Current technology- Molecular fingerprinting of Mycobacterium

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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@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



                                      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
  Strain 1
             DNA
        PCR
        amplification
                           3 repeats      2 repeats



                         MIRU 2        MIRU 4
  Strain 2
             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
              400
 Base Pairs




              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

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




      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 re-
infection/relapse?




                                             HPA North East Laboratory
New infection or relapse?

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

 Isolate   2 2   4    4    2    5    1    7    3       5     3     4
 2002
 Isolate   2 2   4    4    2    5    1    7    3       5     3     4
 2003


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