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

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

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 Response to the Tuberculosis Action Plan 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 4 DNA MIRU 2 PCR amplification Strain 1 3 repeats 2 repeats MIRU 4 DNA MIRU 2 PCR amplification Strain 2 1 repeat2 repeats HPA North East Laboratory

Gel electrophoresis of MIRU PCR products HPA North East Laboratory Repeat numberMM

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 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 HPA North East Laboratory Advantages: Never makes mistakes, never gets bored, doesn’t get RSI. Also not subject to AFC!

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 Data is in the form of retention time on the column Time HPA North East Laboratory

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

Determining the fragment size 346bp = 5 repeats at the M23 locus 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 HPA North East Laboratory However there are disadvantages ­Disposal of the waste buffer (methyl cyanide) ­Data analysis is cumbersome and slow ­Single fragment per column injection

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 Patient A Patient B MIRU locus 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 8798Smear – Culture Positive at 16.3 days 8799Smear + Culture positive at 5.7 days 8801Smear + Culture positive at 9.2 days 8806Smear – Culture positive at 18 days Has there been a cross contamination event? HPA North East Laboratory

Lab cross-contamination? Lab No MIRU locus Four isolates are all different, therefore original culture results were correct HPA North East Laboratory

New infection or relapse? 2002Patient diagnosed with TB, therapy commenced 2003Patient 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? Isolate Isolate MIRU locus 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 Patient IDABC Patient A Patient B Patient C Patient D Patient E Patient F Discussions with the submitting lab identifies that they process a positive control with their patient samples HPA North East Laboratory Locus

The positive control is also indistinguishable! Patient IDABC Patient A Patient B Patient C Patient D Patient E Patient F Positive Control 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 HPA North East Laboratory