TB Methodologies Dr. John G. Magee Regional Reference Centre for Mycobacteriology Health Protection Agency Regional Laboratory, Newcastle upon Tyne

Annual report, tuberculosis cases reported in 2000, England, Wales and Northern Ireland Of 6323 cases ONLY 3350 (53%) were culture confirmed Of 3729 with pulmonary disease: ONLY 2249 (60%) were culture confirmed ONLY 2513 had a smear result! ONLY 1406 (56%) were smear positive

DoH - Getting ahead of the curve - “Tuberculosis Action Plan” The HPA will work with reference laboratories and NHS microbiologists to improve the speed and consistency of laboratory diagnosis by: l providing high quality diagnostic services through a network of suitably equipped and experienced laboratories l standardising methods l establishing quality assurance/performance monitoring programmes covering.. liquid culture for all specimens.. molecular confirmation.. unique typing designation

DoH - Getting ahead of the curve - “Tuberculosis Action Plan” STANDARDS EXPECTED l smear turnaround time - 1 working day l all clinical samples to have access to automated liquid culture performed in experienced centres with large throughput and dedicated facilities and staff l all isolates referred to regional mycobacteriology centre for identification & susceptibility testing

DetectionSmear /CultureMolecular amplification of unique fragments IdentificationPhenotypic tests “Gene probes” PCR Sequencing Susceptibility testing Phenotypic expression Detection of gene mutations Fingerprinting (Typing) Numerical taxonomy RFLP … HIP Spoligotyping VNTR/MIRU Regional Centre for Mycobacteriology, Newcastle upon Tyne Diagnostic and Reference Mycobacteriology

Direct detection using molecular biological tests The problems are have something to do with low organism numbers and much to do with extraction of DNA from clinical samples

Commercial Assays: ç Roche Amplicor (Cobas & “Manual”) ç Abbott LCx (LCR) ç GenProbe Direct (TMA) ç BD ProbeTec ET (SDA) Direct detection using molecular biological tests Regional Centre for Mycobacteriology, Newcastle upon Tyne

In-House Assays: Single PCR; Semi-Nested PCR; Nested PCR…... and now Real-Time PCR Direct detection using molecular biological tests Amplification control (58  C) Target (62  C) Negative control -dF/dT Temp (°C) Regional Centre for Mycobacteriology, Newcastle upon Tyne

Diagnostic and Reference Mycobacteriology Microscopy for AFB, if done well, remains a cheap, simple, fast and effective diagnostic technique Regional Centre for Mycobacteriology, Newcastle upon Tyne

Isolation Regional Centre for Mycobacteriology, Newcastle upon Tyne

Sample M.tuberculosis present l of 330 laboratories 31.8% failed to isolate M.tuberculosis l of 176 UK laboratories 39.2% failed UK NEQAS Distribution Mycobacterium culture Sample M.tuberculosis present l of 331 laboratories 5.1% failed to isolate M.tuberculosis l of 176 UK laboratories 8.5% failed

Isolation: Liquid Media circa 1983 Regional Centre for Mycobacteriology, Newcastle upon Tyne

Isolation: Liquid Media circa 1993 Regional Centre for Mycobacteriology, Newcastle upon Tyne

Continuous Automated Mycobacterial Liquid Culture systems Regional Centre for Mycobacteriology, Newcastle upon Tyne

Cumulative weekly totals of Mtb complex isolates by CAMLiC and LJ slope culture Number of isolates >6 Weeks after inoculation LJ CAMLiC Regional Centre for Mycobacteriology, Newcastle upon Tyne

Detection of Acid-Fast Bacilli:  20 smears per week  1000 per year Mycobacterial Culture:  25 samples per week  1250 per year Susceptibility testing:  20 isolates per week  1000 per year Surety of Competence Regional Centre for Mycobacteriology, Newcastle upon Tyne

UK NEQAS Distribution Mycobacterium culture Sample M.tuberculosis NOT present 8/331 laboratories (2.4%) isolated a mycobacterium! In the last 4 negative samples there were 11,4, 2 & 10 false positives False positivity is probably due to laboratory cross contamination. NEQAS refer us to Breese at al. Arch Pathol Lab Med 2001, 125(9): 1213 BUT see also- de Boer et al. J Clin Microbiol 2002; 40; 4004 They found that labs processing <3000 samples per annum showed a greater risk of cross-contamination

Identification of mycobacteria Regional Centre for Mycobacteriology, Newcastle upon Tyne

Identification of mycobacteria “Gene-Probes” can confirm M.tuberculosis complex in under 2 hours Regional Centre for Mycobacteriology, Newcastle upon Tyne

Identification of mycobacteria “Genetic probes” are NOT amplification procedures - They can identify a limited number of common species: M.tuberculosis complex M.avium complex M.avium M.intracellulare M.kansasii M.gordonae Regional Centre for Mycobacteriology, Newcastle upon Tyne

Characterisation of mycolic acids by HPLC Detection of unique fragments of genomic DNA 16S rRNA sequencingequipment database variances Identification of Mycobacteria [ ] Regional Centre for Mycobacteriology, Newcastle upon Tyne

Susceptibility testing of mycobacteria In the U.K. susceptibility testing of mycobacteria is performed by one of two methods:  The Resistance Ratio Method comparing MICs of test and control strains  The Radiometric or Proportional Method using the Bactec 460 TB The Resistance Ratio Method is reliable & reproducible but laborious and relatively slow The Radiometric Method is faster but suffers from problems inherent in the Bactec 460 Regional Centre for Mycobacteriology, Newcastle upon Tyne

Continuous Automated Mycobacterial Liquid Culture Systems Regional Centre for Mycobacteriology, Newcastle upon Tyne

Molecular detection of drug resistance Drug Putative resistance gene Resistant strains with mutation (%) Isoniazid Rifampicin Pyrazinamide Ethambutol katG inhA ahpC kasA pncA embB rpoB Riska et al. Int J Tuberc Lung Dis 2000; 4(2):S4-10

UK Initial isolates showing resistance to any drug; Isoniazid & MDR resistance (%), Year Number of isolates Percentage Resistant N% Isoniazid resistant% MDR N Regional Centre for Mycobacteriology, Newcastle upon Tyne

DoH - Getting ahead of the curve - “Tuberculosis Action Plan” Develop and implement protocols for DNA fingerprinting taking customers needs into account. Establish a central database … linking fingerprinting and epidemiological data

MTBC Strain Typing Methods IS6110-based fingerprinting Most discriminatory method Slowest method (3-6 weeks) Difficult to compare large numbers of patterns Restriction Fragment Length Polymorphism, RFLP PCR-RFLP Hemi-nested Inverse PCR (HIP) Regional Centre for Mycobacteriology, Newcastle upon Tyne

HIP fingerprinting results Regional Centre for Mycobacteriology, Newcastle upon Tyne

Spoligotyping Spacer Oligonucleotide Typing Less discriminatory than IS6110 typing … BUT... Faster turnaround time Digital results, facilitating comparisons Does not require viable cultures Regional Centre for Mycobacteriology, Newcastle upon Tyne

VNTR - MIRU Variable Number of Tandem Repeats  Measures variability in 6 loci Mycobacterial Interspersed Repetitive Units  Measures variability in 12 loci PCR-based = rapid turnaround Digital results, facilitates comparisons Highly discriminatory Does not require viable cultures High throughput (automated sequence analysers) Regional Centre for Mycobacteriology, Newcastle upon Tyne

Future Genotyping Strategy Primary typing will be high throughput, automated, PCR-based i.e. MIRU/VNTR Secondary typing by IS6110 RFLP when needed for discrimination Regional Centre for Mycobacteriology, Newcastle upon Tyne

Time taken for turnaround of final reports (from date sent by RCM to date final report reaching clinicians) <=1 day2 days3-4 days5-6 days7-8 days9-10 days>10days Time No of samples Regional Centre for Mycobacteriology, Birmingham