Current technology- Molecular fingerprinting of Mycobacterium 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

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

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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 cross contamination

- Potential false positive results

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Stopping Tuberculosis in England An Action Plan from the Chief Medical Officer- Oct 2004

Action 3: High Quality Surveillance Develop and implement protocols for the public health use of laboratory techniques such as DNA fingerprinting and molecular typing, and establish a central database linking fingerprinting and epidemiological data
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Response to the Tuberculosis Action Plan

The HPA has Developed and implemented protocols for prospective fingerprinting of all new isolates of M. tuberculosis
- Detect previously unrecognised transmission events/clusters

Established a central database linking fingerprinting and epidemiological data

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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% <6 bands) Pattern comparison is problematic
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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 between strains (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

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PCR amplification of individual VNTR loci

MIRU 2 Strain 1 DNA
PCR amplification

MIRU 4

3 repeats

2 repeats

Strain 2 DNA
PCR amplification

MIRU 2

MIRU 4

2 repeats

1 repeat

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Gel electrophoresis of MIRU PCR products

M Repeat number M

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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

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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

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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
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Automation of PCR setup

Dedicated PCR set-up robot (Corbett Robotics CAS-1200) Sets up a 96 well plate of PCR reactions in 40 min Performs entire PCR setup Advantages: Never makes mistakes, never gets bored, doesnt get RSI. Also not subject to AFC!

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Automation of fragment sizing

Transgenomic WAVE dHPLC - DNA fragment sizing - No intermediary sample manipulation - Based on novel DNA separation column

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Data from the WAVE instrument

Time

Data is in the form of retention time on the column

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Data from the WAVE instrument

Time

Data is in the form of retention time on the column

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Determining the fragment size

bp = th M
Base Pairs

p ats at l cus

. i s ly. i s

. tim e (m ins) y= .

8.

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Advantages of the WAVE system

Increases the speed and throughput of analysis Removes the ambiguity of gel electrophoresis Reduces the labour input

However there are disadvantages

-Disposal of the waste buffer (methyl cyanide) -Data analysis is cumbersome and slow -Single fragment per column injection

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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

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Application of MIRU-VNTR fingerprinting in the laboratory

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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?

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MIRU-VNTR typing
MIRU locus
2 4 10 3 3 16 3 3 20 2 2 23 5 5 24 1 1 26 7 7 27 3 3 31 4 4 39 4 4 40 3 3

Patient A

2 2

Patient B 2 2

Isolates are indistinguishable, referral lab checks original smears, one patient did not have TB
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Lab cross-contamination?
Four new positive cultures 8798 8799 8801 8806 Smear Smear Smear Smear Culture Positive at 16.3 days Culture positive at 5.7 days Culture positive at 9.2 days Culture positive at 18 days

Has there been a cross contamination event?

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Lab cross-contamination?
MIRU locus
Lab No. 8798 8799 8801 8806 2 4 10 16 20 23 24 26 27 31 39 40 2 2 2 2 2 4 2 2 3 1 2 4 3 3 3 3 2 1 2 2 5 5 5 6 1 1 1 1 6 5 5 5 3 3 3 3 3 3 3 3 2 2 2 2 3 1 2 2

Four isolates are all different, therefore original culture results were correct
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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 reinfection/relapse?

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New infection or relapse?

MIRU locus
2 4 10 4 4 16 4 4 20 2 2 23 5 5 24 1 1 26 7 7 27 3 3 31 5 5 39 3 3 40 4 4

Isolate 2002 Isolate 2003

2 2 2 2

Two strains are indistinguishable, most likely to be the same strain Therefore, relapse or non-compliance
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Six false positives in a week

RCM receives 6 isolates from another lab for ID
Patient ID Patient A Patient B Patient C Patient D Patient E Patient F Source lab No. 767 769 770 771 774 775

Nearly consecutive lab numbers raise suspicion Normally receive very small numbers of isolates per annum
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Fingerprinting finds them all indistinguishable

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

Discussions with the submitting lab identifies that they process a positive control with their patient samples

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The positive control is also indistinguishable!

Patient ID Patient A Patient B Patient C Patient D Patient E Patient F Positive Control A 3 3 3 3 3 3 3 B 2 2 2 2 2 2 2 C 4 4 4 4 4 4 4 2 2 2 2 2 2 2 2 4 2 2 2 2 2 2 2 10 16 20 23 24 26 27 31 39 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 5 5 5 5 5 5 5 1 1 1 1 1 1 1 5 5 5 5 5 5 5 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 40 4 4 4 4 4 4 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
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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 for vigilance and laboratory audit procedures
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Acknowledgements
Regional Centre for Mycobacteriology Newcastle HPA)
Dr John Magee, Anne Barrett, Sara Murray

Regional Centre for Mycobacteriology Birmingham HPA)

Jason Evans, Prof Peter Hawkey

Transgenomic
Phil Eastlake, Helen Lamb

HPA North East Laboratory

Contact details: Andy Sails Health Protection Agency Newcastle Laboratory Institute of Pathology, Newcastle General Hospital Westgate Road, Newcastle upon Tyne, NE4 6BE andrew.sails@hpa.org.uk HPA North East Laboratory