Current technology Molecular fingerprinting of Mycobacterium tuberculosis

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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_at_hpa.org.uk

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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
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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

HPA North East Laboratory
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Stopping Tuberculosis in EnglandAn 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
HPA North East Laboratory
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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

HPA North East Laboratory
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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 lt6 bands)
• Pattern comparison is problematic

HPA North East Laboratory
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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

HPA North East Laboratory
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PCR amplification of individual VNTR loci
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Gel electrophoresis of MIRU PCR products
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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
HPA North East Laboratory
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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

HPA North East Laboratory
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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

HPA North East Laboratory
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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!
HPA North East Laboratory
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Automation of fragment sizing

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

HPA North East Laboratory
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Data from the WAVE instrument
Time

• Data is in the form of retention time on the
  column

HPA North East Laboratory
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Data from the WAVE instrument
Time

• Data is in the form of retention time on the
  column

HPA North East Laboratory
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Determining the fragment size
346bp 5 repeats at the M23 locus
HPA North East Laboratory
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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

HPA North East Laboratory
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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)
• NB. This does not include capital, labour,
  overheads etc.
• Throughput 6 plates week gt1,000 isolates annum

HPA North East Laboratory
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Application of MIRU-VNTR fingerprinting in the
laboratory
HPA North East Laboratory
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Lab cross-contamination with MDR TB?

• 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
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MIRU-VNTR typing
MIRU locus
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 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
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Lab cross-contamination?
MIRU locus
Four isolates are all different, therefore
original culture results were correct
HPA North East Laboratory
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New infection or relapse?

• 2002 Patient diagnosed with TB, therapy commenced
• 2003 Patient again presents with active TB
• Has the patient acquired a new infection or is
  it re-infection/relapse?

HPA North East Laboratory
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New infection or relapse?
MIRU locus
Two strains are indistinguishable, most likely to
be the same strain Therefore, relapse or
non-compliance
HPA North East Laboratory
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Six false positives in a week

• RCM receives 6 isolates from another lab for ID

• Nearly consecutive lab numbers raise suspicion
• Normally receive very small numbers of isolates
  per annum

HPA North East Laboratory
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Fingerprinting finds them all indistinguishable
Locus
Discussions with the submitting lab identifies
that they process a positive control with their
patient samples
HPA North East Laboratory
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The positive control is also indistinguishable!
The profile has not previously been recognised in
our local database (gt1,500 strains) Also not
present in the national database ?WHO strain from
a QC distribution
HPA North East Laboratory
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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

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

HPA North East Laboratory