed-linker PCR100%81 Spoligotyping94%61 DRE – PCR58%63 ETR – VNTR97%56 MIRU – VNTR100%78 Mostrom et al., 2002 CMI 8 694-704 Combined ETR and MIRU - VNTR = close correlation with RFLP (Hawkey et al., 2003 JCM)

13. Variable number of tandem repeats (VNTR) Genetic loci (minisatellite) –Tandem repeat repeating sequence/ adjacent TAAGGGCCATAAGGGCCA –1500 potential regions –5-100bp –The number of repeats at a loci varies = VNTR –PCR using primers flanking the locus –PCR products whose size is dependent on the number of repeats Exact tandem repeat (ETR-VNTR) –ETR A – E (Frothingham 1998) Mycobacterial interspersed repetitive units (MIRU-VNTR) –MIRU 2, 4, 10, 16, 20, 23, 24, 26, 27, 31, 39 and 40 (Supply 2000)

14. Typical ETR A and B results M 2 4 4 3 7 9 3 2 M 2 2 2 2 1 3 2 1 M

15. Typical ETR/MIRU VNTR codes ETRMIRU IsolateABC2410162023242627313940 1844234226223513 2422226325173533 3323223325153224 4422225425153533 5324223326153323 6424223325153533 7324224325153323 8324223125153224 Level of discrimination: Outbreak investigations: 15 MIRU VNTR performance = RFLP Unlinked populations: 15 MIRU VNTR = False clusters Increasing the MIRU VNTR panel size < False clustering 24 loci typing (Supply et al., 2006 JCM)

16. 24 loci MIRU VNTR Multiplex PCR –D, 4156 –16, 39 –20, 424 –A, 3171 –2, 24 –31, 40 –27, 2347 –2401, 2163b –10, 1955 –B, C –26, 3690 –23, 4052 - Fluorescently labelled primers

17. Typical MIRU VNTR results

18. Prospective typing April 2005 – To date: –All Wales, South West England, Channel Islands, Isle of Man and Regions of Ireland –>2000 isolates –Approximately 1050 different profiles –803 profiles contain a single isolate –Largest cluster: 42 isolates –>20 possible outbreaks –5 laboratory contamination investigations –10 year profile of TB in Cardiff

19. Lab contamination investigation Laboratory perspective –Confirm the contamination –Possibly identify the source Route of contamination Implement controls to prevent future contamination –True cases may be masked by a contamination issue Identify these true cases Public Health implications –August 2010 –Contamination issue involving 17 cultures –16 generated the same profile –Only 1 previous isolates with this profile: July 2010 Source Linked to a true case in August, also the source –1 isolate with a different profile was linked to cluster that has been epidemiologically confirmed

20. A 10 year profile of TB in Cardiff - Summary 378 cases 243 profiles 77.8% of profiles contain a single isolate Largest cluster: 33 isolates Links between strains and geographical areas Areas with no dominant strain 4 of the 5 most prevalent strains in Cardiff are not in the top 20 strains in the UK Identified strains that have a link to gender and age Identified strains that have an increased level of resistance

21. Hospital Acquired TB Index Case: –Cancer/TB on admission? –Risk Stratification performed: Low Risk –Not felt to have TB –However, sputum sample taken –Nightingale Ward –TB cultured –Typing linked to an established Cardiff Outbreak –High Risk strain –On going outbreak in a Cardiff hostel –Epidemiological links with outbreak established Secondary Case: –Later Case presented with TB –Typing indicated it to be the same strain –On same ward as index –Pneumoconiosis Investigation by Infection Control –sample induced by a nebuliser –Patient was not isolated during procedure –Risk stratification focussed on index case

22. Initiating an outbreak investigation Autumn 2010 WCM notified the Carmarthen HPT of 4 cases of TB with the same typing profile. Low risk area for TB: 3 year mean 2 per 100000 Case 1: 83yo male, TB notification in 2005 Case 2: 72yo female, TB notification in March 2010 Case 3: 49yo male, TB notification in July 2010 Case 4: 41yo male, TB notification August 2010. Initial symptoms Nov 2009 Epidemiological investigation: –Case 1: no obvious links with other cases, lived 10 miles from other cases, died 2006. –Cases 2-4: Live within 2 miles of each other –Cases 3 and 4: Live within 0.5 miles of each other –Case 4: Publican –Cases 2 and 3: Regular drinkers in pub Outbreak control

23. Screening of Case 2 –Negative Screening of Case 3 –Son was Quantiferon positive: latent TB, received chemoprophylaxis Screening of Case 4 –Son was Quantiferon positive: latent TB, received chemoprophylaxis –Three pub workers were Quantiferon positive: latent TB, received chemoprophylaxis –Wife was quantiferon negative, mantoux 6-15mm (previous BCG), asymptomatic and negative microscopy and culture in sputum Wife of Case 4 –Previously diagnosed with Breast Cancer –Still immuno-compromised –Radiological changes on CXR –Clinicians considering re-initiating cancer therapy –Targeted bronchoscopy: washings grew MTB with same profile –Case 5

24. Further developments December 2010: Two further cases in area –Case 6: 61yo male with COPD, drinks in the local pubs, but not pub belonging to Case 4! Lives within 0.5 miles of Cases 3-5! Knew Case 3 but had not had contact for 2yrs! –Case 7: 70yo male with H1N1 Flu? Lived 14 miles from other cases. Did not visit pubs and no links with outbreak established. Brother-in-law had TB 5-10yrs earlier! Cross contamination with a different strain - excluded January 2011: –Cases 4 and 5 inform HPT they have children (grandchildren) with previous partners –Regular contact –Case 8: 15 month old female with recurrent “chestiness” and multiple Abx in 2010 GP dismisses TB and not initially investigated. –HPT screen: Quantiferon +tive, Mantoux >15mm, Changes on CXR –Active TB

25. TB typing in a low incidence area Prompted investigation by HPT Real-time determination of an outbreak Identified the focus of the outbreak Excluded cross contamination Helped to focus investigation –Identify cases not thought to be active TB –Asymptomatic patient additional investigations required to determine diagnosis –Identify cases of latent TB –Overcome limitations of epidemiological investigations Extensive awareness of TB to clinicians and public Targeted BCG vaccination

26. Acknowledgements WCM –Dr Michael Ruddy –Rhian Williams –Kay Parry –Gwyneth Samuel –Dave Tucker Molecular Unit –Dr Sally Corden –Michael Perry –Joanne Watkins –Puwfun Wong Outbreak control team – Dr Mac Walapu – Dr Speed – Dr Keir Lewis – Dr Mark Temple – Helen Bartlett – Jennifer Murphy – Chris Hayes – Polly Leet – Lon Jon – Denise Western – Sue Morgan