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

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

WSLH Initial Smear Positive Primary Specimens 2012, n=89

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

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

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) 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

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 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

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

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

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

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 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 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

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%

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 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 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.

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