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MAC PCR at WSLH

MAC PCR at WSLH. Julie Tans-Kersten, MS, BS-MT (ASCP) Tuberculosis Laboratory Program Coordinator Wisconsin State Laboratory of Hygiene tanskejl@mail.slh.wisc.edu (608) 263-5364. Outline. Background Validation Testing algorithm Fee exempt testing Recent test results.

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MAC PCR at WSLH

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  1. MAC PCR at WSLH Julie Tans-Kersten, MS, BS-MT (ASCP) Tuberculosis Laboratory Program Coordinator Wisconsin State Laboratory of Hygiene tanskejl@mail.slh.wisc.edu (608) 263-5364

  2. Outline • Background • Validation • Testing algorithm • Fee exempt testing • Recent test results

  3. State-wide SurveillanceWI Mycobacteria Isolates, 2012n=1638 (3.4%) (57.7%)

  4. WSLH Initial Smear Positive Primary Specimens 2012, n=89

  5. WSLH TB/MAC PCR Goal • Identify smear positive TB patients within 48 hours (HP 2020 Goal) • Respiratory isolation • Start therapy • Identify smear positive MAC patients • Release from isolation • Alter therapy decisions • Presumptive rapid results for 59% of smear positive patients

  6. Real-Time MAC PCR Assay • New York State Department of Health Wadsworth Center Real-Time multiplex PCR Assay on ABI 7500 • Targets the 16S-23S internal transcribed spacer (ITS) region • 1 forward primer • 5 reverse primers • 2 probes • WSLH running singleplex assay on Roche LightCycler

  7. Real-Time MAC PCR Assay Amplifies species within the M. avium complex: M. avium M. avium subsp. avium M. avium subsp. paratuberculosis M. intracellulare M. chimaera M. arosiense M. colombiense M. marseillense M. bouchedurhonense M.timonense.

  8. Real-Time PCR Assay (“Taqman”) • If MTBC DNA is present, 16S-23S ITS target will be amplified • Taqman probes are designed to hybridize within target. • As Taq polymerase amplifies target, it also separates fluorescent tag from quencher of Taqman probe. • More target amplified = more fluorescence

  9. Real-time MAC PCR Assay As more target is amplified, fluorescence increases above the baseline.

  10. Real-Time PCR Assay: Validation • Adapted assay for the Roche LightCycler, evaluated reaction and cycling conditions • Evaluated extraction methods • Limit of detection • Specificity • Reproducibility • Comparison of PCR results to culture

  11. Extraction Method (same as TB PCR) • Add 100 ul of sample to the lysis tube (yellow cap). Close tightly. • Vortex at high speed for 5 minutes (use HPLC tube vortex) • Centrifuge the lysis tube at low speed for 2-5 seconds to bring the contents to the bottom of the tube. • Heat at 90-100°C for 10 minutes (heat block) • Centrifuge for 2-3 minutes at 10,000 X g • Use supernatant for amplification

  12. Validation: Limit of Detection, MAC Assay compared to TB Assay

  13. MAC PCR Validation:Sensitivity based on Smear Status (*) Specimens tested were culture-positive for MAC

  14. Limitations of MAC PCR • Validation only includes respiratory specimens at this time • Sensitivity is limited; testing will be performed on smear positive specimens only

  15. TB/MAC PCR Testing

  16. Primary Specimens for TB/MAC PCR • Submit 3-10 ml of a primary respiratory specimen (sputum, tracheal aspirate, bronchial washing, BAL). • For primary, non-respiratory specimens: • Body fluids 5-10 ml • CSF at least 1 ml • Tissue: 1 g or 0.5 ml homogenized and concentrated • For primary specimens from non-sterile sites, WSLH will perform decontamination

  17. Specimens for TB/MAC PCR • 0.5 ml of a decontaminated sediment • Specimens with visible blood will be accepted • TB PCR testing must be performed within 7 days of specimen collection.

  18. Submitting Specimens for TB/MAC PCR • All specimens should be submitted in sterile, leak-proof containers. • Specimens should be stored at 2-8°C before and during transport • Notify the WI DPH TB program or Local Public Health Dept of new TB suspect • (Recommended): Notify WSLH of incoming specimen • Dunham Express courier: 1-800-236-7127, account 7271

  19. Preliminary Data, MAC PCRPrimary Specimens (25 culture confirmed)

  20. Summary • Fee-exempt MAC PCR testing is available at WSLH for smear positive respiratory specimens • WSLH will continue to validate testing for non-respiratory specimens • If MAC PCR positive, this testing can be used to presumptively rule out TB and release patients from isolation.

  21. For More Information Julie Tans-Kersten Wisconsin State Lab of Hygiene (608) 263-5364 Fax: (608) 890-2548 julie.tanskersten@slh.wisc.edu TB (Mycobacteriology) Lab: (608) 262-1618 Lorna Will, Philip Wegner, Pa Vang WI State TB Program 608-261-6319

  22. References • Combined real-time PCR and rpoB gene pyrosequencing for rapid identification of Mycobacterium tuberculosis and determination of rifampin resistance directly in clinical specimens. Halse TA, Edwards J, Cunningham PL, Wolfgang WJ, Dumas NB, Escuyer VE, Musser KA. J Clin Microbiol. 2010 Apr;48(4):1182-8.

  23. WSLH Laboratory Team Nate Youngmi and Ana Dave Don Julie B. Julie TK

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