Andy Sails[1]
Transcript of Andy Sails[1]
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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
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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
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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 crosscontamination
- Potential false positive results
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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
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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%
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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 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
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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
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Gel electrophoresis of MIRU PCR products
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Repeat numberM 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 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
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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
Data is in the form of retention time on the column
Time
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Data from the WAVE instrument
Data is in the form of retention time on the column
Time
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Determining the fragment size
y = .
- 8. + .
. . . . . .
time (mins)
BasePair
s
i s
ly. i s
bp = p ats at
th M l cus
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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
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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
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
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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?
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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
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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 re-
infection/relapse?
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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
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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
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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
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Locus
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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
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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 forvigilance 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 BirminghamHPA)
Jason Evans, Prof Peter Hawkey
TransgenomicPhil Eastlake, Helen Lamb
HPA North East LaboratoryHPA North East Laboratory
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Contact details: Andy Sails
Health Protection Agency Newcastle Laboratory
Institute ofPathology, NewcastleGeneral Hospital
Westgate Road, Newcastle upon Tyne, NE4 6BE
[email protected] HPA North East Laboratory