TB typing and its impact on Public health in Wales

Dr P. Lewis WhitePublic Health WalesMicrobiology Cardiff

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

Modified from HPA annual report on TB in UK 2010

2

4

6

8

10

12

14

16

18

1999 2001 2003 2005 2007 2009

Rat

e p

er 1

0000

0 p

op

ula

tio

n

year

UK rates of TB

UK

England

Scotland

Wales

Northern Ireland

HPA annual report on TB in UK 2010

19 High-risk PCTs

TB rates across the UK

Modified from HPA annual report on TB in UK 2010

16

3.19.3 7.1

12.38.4

44.4

6.511.8 9.3

6.1

18.713.4

10 1114

05

101520253035404550

Rate

per

100

000

popu

lati

on

14

7

57

4

5

2

Three year average rate in Wales

Cardiff Distribution – 10yr mean IR

00

0

08.7

<1

80.241.1

12.4

23.4

30.0

15.19.9

6.8

Wong MSc Thesis 2010

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.

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

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

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

The alternatives

Method Reproducibility No. of types

RFLP 100% 84

Mixed-linker PCR 100% 81

Spoligotyping 94% 61

DRE – PCR 58% 63

ETR – VNTR 97% 56

MIRU – VNTR 100% 78

Mostrom et al., 2002 CMI 8 694-704

Combined ETR and MIRU - VNTR = close correlation with RFLP (Hawkey et al., 2003 JCM)

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)

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

Typical ETR/MIRU VNTR codes

ETR MIRU

Isolate A B C 2 4 10 16 20 23 24 26 27 31 39 40

1 8 4 4 2 3 4 2 2 6 2 2 3 5 1 3

2 4 2 2 2 2 6 3 2 5 1 7 3 5 3 3

3 3 2 3 2 2 3 3 2 5 1 5 3 2 2 4

4 4 2 2 2 2 5 4 2 5 1 5 3 5 3 3

5 3 2 4 2 2 3 3 2 6 1 5 3 3 2 3

6 4 2 4 2 2 3 3 2 5 1 5 3 5 3 3

7 3 2 4 2 2 4 3 2 5 1 5 3 3 2 3

8 3 2 4 2 2 3 1 2 5 1 5 3 2 2 4

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)

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

Typical MIRU VNTR results

0

5000

10000

15000

20000

25000

30000

35000

0 100 200 300 400 500 600 700

4 3 0 7 A .B 1 1 _ 0 5 0 7 0 5 1 4 A 8

Size (nt)

Dye S

ignal

2","2MIRU

24","2MIRU

10","4MIRU

4","5MIRU

16","3MIRU

23","10MIRU

0

10000

20000

30000

40000

50000

60000

70000

0 50 100 150 200 250 300 350 400 450 500 550 600 650 700

4307 b .B 12_05070515M I

Size (nt)

Dye S

ignal

31","5MIRU

39","3MIRU

26","2MIRU

20","2MIRU

40","3MIRU

27","3MIRU

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

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

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

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

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

Outbreak control

• 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

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

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

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