Current technology- Molecular fingerprinting of Mycobacterium tuberculosis

1. 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

2. 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

3. 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

4. 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

5. 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

6. 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

7. VNTR fingerprinting Variable Number Tandem Repeat sequences have been found in the genomes of bacterial pathogens The number of copies of repeat sequencescan 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

8. 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 PCR amplification of individual VNTR loci HPA North East Laboratory

9. M Repeat number M Gel electrophoresis of MIRU PCR products HPA North East Laboratory

10. 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

11. 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

12. 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

13. 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

14. Automation of fragment sizing • Transgenomic WAVE dHPLC • - DNA fragment sizing • - No intermediary sample manipulation • - Based on novel DNA separation column HPA North East Laboratory

15. Data from the WAVE instrument • Data is in the form of retention time on the column Time HPA North East Laboratory

16. Data from the WAVE instrument • Data is in the form of retention time on the column Time HPA North East Laboratory

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

18. 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

19. 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

20. Application of MIRU-VNTR fingerprinting in the laboratory HPA North East Laboratory

21. 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

22. MIRU-VNTR typing MIRU locus Isolates are indistinguishable, referral lab checks original smears, one patient did not have TB HPA North East Laboratory

23. 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

24. Lab cross-contamination? MIRU locus Four isolates are all different, therefore original culture results were correct HPA North East Laboratory

25. 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

26. 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

27. 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

28. 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

29. The positive control is also indistinguishable! 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

30. 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

31. 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

32. 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