8/6/2019 Andy Sails[1]

1/32

Current technology- Molecular fingerprinting

ofMycobacterium tuberculosis

Andy Sails

Regional Centre for Mycobacteriology

Health Protection Agency Newcastle Laboratory

Institute of Pathology, Newcastle General Hospital

Westgate Road, Newcastle upon Tyne, NE4 6BE

andrew.sails@hpa.org.uk

8/6/2019 Andy Sails[1]

2/32

Overview

Why fingerprint M. tuberculosis?

How do we fingerprint M. tuberculosis?

Application of new technology to streamline the

process

Examples of the usefulness of fingerprinting

HPA North East Laboratory

8/6/2019 Andy Sails[1]

3/32

Why fingerprint M. tuberculosis?

Epidemiological studies of defined geographic

regions or population groups

Contact tracing and outbreak investigations

- Confirm or refute suspected links between patients

Investigate potential laboratory crosscontamination

- Potential false positive results

HPA North East Laboratory

8/6/2019 Andy Sails[1]

4/32

8/6/2019 Andy Sails[1]

5/32

The HPA has-

Developed and implemented protocols for prospective

fingerprinting of all new isolates ofM. tuberculosis

- Detect previously unrecognised transmission events/clusters

Established a central database linking fingerprinting

and epidemiological data

Response to the Tuberculosis Action Plan

HPA North East Laboratory

8/6/2019 Andy Sails[1]

6/32

IS-6110 RFLP The gold standard

Advantages

Highly discriminatory method

Disadvantages

Technically demanding/cumbersome

Slow - poor in outbreak situations

Poor discrimination with low copy number isolates (25%

8/6/2019 Andy Sails[1]

7/32

VNTR fingerprinting

Variable Number Tandem Repeat sequences have been found in

the genomes of bacterial pathogens

The number of copies of repeat sequences can vary betweenstrains (however some are conserved and do not vary)

Demonstrated to be very useful for typing clonal pathogens

e.g. B. anthracis

More than 40 VNTR loci have been identified in M. tuberculosis

HPA North East Laboratory

8/6/2019 Andy Sails[1]

8/32

PCR amplification of individual VNTR loci

MIRU 4

DNA

MIRU 2

PCR

amplification

Strain 1

3 repeats 2 repeats

MIRU 4

DNA

MIRU 2

PCRamplification

Strain 2

1 repeat2 repeats

HPA North East Laboratory

8/6/2019 Andy Sails[1]

9/32

Gel electrophoresis of MIRU PCR products

HPA North East Laboratory

Repeat numberM M

8/6/2019 Andy Sails[1]

10/32

MIRU-VNTR protocol

Extract DNA from isolate

PCR amplification of the MIRU VNTR loci

Agarose gel electrophoresis to determine the

number of repeats

Combine the numbers of repeats at each locus into a digital

profile e.g. 2.3.3.2.2.6.1.3.3.3.2.1

HPA North East Laboratory

8/6/2019 Andy Sails[1]

11/32

MIRU-VNTR typing

Advantages

PCR-based therefore rapid turnaround

Do not require a viable culture

As discriminatory as IS6110 RFLP typing

Yields digital results, facilitates comparisons

Disadvantages

Labor intensive

Gel electrophoresis - cumbersome/can be difficult to interpret

HPA North East Laboratory

8/6/2019 Andy Sails[1]

12/32

Streamlining the process

Why?

- Each test requires 15 PCR reactions, 15 lanes on a gel!

- Approximately 1,000 isolates per annum

- Highly labour intensive process

- Potential to introduce errors may lead to an incorrect

assignment of profile

Which steps can we automate?

- PCR set-up

- Analysis of PCR products

HPA North East Laboratory

8/6/2019 Andy Sails[1]

13/32

Automation ofPCR setup

Dedicated PCR set-up robot (CorbettRobotics CAS-1200)

Sets up a 96 well plate of PCR reactionsin 40 min

Performs entire PCR setup

HPA North East Laboratory

Advantages: Never makes mistakes, never gets bored,

doesnt get RSI.

Also not subject to AFC!

8/6/2019 Andy Sails[1]

14/32

Automation of fragment sizing

Transgenomic WAVE

dHPLC

- DNA fragment sizing

- No intermediary sample

manipulation

- Based on novel DNA

separation column

HPA North East Laboratory

8/6/2019 Andy Sails[1]

15/32

Data from the WAVE instrument

Data is in the form of retention time on the column

Time

HPA North East Laboratory

8/6/2019 Andy Sails[1]

16/32

Data from the WAVE instrument

Data is in the form of retention time on the column

Time

HPA North East Laboratory

8/6/2019 Andy Sails[1]

17/32

Determining the fragment size

y = .

- 8. + .

. . . . . .

time (mins)

BasePair

s

i s

ly. i s

bp = p ats at

th M l cus

HPA North East Laboratory

8/6/2019 Andy Sails[1]

18/32

Advantages of the WAVE system

Increases the speed and throughput of analysis

Removes the ambiguity of gel electrophoresis

Reduces the labour input

HPA North East Laboratory

However there are disadvantages

-Disposal of the waste buffer (methyl cyanide)

-Data analysis is cumbersome and slow

-Single fragment per column injection

8/6/2019 Andy Sails[1]

19/32

Cost of fingerprinting

PCR costs: reagents and plastic consumables:

- 20.25 per isolate (15 loci)

Fragment size analysis on the WAVE system:

- 16.50 per isolate (15 loci)

Total reagent and consumables costs per isolate

- 36.50 (inc. VAT)

N . This does not include capital, labour, overheads etc.

Throughput: 6 plates week = >1,000 isolates annum

HPA North East Laboratory

8/6/2019 Andy Sails[1]

20/32

Application of MIRU-VNTR

fingerprinting in the laboratory

HPA North East Laboratory

8/6/2019 Andy Sails[1]

21/32

Lab cross-contamination with MDR T ?

The story:

Two isolates referred from source lab 2 patients

RCM susceptibility testing determines them to be multi

drug resistant MDR

Our lab notes that they have consecutive source lab

numbers unlikely to have 2 MDRs

One sample pulmonary the second one a urine

Has the source lab cross-contaminated these

two specimens?

HPA North East Laboratory

8/6/2019 Andy Sails[1]

22/32

MIRU-VNTR typing

2 4 10 16 20 23 24 26 27 31 39 40

Patient A 2 2 3 3 2 5 1 7 3 4 4 3

Patient B 2 2 3 3 2 5 1 7 3 4 4 3

MIRU locus

Isolates are indistinguishable, referral lab

checks original smears, one patient did not

have TB

HPA North East Laboratory

8/6/2019 Andy Sails[1]

23/32

Lab cross-contamination?

Four new positive cultures

8798 Smear Culture Positive at 16.3 days

8799 Smear Culture positive at 5.7 days

8801 Smear Culture positive at 9.2 days

8806 Smear Culture positive at 18 days

Has there been a cross contamination event?

HPA North East Laboratory

8/6/2019 Andy Sails[1]

24/32

Lab cross-contamination?

Lab No. 2 4 10 16 20 23 24 26 27 31 39 40

8798 2 2 2 3 2 5 1 6 3 3 2 3

8799 2 2 4 3 1 5 1 5 3 3 2 1

8801 2 2 3 3 2 5 1 5 3 3 2 2

8806 1 2 4 3 2 6 1 5 3 3 2 2

MIRU locus

Four isolates are all different, therefore

original culture results were correct

HPA North East Laboratory

8/6/2019 Andy Sails[1]

25/32

New infection or relapse?

2002 Patient diagnosed with T , therapy commenced

2003 Patient again presents with active T

Has the patient acquired a new infection or is it re-

infection/relapse?

HPA North East Laboratory

8/6/2019 Andy Sails[1]

26/32

New infection or relapse?

2 4 10 16 20 23 24 26 27 31 39 40

Isolate

2002

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

Isolate

2003

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

MIRU locus

Two strains are indistinguishable, mostlikely to be the same strain

Therefore, relapse or non-compliance

HPA North East Laboratory

8/6/2019 Andy Sails[1]

27/32

Six false positives in a week

RCM receives 6 isolates from another lab for ID

Patient ID Source lab No.

Patient A 767

Patient B 769

Patient C 770

Patient D 771

Patient E 774

Patient F 775

Nearly consecutive lab numbers raise suspicion

Normally receive very small numbers of isolates

per annum

HPA North East Laboratory

8/6/2019 Andy Sails[1]

28/32

Fingerprinting finds them all indistinguishable

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

Patient A 3 2 4 2 2 2 2 2 5 1 5 3 3 2 4

Patient B 3 2 4 2 2 2 2 2 5 1 5 3 3 2 4

Patient C 3 2 4 2 2 2 2 2 5 1 5 3 3 2 4

Patient D 3 2 4 2 2 2 2 2 5 1 5 3 3 2 4Patient E 3 2 4 2 2 2 2 2 5 1 5 3 3 2 4

Patient F 3 2 4 2 2 2 2 2 5 1 5 3 3 2 4

Discussions with the submitting lab identifies

that they process a positive control with theirpatient samples

HPA North East Laboratory

Locus

8/6/2019 Andy Sails[1]

29/32

The positive control is also indistinguishable!

Patient ID A B C 2 4 10 16 20 23 24 26 27 31 39 40Patient A 3 2 4 2 2 2 2 2 5 1 5 3 3 2 4

Patient B 3 2 4 2 2 2 2 2 5 1 5 3 3 2 4

Patient C 3 2 4 2 2 2 2 2 5 1 5 3 3 2 4

Patient D 3 2 4 2 2 2 2 2 5 1 5 3 3 2 4

Patient E 3 2 4 2 2 2 2 2 5 1 5 3 3 2 4Patient F 3 2 4 2 2 2 2 2 5 1 5 3 3 2 4

Positive Control 3 2 4 2 2 2 2 2 5 1 5 3 3 2 4

The profile has not previously been recognised in

our local database (>1,500 strains)

Also not present in the national database

?WHO strain from a QC distribution

HPA North East Laboratory

8/6/2019 Andy Sails[1]

30/32

Conclusions

Overview of current technology and practice for

fingerprinting

Demonstrated the usefulness of MIRU in the

laboratory

Fingerprinting can rapidly confirm suspected cases of

cross-contamination

MIRU-VNTR typing can also validate culture results

Highlighted the need forvigilance and laboratory audit

procedures

HPA North East Laboratory

8/6/2019 Andy Sails[1]

31/32

Acknowledgements

Regional Centre for Mycobacteriology Newcastle

HPA)

Dr John Magee, Anne Barrett, Sara Murray

Regional Centre for Mycobacteriology BirminghamHPA)

Jason Evans, Prof Peter Hawkey

TransgenomicPhil Eastlake, Helen Lamb

HPA North East LaboratoryHPA North East Laboratory

8/6/2019 Andy Sails[1]

32/32

Contact details: Andy Sails

Health Protection Agency Newcastle Laboratory

Institute ofPathology, NewcastleGeneral Hospital

Westgate Road, Newcastle upon Tyne, NE4 6BE

andrew.sails@hpa.org.uk HPA North East Laboratory