2. MAC PCR at WSLH Julie Tans-Kersten, MS, BS-MT (ASCP)
Tuberculosis Laboratory Program Coordinator
Wisconsin State Laboratory of Hygiene
(608)

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

4. State-wide Surveillance WI Mycobacteria Isolates, 2012 n=1638
(3.4%)
(57.7%)

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

6. Identify smear positive TB patients within 48 hours (HP 2020 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

7. 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

8. 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.

9. 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

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

11. 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

12. 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 °C for 10 minutes (heat block)
Centrifuge for 2-3 minutes at 10,000 X g
Use supernatant for amplification

13. Validation: Limit of Detection, MAC Assay compared to TB Assay
Strain
Limit of Detection: cells per reaction
Limit of Detection (cfu/ml)
MAV
ATCC
14.25
2,800
TMC 706
37.66
7,500
M. Intracellulare
ATCC 1463
34.66
6,900
MTBC (IS6110)
ATCC 27294
0.75
150
Cell suspensions (known concentration) diluted in pooled sputa
LOD = highest dilution that can still be detected in 19/20 specimens

14. MAC PCR Validation: Sensitivity based on Smear Status
Smear Result
Number Tested*
Number MAC PCR Positive
Sensitivity
1-9/100 (1+) 10 1
10%
1-9/10 (2+) 15
66.6%
1-9/field (3+) 13 12
92%
>9/field (4+)
100%
Real smear positive patient respiratory sediments
(*) Specimens tested were culture-positive for MAC

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

16. TB/MAC PCR Testing TB/MAC PCR Panel MM00256 MM00260
Specimen Type
Billing
TB/MAC PCR Panel
MM00256
MM00260
Initial smear positive respiratory specimens
Same day, fee-exempt testing
TB PCR alone
Smear negative specimens (respiratory and non-respiratory)
Fee exempt testing is performed with pre-approval for the WI TB Program
MAC PCR alone
By special request on initial smear positive respiratory specimens for which TB has been ruled out
$100
CPT code 87556

17. 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

18. 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.

19. 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: , account 7271

20. Preliminary Data, MAC PCR Primary Specimens (25 culture confirmed)
Smear
Number of Specimens Tested
Number of Specimens that were MAC culture positive
Number of Specimens that were MAC PCR positive
Sensitivity (%)
Specificity (%)
Positive 4 2
100 1+ 6 3
75% 2+ 5 1
33% 3+ 0% 4+ 7
100%

21. 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.

22. For More Information Julie Tans-Kersten Wisconsin State Lab of Hygiene
(608)
Fax: (608)
TB (Mycobacteriology) Lab: (608)
Lorna Will, Philip Wegner, Pa Vang
WI State TB Program

23. 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 Apr;48(4):

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