Wisconsin State Laboratory of Hygiene
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
WISCONSIN STATE
LABORATORY OF HYGIENE
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Outline
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Background
Validation
Testing algorithm
Fee exempt testing
Recent test results
WISCONSIN STATE
LABORATORY OF HYGIENE
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State-wide Surveillance
WI Mycobacteria Isolates, 2012
n=1638
(3.4%)
(57.7%)
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LABORATORY OF HYGIENE
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WSLH Initial Smear Positive Primary
Specimens 2012, n=89
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LABORATORY OF HYGIENE
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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
WISCONSIN STATE
LABORATORY OF HYGIENE
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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
WISCONSIN STATE
LABORATORY OF HYGIENE
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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.
WISCONSIN STATE
LABORATORY OF HYGIENE
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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
WISCONSIN STATE
LABORATORY OF HYGIENE
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Real-time MAC PCR Assay
As more target is
amplified, fluorescence
increases above the
baseline.
WISCONSIN STATE
LABORATORY OF HYGIENE
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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
WISCONSIN STATE
LABORATORY OF HYGIENE
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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
WISCONSIN STATE
LABORATORY OF HYGIENE
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Validation: Limit of Detection,
MAC Assay compared to TB Assay
Strain
Limit of
Detection: cells
per reaction
Limit of
Detection
(cfu/ml)
MAV
ATCC 700898
14.25
2,800
MAV
TMC 706
37.66
7,500
M. Intracellulare
ATCC 1463
34.66
6,900
MTBC (IS6110)
ATCC 27294
0.75
150
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LABORATORY OF HYGIENE
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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
10
66.6%
1-9/field (3+)
13
12
92%
>9/field (4+)
10
10
100%
(*) Specimens tested were culture-positive for MAC
WISCONSIN STATE
LABORATORY OF HYGIENE
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Limitations of MAC PCR
• Validation only includes
respiratory specimens at this time
• Sensitivity is limited; testing will
be performed on smear positive
specimens only
WISCONSIN STATE
LABORATORY OF HYGIENE
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TB/MAC PCR Testing
Test
Specimen Type
Billing
TB/MAC PCR Panel
MM00256
MM00260
Initial smear positive
respiratory specimens
Same day, fee-exempt
testing
TB PCR alone
MM00256
Smear negative
specimens (respiratory
and non-respiratory)
Fee exempt testing is
performed with preapproval for the WI TB
Program
MAC PCR alone
MM00260
By special request on
initial smear positive
respiratory specimens
for which TB has been
ruled out
$100
CPT code 87556
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LABORATORY OF HYGIENE
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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
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LABORATORY OF HYGIENE
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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.
WISCONSIN STATE
LABORATORY OF HYGIENE
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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
WISCONSIN STATE
LABORATORY OF HYGIENE
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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
2
100
100
1+
6
4
3
75%
100
2+
5
3
1
33%
100
3+
3
1
0
0%
100
4+
7
2
2
100%
100
WISCONSIN STATE
LABORATORY OF HYGIENE
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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.
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LABORATORY OF HYGIENE
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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
WISCONSIN STATE
LABORATORY OF HYGIENE
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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LABORATORY OF HYGIENE
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WSLH Laboratory
Team
Nate
Dave
Julie B.
WISCONSIN STATE
LABORATORY OF HYGIENE
Don
Youngmi and Ana
Julie TK
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