Transcript Current technology- Molecular fingerprinting of

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
[email protected]
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
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
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”
HPA North East Laboratory
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
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
HPA North East Laboratory
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
PCR amplification of individual VNTR loci
MIRU 2
MIRU 4
3 repeats
2 repeats
Strain 1
DNA
PCR
amplification
MIRU 2
Strain 2
MIRU 4
DNA
PCR
amplification
2 repeats
1 repeat
HPA North East Laboratory
Gel electrophoresis of MIRU PCR products
M
Repeat number
M
HPA North East Laboratory
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
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
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
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,
doesn’t get RSI.
Also not subject to AFC!
HPA North East Laboratory
Automation of fragment sizing
Transgenomic WAVE
dHPLC
- DNA fragment sizing
- No intermediary sample
manipulation
- Based on novel DNA
separation column
HPA North East Laboratory
Data from the WAVE instrument
Time
Data is in the form of retention time on the column
HPA North East Laboratory
Data from the WAVE instrument
Time
Data is in the form of retention time on the column
HPA North East Laboratory
Determining the fragment size
600
500
346bp = 5 repeats at
the M23 locus
Base Pairs
400
300
200
100
0
3.50
4.00
4.50
5.00
5.50
6.00
tim e (m ins)
Series1
Poly. (Series1)
y = 46.616x 2 - 238.43x + 342.52
HPA North East Laboratory
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
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 = >1,000 isolates annum
HPA North East Laboratory
Application of MIRU-VNTR
fingerprinting in the laboratory
HPA North East Laboratory
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 MDR’s)
• One sample pulmonary the second one a urine
Has the source lab cross-contaminated these
two specimens?
HPA North East Laboratory
MIRU-VNTR typing
MIRU locus
2 4
10
16
20
23
24
26
27
31
39
40
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
Patient A
Isolates are indistinguishable, referral lab
checks original smears, one patient did not
have TB
HPA North East Laboratory
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
Lab cross-contamination?
MIRU locus
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
Four isolates are all different, therefore
original culture results were correct
HPA North East Laboratory
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 reinfection/relapse?
HPA North East Laboratory
New infection or relapse?
MIRU locus
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
Two strains are indistinguishable, most
likely to be the same strain
Therefore, relapse or non-compliance
HPA North East Laboratory
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
Fingerprinting finds them all indistinguishable
Locus
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
4
Patient 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 their
patient samples
HPA North East Laboratory
The positive control is also 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
4
Patient 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
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
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
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
[email protected]
HPA North East Laboratory