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MAC PCR at WSLH

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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

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
Outline
  • Background
  • Validation
  • Testing algorithm
  • Fee exempt testing
  • Recent test results
wslh tb mac pcr goal
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
real time mac pcr assay
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
real time mac pcr assay1
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.

real time pcr assay taqman
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
real time mac pcr assay2
Real-time MAC PCR Assay

As more target is amplified, fluorescence increases above the baseline.

real time pcr assay validation
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
extraction method same as tb pcr
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
mac pcr validation sensitivity based on smear status
MAC PCR Validation:Sensitivity based on Smear Status

(*) Specimens tested were culture-positive for MAC

limitations of mac pcr
Limitations of MAC PCR
  • Validation only includes respiratory specimens at this time
  • Sensitivity is limited; testing will be performed on smear positive specimens only
primary specimens for tb mac pcr
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
specimens for tb mac pcr
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.
submitting specimens for tb mac pcr
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
summary
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.
for more information
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

references
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.
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WSLH Laboratory Team

Nate

Youngmi and Ana

Dave

Don

Julie B.

Julie TK