FOR IN VITRO DIAGNOSTIC USE

Transcription

1 For the qualitative detection and identification of herpes simplex virus types 1 and 2 and varicella zoster virus from cutaneous or mucocutaneous lesion swab specimens. FOR IN VITRO DIAGNOSTIC USE Contents INTENDED USE... 3 SUMMARY AND EXPLANATION... 3 PRINCIPLE OF THE PROCEDURE... 4 MATERIALS PROVIDED... 4 MATERIALS REQUIRED BUT NOT PROVIDED... 5 WARNINGS AND PRECAUTIONS... 5 STORAGE AND HANDLING OF KIT REAGENTS... 6 SPECIMEN COLLECTION, STORAGE, AND HANDLING... 6 PREPARED SAMPLE STORAGE... 6 INITIAL THERMOCYCLER PROGRAMMING FOR HSV 1 + 2/VZV DETECTION AND IDENTIFICATION... 6 Life Technologies QuantStudio Dx Programming Instructions... 6 Applied Biosystems 7500 Fast and Fast Dx Programming Instructions... 6 Cepheid SmartCycler II System Programming Instructions... 9 Initial Thermocycler Programming for HSV detection and identification Life Technologies QuantStudio Dx Programming Instructions Applied Biosystems 7500 Fast and Fast Dx Programming Instructions Cepheid SmartCycler II System Programming Instructions Initial Thermocycler Programming for VZV detection Life Technologies QuantStudio Dx Programming Instructions Applied Biosystems 7500 Fast and Fast Dx Programming Instructions Cepheid SmartCycler II System Programming Instructions ASSAY PROCEDURE Lyra Direct HSV 1+2/VZV Assay Page 1 of 50

2 Sample Prep Procedure Master Mix Rehydration Procedure HSV 1 + 2/VZV PCR Set-up Procedure AMPLIFICATION PROTOCOL FOR HSV 1 + 2/VZV DETECTION AND IDENTIFICATION Amplification Protocol on the Life Technologies QuantStudio Dx Amplification Protocol on the Applied Biosystems 7500 Fast and Fast Dx Amplification Protocol on the Cepheid SmartCycler II System AMPLIFICATION PROTOCOL FOR HSV DETECTION AND IDENTIFICATION Amplification Protocol on the Life Technologies QuantStudio Dx Amplification Protocol on the Applied Biosystems 7500 Fast and Fast Dx Amplification Protocol on the Cepheid SmartCycler II System AMPLIFICATION PROTOCOL FOR VZV DETECTION Amplification Protocol on the Life Technologies QuantStudio Dx Amplification Protocol on the Applied Biosystems 7500 Fast and Fast Dx Amplification Protocol on the Cepheid SmartCycler II System INTERPRETATION OF RESULTS Interpretation of Results using the Life Technologies QuantStudio Dx Interpretation of Results using the Applied Biosystems 7500 Fast and Fast Dx Interpretation of Results using the Cepheid SmartCycler II System QUALITY CONTROL LIMITATIONS EXPECTED VALUES Life Technologies QuantStudio Dx Applied Biosystems 7500 Fast Dx Cepheid SmartCycler II System CLINICAL PERFORMANCE Life Technologies QuantStudio Dx Cutaneous Lesions Mucocutaneous Lesions Applied Biosystems 7500 Fast Dx Cutaneous Lesions Mucocutaneous Lesions Cepheid SmartCycler II System Cutaneous Lesions Mucocutaneous Lesions ANALYTICAL PERFORMANCE Level of Detection Lyra Direct HSV 1+2/VZV Assay Page 2 of 50

3 Precision Repeatability Analytical Reactivity (Inclusivity) Reproducibility Study Analytical Specificity Interfering or Cross-Reactive Microorganism Analytical Specificity Interfering or Cross-Reactive Substances Carryover and Cross-Contamination Studies CUSTOMER AND TECHNICAL ASSISTANCE INTELLECTUAL PROPERTY REFERENCES GLOSSARY INTENDED USE The Lyra Direct HSV 1+2/VZV Assay is an in vitro multiplex Real-Time PCR test for qualitative detection and differentiation of herpes simplex virus type 1, herpes simplex virus type 2, and varicella-zoster virus DNA isolated and purified from cutaneous or mucocutaneous lesion samples obtained from symptomatic patients suspected of active herpes simplex virus 1, herpes simplex virus 2 and/or varicella-zoster infection. The Lyra Direct HSV 1+2/VZV Assay is intended to aid in the diagnosis of herpes simplex virus 1, herpes simplex virus 2 and varicella-zoster virus active cutaneous or mucocutaneous infections. Negative results do not preclude herpes simplex virus 1, herpes simplex virus 2 and varicellazoster virus infections and should not be used as the sole basis for diagnosis, treatment or other management decisions. The Lyra Direct HSV 1+2/VZV Assay is not intended for use with cerebrospinal fluid or to aid in the diagnosis of HSV or VZV infections of the central nervous system (CNS). The Lyra Direct HSV 1+2/VZV Assay is not intended for use in prenatal screening. The device is not intended for point-of-care use. Warning: The Lyra Direct HSV 1+2/VZV Assay is not intended for use with cerebrospinal fluid or to aid in the diagnosis of HSV or VZV infections of the central nervous system (CNS). The Lyra Direct HSV 1+2/VZV Assay is not intended for use in prenatal screening. SUMMARY AND EXPLANATION HSV-1 and HSV-2: Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), also known as Human herpes virus 1 and 2 (HHV-1 and HHV-2), are DNA viruses of the family Herpesviridae. HSV infections in humans can cause lesions at a variety of cutaneous and mucocutaneous sites. These lesions can be a result of the primary infection by the virus or they can result from a reactivation of the latent virus, causing recurrent episodes of the disease. HSV-1 and HSV-2 are genetically and antigenically distinct forms of HSV. HSV-2 is the most common cause of genital infections, due to venereal transmission; HSV-1 is commonly associated with other disease locations although both serotypes have been shown to cause disease in all locations of the body. Studies have shown an increasing prevalence of genital HSV infections with a concomitant increase of the disease in neonates. VZV: Varicella-zoster virus (VZV), also known as Human herpes virus 3 (HHV-3), is a DNA virus of the family Herpesviridae. Primary VZV infection results in chickenpox (varicella), which may rarely result in complications including encephalitis or pneumonia. Even when clinical symptoms of chickenpox have resolved, VZV remains dormant in the nervous system of the infected person (virus latency). In approximately 10% to 20% of cases, VZV reactivates later in life producing shingles. Serious complications of shingles include postherpetic neuralgia, zoster multiplex, myelitis, herpes ophthalmicus, or zoster sine herpete. Lyra Direct HSV 1+2/VZV Assay Page 3 of 50

4 PRINCIPLE OF THE PROCEDURE The assay detects viral nucleic acids from a patient sample. A multiplex Real-time PCR reaction is carried out under optimized conditions in a single tube or well generating amplicons for HSV-1, HSV-2, VZV, and the Process Control (PRC). Identification of HSV-1, HSV-2, VZV, and the PRC occurs by the use of target-specific primers and fluorescent-labeled probes that hybridize to conserved regions in the genomes of HSV-1, HSV-2, VZV and the PRC. The following is a summary of the procedure: Lyra Direct Probe Labels Target Dye HSV-1 FAM HSV-2 CAL Fluor Orange 560 VZV CAL Fluor Red 610 PRC Quasar Sample Collection: Obtain lesion swabs using standard techniques from symptomatic patients. Transport, store, and process these specimens according to established laboratory procedures Sample Preparation: Remove 100 µl of each clinical specimen from the original collection tube and place into a clean microcentrifuge tube. Heat at 60 C for 5 minutes. Remove from heat and add 25 µl of Process Buffer within 60 minutes according to the detailed instructions and using the appropriate reagents (See Materials Required but Not Provided). The Process Buffer contains a buffer and diluents as well as the PRC. The PRC serves to monitor inhibitors in the prepared specimen, assures that adequate amplification has taken place, and confirms that the overall process was performed correctly. 3. Rehydration of Master Mix: Rehydrate the lyophilized Master Mix using 135 µl of the Rehydration Solution. The Master Mix contains oligonucleotide primers, fluorophore and quencher-labeled probes targeting conserved regions of HSV-1, HSV-2, and VZV, as well as the PRC. 4. Nucleic Acid Amplification and Detection: Add 15 µl of the rehydrated Master Mix to each reaction tube or plate well. Then add 5 µl of nucleic acids (specimen with PRC) to the plate well or appropriately labeled reaction tube. Place the plate or tube into either the Life Technologies QuantStudio Dx, Applied Biosystems 7500 Fast Dx, Applied Biosystems 7500 Fast or Cepheid SmartCycler II instruments, respectively. Once the reaction tube or plate is added to the instrument, initiate the assay protocol. This protocol initiates amplification of the DNA target amplicons. The Lyra Direct HSV 1+2/VZV assay is based on TaqMan chemistry and uses an enzyme with DNA polymerase, and 5-3 exonuclease activities. During DNA amplification, this enzyme cleaves the probe bound to the complementary DNA sequence, separating the quencher dye from the reporter dye. This step generates an increase in fluorescent signal upon excitation by a light source of the appropriate wavelength. With each cycle, additional dye molecules are separated from their quenchers resulting in additional signal. If sufficient fluorescence is achieved on the Dx, 7500 Fast Dx, 7500 Fast or the SmartCycler II System, the sample is reported as positive for the detected nucleic acid. MATERIALS PROVIDED Cat. #M102 Detection Kit (96 Reactions) Store at 2 C to 8 C Component Rehydration Solution Lyra Direct HSV 1+2/VZV Master Mix Lyophilized Contents: DNA polymerase enzyme Primers and probes Quantity 1 vial/kit 1.9 ml 12 vials/kit, 8 reactions/vial Lyra Direct HSV 1+2/VZV Assay Page 4 of 50

5 Component dntps Stabilizers Process Buffer Quantity 2 vials/kit 1.5 ml each MATERIALS REQUIRED BUT NOT PROVIDED controls for HSV-1, HSV-2 and VZV (i.e. the laboratory s own internal control materials from isolated and characterized clinical specimen previously submitted for interpretation which serves as an external assay control or Quidel Molecular HSV 1+2/VZV Control Set, Cat. #M121, which contains positive and negative controls, serves as an external processing control) Micropipettors (range between 2 to 20 μl and 20 to 200 μl) Non-aerosol pipette tips Life Technologies QuantStudio Dx with software version 1.0 or later Applied Biosystems 7500 Fast Dx or 7500 Fast with software version or later Applied Biosystems Fast Dx 96 well PCR plate Optical plate films Plate centrifuge for ABI 96 well plate Dry heating block, capable of heating 1.5 ml tubes at 60 C for 5 minutes Empty microcentrifuge tubes Or Micropipettors (range between 2 to 20 μl and 20 to 200 μl) Non-aerosol pipette tips Cepheid SmartCycler II with software version 3.0b or later SmartCycler disposables SmartCycler centrifuge Smartcycler reaction tube racks Dry heating block, capable of heating 1.5 ml tubes at 60 C for 5 minutes Empty microcentrifuge tubes WARNINGS AND PRECAUTIONS Treat all specimen/samples as potentially infectious. Follow universal precautions when handling samples, this kit, and its contents. Proper sample collection, storage, and transport are essential for correct results. Store assay reagents as indicated on their individual labels. Wear suitable protective clothing, gloves, eye, and face protection when using this kit. For accurate results, pipette carefully using only calibrated equipment. Thoroughly clean and disinfect all surfaces with a 10% bleach solution followed by molecular grade water. Use micropipettes with an aerosol barrier or positive displacement tips for all procedures. Avoid microbial and cross contamination of the kit reagents. Follow Good Laboratory Procedures. Do not mix reagents from kits with different lot numbers. Do not use reagents from other manufacturers with this kit. Do not use product after its expiration date. Proper workflow planning is essential to minimize contamination risk. Always plan laboratory workflow in a unidirectional manner, beginning with pre-amplification and moving through amplification and detection. Use dedicated supplies and equipment in pre-amplification and amplification areas. Do not allow cross movement of personnel or equipment between areas. Keep amplification supplies separate from pre-amplification supplies at all times. Do not open sample tubes or unseal plates post amplification. Dispose of amplified material carefully and in accordance with local laws and regulations in order to minimize the risk of amplicon contamination. Lyra Direct HSV 1+2/VZV Assay Page 5 of 50

6 Do not use supplies dedicated for reagent or sample preparation for processing target nucleic acid. Wash hands thoroughly after handling. For additional information on hazard symbols, safety, handling and disposal of the components within this kit, please refer to the Safety Data Sheet (SDS) located at quidel.com. STORAGE AND HANDLING OF KIT REAGENTS Store the unopened kit at 2 C to 8 C until the expiration date listed on the outer kit box. The rehydrated Master Mix may be kept at room temperature (20 C to 25 C) for up to 1 hour, or at 2 C to 8 C for up to 9 hours or at 20 C for up to 3 days. The rehydrated Master Mix should be recapped, sealed with parafilm, and stored in an upright position. Protect the Master Mix from light during storage. Indications of Instability or Deterioration of Reagents: Cloudiness of the Rehydration Solution may indicate deterioration of this reagent. Contact Quidel Technical Support for a replacement. SPECIMEN COLLECTION, STORAGE, AND HANDLING Specimens used for the validation of the Lyra Direct HSV 1+2/VZV Assay were obtained using standard techniques from patients with lesion infection symptoms. These specimens were collected, transported, stored, and processed according to CLSI M41-A. 2 Samples can be stored at 2 C to 8 C or 20 C for up to 7 days prior to processing. A series of studies were preformed evaluating a number of routinely used viral transport medium at a volume of 2 ml: M4, M4-RT, M5, M6, and UTM. No significant difference in assay performance was seen between the five different types of viral transport media. PREPARED SAMPLE STORAGE Prepared samples can be stored for 48 hours when stored at 2 C to 8 C, 31 days when stored at either 20 C or 70 C. INITIAL THERMOCYCLER PROGRAMMING FOR HSV 1+2/VZV DETECTION AND IDENTIFICATION Life Technologies QuantStudio Dx Programming Instructions Lyra provides pre-defined templates for each assay that must be uploaded to the Life Technologies QuantStudio Dx. These templates contain the run parameters such that no instrument programming is needed to get started. To install a test definition document: 1. From the Life Technologies QuantStudio Dx Software Home tab, click Manage Test in the Tools panel. 2. From the Test Menu, click Install. 3. Navigate to your test definition document (.tdd) file, select the Lyra HSV+VZV file, and click Open. The Life Technologies QuantStudio Dx Software automatically adds the selected test to the Test Menu. 4. Click Close to close the Test Menu and save your changes. Applied Biosystems 7500 Fast and Fast Dx Programming Instructions 1. Launch the Applied Biosystems 7500 Fast or Fast Dx software package. 2. The Quick Startup document dialog window will open. Select the Create New Document button to start the New Document Wizard. Follow each step to initiate the Lyra Direct HSV+VZV protocol. a. Define Document: Most of the following should be the default setting. If not, change accordingly. i. Confirm or enter the following information Assay: Standard Curve (Absolute Quantitation) Container: 96-Well Clear Template: Blank Document Lyra Direct HSV 1+2/VZV Assay Page 6 of 50

7 Run Mode: Fast 7500 Operator: your operator name Comments: SDS v1.4.1 Plate Name: Lyra Direct HSV+VZV ii. Select the Next button. iii. Select Detectors: New detectors for HSV-1, HSV-2, VZV and the process control (PRC), must be added. For each target, select the New Detector button to open the New Detector pop-up window. Alternatively, use the Create Another button from within the New Detector pop-up window for the last two detectors Enter the following information for each detector: Name Reporter Dye Quencher Dye Color HSV-1 FAM (none) (Select) HSV-2 JOE (none) (Select) VZV Texas Red (none) (Select) PRC Cy5 (none) (Select) iv. Select a unique color to represent each detector. v. Highlight the new detectors and add to the Detectors in Document column using the Add button. vi. Select (none) from the Passive Reference drop-down menu. vii. Select the Next button. viii. Select the Finish button without setting any wells. b. The wizard will close, and the software will open, starting with the Setup tab. This will show the sample plate that was set up during the quick start. For the initial set up, nothing needs to be changed here. c. Define the Thermocycler Protocol: i. Select the Instrument tab to set up the Lyra Direct HSV 1+2/VZV PCR cycling times and temperatures. ii. Under Thermal Profile there should be a default 2-stage protocol. Each stage will have 3 usereditable text boxes. The top box value represents the number of reps or cycles for that stage. The middle box value represents the temperature ( C), and the lowest box value represents the time (minutes: seconds). iii. Make the following changes to the default Thermal Cycler Protocol: 1. Stage 1 a. Reps: 1 b. Temp: 95 c. Time: 2:00 2. Stage 2 (2-Step Amplification Stage) a. Reps: 40 Step 1 i. Temp: 95 ii. Time: 0:05 b. Step 2 i. Temp: 58 ii. Time: 0:25 3. If a wrong stage is added, the stage may be removed by pressing the Delete button after highlighting the stage between the vertical lines. iv. Under Settings, enter the following: Sample Volume (μl): 20 (default) Run Mode: Fast 7500 (default) Data Collection: Stage 2, Step 2 0:25) NOTE: Do NOT check the check box next to Expert Mode. Lyra Direct HSV 1+2/VZV Assay Page 7 of 50

8 v. Final protocol d. Set threshold for each analyte as follows: i. Select the Results tab. ii. Select the Amplification Plot tab. iii. Select HSV-1 from the Detector tab in the top right corner. iv. In the Analysis Settings block, set the Threshold to 1.0e5. v. Select the Auto Baseline radio button. vi. Repeat iii-v for HSV-2 setting the Threshold to 1.5e4. vii. Repeat iii-v for VZV setting the Threshold to 1.0e5. viii. Repeat iii-v for PRC setting the Threshold to 1.5e4. e. Save the new protocol as a template for future uses. i. At the top of the screen select File and then Save As. ii. Save In: D:\Applied Biosystems\7500 Fast System\Templates\. iii. File name: Lyra Direct HSV+VZV. iv. Save as type: SDS Templates (*.sdt). f. Exit the software. Lyra Direct HSV 1+2/VZV Assay Page 8 of 50

9 Cepheid SmartCycler II System Programming Instructions 1. Launch the Cepheid SmartCycler II System Version 3.0b software package. 2. Create the Lyra Direct HSV+VZV assay. a. Select the Define Assays button from the top of the screen. b. Name the assay. i. Select the New button at the bottom left corner of the screen. ii. Type in Lyra Direct HSV+VZV and select OK. iii. Lyra Direct HSV+VZV will be added to the top of the Assay Name list located on the upper lefthand of the screen. c. Set the analysis values: Under the Assay Type: Research section, select the Analysis Settings tab, and make sure the following specifications are set: i. Select FCTC25 from the Dye Set drop-down menu. ii. The Analysis Type drop-down menu should be set to Qualitative (Default setting). iii. In the Channel Name column, enter HSV-1 for Channel 1, HSV-2 for Channel 2, VZV for Channel 3, and PRC for Channel 4. iv. In the Usage column, select Target from the drop down menus for HSV-1, HSV-2, and VZV, and select Internal Control for PRC. When selecting Internal Control, a window below will pop up. Select the Yes button. v. In the Curve Analysis column, enter Primary Curve for each channel (HSV-1, HSV-2, VZV, and PRC) (Default setting). vi. In the Thresh Setting column, enter Manual Threshold for each channel (HSV-1, HSV-2, VZV, and PRC) (Default setting). vii. In the Manual Thresh Fluor Units column, enter the following thresholds: a. HSV-1: 30.0 b. HSV-2: 30.0 c. VZV: 30.0 d. PRC: 8.0 viii. In the Valid Min Cycle column (scroll to the right if not immediately visible), enter 5 for each channel (HSV-1, HSV-2, VZV, and PRC). ix. In the Valid Max Cycle column (scroll to the right if not immediately visible), enter 45 for each channel (HSV-1, HSV-2, VZV, and PRC). x. In the Bkgnd Sub column, use ON for each channel (HSV-1, HSV-2, VZV, and PRC). (Default setting). xi. In the Bkgnd Min Cycle column, enter 5 for each channel (HSV-1, HSV-2, VZV, and PRC). xii. In the Bkgnd Max Cycle column, enter 45 for both HSV1 and HSV2 channels. xiii. In the Bkgnd Max Cycle column, enter 30 for VZV channel. xiv. In the Bkgnd Max Cycle column, enter 25 for PRC channel. xv. In the Boxcar Avg cycles column, keep 0 for each channel (HSV-1, HSV-2, VZV, and PRC). (Default setting). xvi. In the End Pt Threshold column, enter 30 for each channel (HSV-1, HSV-2, and VZV) and 8 for PRC. (Default setting). xvii. In the NC IC% column, keep NA for PRC channel (Default setting). xviii. In the IC Delta column, keep NA for each HSV-1, HSV-2, and VZV channels (Default setting). xix. In the Customize Result Text section (below the table), select Organism Based Result Text from the drop-down menu. The warning window below will pop up. Select Yes. Lyra Direct HSV 1+2/VZV Assay Page 9 of 50

10 xx. Select the Customize button to open the Organism-Based Result Text dialog window. Select the Add button, enter HSV-1 in the Organism Name column and check the HSV-1 box. Select the Add button again, enter HSV-2 in the Organism Name column and check the HSV-2 box. Select the Add button again, enter VZV in the Organism Name column and check the VZV box. Click OK at the bottom of the pop up window. d. Set the PCR cycling times and temperatures as follows: i. Stage 1 1. Hold 2. Temp: Secs: Optics: OFF ii. Stage Temperature Cycle 2. Times to Repeat: First Temperature Row: a. Temp: 95.0 b. Secs: 10 c. Optics: OFF 4. Second Temperature Row a. Temp: 58.0 b. Secs: 15 c. Optics: ON 5. Third Temperature Row a. Temp: 68.0 b. Secs: 10 c. Optics: OFF 3. Save the protocol by selecting the Save button at the bottom of the screen. Lyra Direct HSV 1+2/VZV Assay Page 10 of 50

11 Figure of the completed Lyra Direct HSV+VZV Protocol: INITIAL THERMOCYCLER PROGRAMMING FOR HSV 1+2 DETECTION AND IDENTIFICATION Life Technologies QuantStudio Dx Programming Instructions Lyra provides pre-defined templates for each assay that must be uploaded to the Life Technologies QuantStudio Dx. These templates contain the run parameters such that no instrument programming is needed to get started. To install a test definition document: 1. From the Life Technologies QuantStudio Dx Software Home tab, click Manage Test in the Tools panel. 2. From the Test Menu, click Install. 3. Navigate to your test definition document (.tdd) file, select the Lyra HSV file, and click Open. The Life Technologies QuantStudio Dx Software automatically adds the selected test to the Test Menu. 4. Click Close to close the Test Menu and save your changes. Applied Biosystems 7500 Fast and Fast Dx Programming Instructions 1. Launch the Applied Biosystems 7500 Fast or Fast Dx software package. 2. The Quick Startup document dialog window will open. Select the Create New Document button to start the New Document Wizard. Follow each step to initiate the Lyra Direct HSV only protocol. a. Define Document: Most of the following should be the default setting. If not, change accordingly. i. Confirm or enter the following information Assay: Standard Curve (Absolute Quantitation) Container: 96-Well Clear Template: Blank Document Run Mode: Fast 7500 Operator: your operator name Comments: SDS v1.4.1 Plate Name: Lyra Direct HSV ii. Select the Next button. iii. Select Detectors: New detectors for HSV-1, HSV-2 and the process control (PRC), must be added. For each target, select the New Detector button to open the New Detector pop-up window. Alternatively, use the Create Another button from within the New Detector pop-up window for the last two detectors Enter the following information for each detector: Lyra Direct HSV 1+2/VZV Assay Page 11 of 50

12 Name Reporter Dye Quencher Dye Color HSV-1 FAM (none) (Select) HSV-2 JOE (none) (Select) PRC Cy5 (none) (Select) iv. Select a unique color to represent each detector. v. Highlight the new detectors and add to the Detectors in Document column using the Add button. vi. Select (none) from the Passive Reference drop-down menu. vii. Select the Next button. viii. Select the Finish button without setting any wells. b. The wizard will close, and the software will open, starting with the Setup tab. This will show the sample plate that was set up during the quick start. For the initial set up, nothing needs to be changed here. c. Define the Thermocycler Protocol: i. Select the Instrument tab to set up the Lyra Direct HSV 1+2/VZV PCR cycling times and temperatures. ii. Under Thermal Profile there should be a default 2-stage protocol. Each stage will have 3 usereditable text boxes. The top box value represents the number of reps or cycles for that stage. The middle box value represents the temperature ( C), and the lowest box value represents the time (minutes: seconds). iii. Make the following changes to the default Thermal Cycler Protocol: 1. Stage 1 a. Reps: 1 b. Temp: 95 c. Time: 2:00 2. Stage 2 (2-Step Amplification Stage) a. Reps: 40 Step 1 i. Temp: 95 ii. Time: 0:05 b. Step 2 i. Temp: 58 ii. Time: 0:25 3. If a wrong stage is added, the stage may be removed by pressing the Delete button after highlighting the stage between the vertical lines. iv. Under Settings, enter the following: Sample Volume (μl): 20 (default) Run Mode: Fast 7500 (default) Data Collection: Stage 2, Step 2 0:25) NOTE: Do NOT check the check box next to Expert Mode. Lyra Direct HSV 1+2/VZV Assay Page 12 of 50

13 v. Final protocol d. Set threshold for each analyte as follows: i. Select the Results tab. ii. Select the Amplification Plot tab. iii. Select HSV-1 from the Detector tab in the top right corner. iv. In the Analysis Settings block, set the Threshold to 1.0e5. v. Select the Auto Baseline radio button. vi. Repeat iii-v for HSV-2 setting the Threshold to 1.5e4. vii. Repeat iii-v for PRC setting the Threshold to 1.5e4. e. Save the new protocol as a template for future uses. i. At the top of the screen select File and then Save As. ii. Save In: D:\Applied Biosystems\7500 Fast System\Templates\. iii. File name: Lyra Direct HSV. iv. Save as type: SDS Templates (*.sdt). f. Exit the software. Lyra Direct HSV 1+2/VZV Assay Page 13 of 50

14 Cepheid SmartCycler II System Programming Instructions 1. Launch the Cepheid SmartCycler II System Version 3.0b software package. 2. Create the Lyra Direct HSV assay. a. Select the Define Assays button from the top of the screen. b. Name the assay. i. Select the New button at the bottom left corner of the screen. ii. Type in Lyra Direct HSV and select OK. iii. Lyra Direct HSV will be added to the top of the Assay Name list located on the upper left-hand of the screen. c. Set the analysis values: Under the Assay Type: Research section, select the Analysis Settings tab, and make sure the following specifications are set: i. Select FCTC25 from the Dye Set drop-down menu. ii. The Analysis Type drop-down menu should be set to Qualitative (Default setting). iii. In the Channel Name column, enter HSV-1 for Channel 1, HSV-2 for Channel 2, and PRC for Channel 4. Channel 3 will not be used. iv. In the Usage column, select Target from the drop down menus for HSV-1, HSV-2, select Internal Control for PRC, and select Unused for Channel 3. When selecting Internal Control, a window below will pop up. Select the Yes button. v. In the Curve Analysis column, enter Primary Curve for each channel (HSV-1, HSV-2, and PRC) (Default setting). vi. In the Thresh Setting column, enter Manual Threshold for each channel (HSV-1, HSV-2, and PRC) (Default setting). vii. In the Manual Thresh Fluor Units column, enter the following thresholds: a. HSV-1: 30.0 b. HSV-2: 30.0 c. PRC: 8.0 viii. In the Valid Min Cycle column (scroll to the right if not immediately visible), enter 5 for each channel (HSV-1, HSV-2, and PRC). ix. In the Valid Max Cycle column (scroll to the right if not immediately visible), enter 45 for each channel (HSV-1, HSV-2, and PRC). x. In the Bkgnd Sub column, use ON for each channel (HSV-1, HSV-2, and PRC). (Default setting). xi. In the Bkgnd Min Cycle column, enter 5 for each channel (HSV-1, HSV-2, and PRC). xii. In the Bkgnd Max Cycle column, enter 45 for both HSV-1 and HSV-2 channels xiii. In the Bkgnd Max Cycle column, enter 25 for PRC channel xiv. In the Boxcar Avg cycles column, keep 0 for each channel (HSV-1, HSV-2, and PRC). (Default setting). xv. In the End Pt Threshold column, enter 30 for each channel (HSV-1 and HSV-2) and 8 for PRC. (Default setting). xvi. In the NC IC% column, keep NA for PRC channel (Default setting). xvii. In the IC Delta column, keep NA for each HSV-1 and HSV-2 channels (Default setting). xviii. In the Customize Result Text section (below the table), select Organism Based Result Text from the drop-down menu. The warning window below will pop up. Select Yes. Lyra Direct HSV 1+2/VZV Assay Page 14 of 50

15 xix. Select the Customize button to open the Organism-Based Result Text dialog window. Select the Add button, enter HSV-1 in the Organism Name column and check the HSV-1 box. Select the Add button again, enter HSV-2 in the Organism Name column and check the HSV-2 box. Click OK at the bottom of the pop up window. d. Set the PCR cycling times and temperatures as follows: i. Stage 1 1. Hold 2. Temp: Secs: Optics: OFF ii. Stage Temperature Cycle 2. Times to Repeat: First Temperature Row: a. Temp: 95.0 b. Secs: 10 c. Optics: OFF 4. Second Temperature Row a. Temp: 58.0 b. Secs: 15 c. Optics: ON 5. Third Temperature Row a. Temp: 68.0 b. Secs: 10 c. Optics: OFF 3. Save the protocol by selecting the Save button at the bottom of the screen. Lyra Direct HSV 1+2/VZV Assay Page 15 of 50

16 Figure of the completed Lyra Direct HSV Protocol: INITIAL THERMOCYCLER PROGRAMMING FOR VZV DETECTION Life Technologies QuantStudio Dx Programming Instructions Lyra provides pre-defined templates for each assay that must be uploaded to the Life Technologies QuantStudio Dx. These templates contain the run parameters such that no instrument programming is needed to get started. To install a test definition document: 1. From the Life Technologies QuantStudio Dx Software Home tab, click Manage Test in the Tools panel. 2. From the Test Menu, click Install. 3. Navigate to your test definition document (.tdd) file, select the Lyra VZV file, and click Open. The Life Technologies QuantStudio Dx Software automatically adds the selected test to the Test Menu. 4. Click Close to close the Test Menu and save your changes. Applied Biosystems 7500 Fast and Fast Dx Programming Instructions 1. Launch the Applied Biosystems 7500 Fast or Fast Dx software package. 2. The Quick Startup document dialog window will open. Select the Create New Document button to start the New Document Wizard. Follow each step to initiate the Lyra Direct VZV protocol. a. Define Document: Most of the following should be the default setting. If not, change accordingly. i. Confirm or enter the following information Assay: Standard Curve (Absolute Quantitation) Container: 96-Well Clear Template: Blank Document Run Mode: Fast 7500 Operator: your operator name Comments: SDS v1.4.1 Plate Name: Lyra Direct VZV ii. Select the Next button. iii. Select Detectors: New detectors for VZV and the process control (PRC) must be added. For each target, select the New Detector button to open the New Detector pop-up window. Alternatively, use the Create Another button from within the New Detector pop-up window for the last two detectors Enter the following information for each detector: Lyra Direct HSV 1+2/VZV Assay Page 16 of 50

17 Name Reporter Dye Quencher Dye Color VZV Texas Red (none) (Select) PRC Cy5 (none) (Select) iv. Select a unique color to represent each detector. v. Highlight the new detectors and add to the Detectors in Document column using the Add button. vi. Select (none) from the Passive Reference drop-down menu. vii. Select the Next button. viii. Select the Finish button without setting any wells. b. The wizard will close, and the software will open, starting with the Setup tab. This will show the sample plate that was set up during the quick start. For the initial set up, nothing needs to be changed here. c. Define the Thermocycler Protocol: i. Select the Instrument tab to set up the Lyra Direct VZV PCR cycling times and temperatures. ii. Under Thermal Profile there should be a default 2-stage protocol. Each stage will have 3 usereditable text boxes. The top box value represents the number of reps or cycles for that stage. The middle box value represents the temperature ( C), and the lowest box value represents the time (minutes: seconds). iii. Make the following changes to the default Thermal Cycler Protocol: 1. Stage 1 a. Reps: 1 b. Temp: 95 c. Time: 2:00 2. Stage 2 (2-Step Amplification Stage) a. Reps: 40 Step 1 i. Temp: 95 ii. Time: 0:05 b. Step 2 i. Temp: 58 ii. Time: 0:25 3. If a wrong stage is added, the stage may be removed by pressing the Delete button after highlighting the stage between the vertical lines. iv. Under Settings, enter the following: Sample Volume (μl): 20 (default) Run Mode: Fast 7500 (default) Data Collection: Stage 2, Step 2 0:25) NOTE: Do NOT check the check box next to Expert Mode. v. Final protocol Lyra Direct HSV 1+2/VZV Assay Page 17 of 50

18 d. Set threshold for each analyte as follows: i. Select the Results tab. ii. Select the Amplification Plot tab. iii. Select VZV from the Detector tab in the top right corner. iv. In the Analysis Settings block, set the Threshold to 1.0e5. v. Select the Auto Baseline radio button. vi. Repeat iii-v for PRC setting the Threshold to 1.5e4. e. Save the new protocol as a template for future uses. i. At the top of the screen select File and then Save As. ii. Save In: D:\Applied Biosystems\7500 Fast System\Templates\. iii. File name: Lyra Direct VZV. iv. Save as type: SDS Templates (*.sdt). f. Exit the software. Cepheid SmartCycler II System Programming Instructions 1. Launch the Cepheid SmartCycler II System Version 3.0b software package. 2. Create the Lyra Direct VZV assay. a. Select the Define Assays button from the top of the screen. b. Name the assay. i. Select the New button at the bottom left corner of the screen. ii. Type in Lyra Direct VZV and select OK. iii. Lyra Direct VZV will be added to the top of the Assay Name list located on the upper left-hand of the screen. c. Set the analysis values: Under the Assay Type: Research section, select the Analysis Settings tab, and make sure the following specifications are set: i. Select FCTC25 from the Dye Set drop-down menu. ii. The Analysis Type drop-down menu should be set to Qualitative (Default setting) iii. In the Channel Name column, enter VZV for Channel 3 and PRC for Channel 4. Channels 1 and 2 will not be used in this programming. iv. In the Usage column, select Target from the drop down menus for VZV, select Internal Control for PRC, and select Unused for Channels 1 and 2. When selecting Internal Control, a window below will pop up. Select the Yes button. Lyra Direct HSV 1+2/VZV Assay Page 18 of 50

19 v. In the Curve Analysis column, enter Primary Curve for each channel (VZV, and PRC) (Default setting). vi. In the Thresh Setting column, enter Manual Threshold for each channel (VZV, and PRC) (Default setting). vii. In the Manual Thresh Fluor Units column, enter the following thresholds: a. VZV: 30.0 b. PRC: 8.0 viii. In the Valid Min Cycle column (scroll to the right if not immediately visible), enter 5 for each channel (VZV, and PRC). ix. In the Valid Max Cycle column (scroll to the right if not immediately visible), enter 45 for each channel (VZV, and PRC). x. In the Bkgnd Sub column, use ON for each channel (VZV, and PRC). (Default setting). xi. In the Bkgnd Min Cycle column, enter 5 for each channel (VZV, and PRC). xii. In the Bkgnd Max Cycle column, enter 30 for VZV channel. xiii. In the Bkgnd Max Cycle column, enter 25 for PRC channel. xiv. In the Boxcar Avg cycles column, keep 0 for each channel (VZV, and PRC). (Default setting). xv. In the End Pt Threshold column, enter 30 for VZV channel and 8 for PRC. (Default setting). xvi. In the NC IC% column, keep NA for PRC channel (Default setting). xvii. In the IC Delta column, keep NA for the VZV channels (Default setting). xviii. In the Customize Result Text section (below the table), select Organism Based Result Text from the drop-down menu. The warning window below will pop up. Select Yes. xix. Select the Customize button to open the Organism-Based Result Text dialog window. Select the Add button, enter VZV in the Organism Name column and check the VZV box. Click OK at the bottom of the pop up window. Lyra Direct HSV 1+2/VZV Assay Page 19 of 50

20 d. Set the PCR cycling times and temperatures as follows: i. Stage 1 1. Hold 2. Temp: Secs: Optics: OFF ii. Stage Temperature Cycle 2. Times to Repeat: First Temperature Row: a. Temp: 95.0 b. Secs: 10 c. Optics: OFF 4. Second Temperature Row a. Temp: 58.0 b. Secs: 15 c. Optics: ON 5. Third Temperature Row a. Temp: 68.0 b. Secs: 10 c. Optics: OFF 3. Save the protocol by selecting the Save button at the bottom of the screen. Figure of the completed Lyra Direct VZV Protocol: ASSAY PROCEDURE Run the following procedures at the controlled room temperature of 20 C to 25 C. Sample Prep Procedure 1. Remove 100 µl of liquid from lesion swab specimen tube, and place into clean pre-labeled 1.5 ml micro centrifuge tube. 2. Heat at 60 C in dry heat block for 5 minutes. 3. Remove from heat and add 25 µl of Process Buffer within 60 minutes. Mixing is not required. Lyra Direct HSV 1+2/VZV Assay Page 20 of 50

21 Master Mix Rehydration Procedure 1. Determine the number of specimens to be tested, and obtain the correct number of eight-test lyophilized Master Mix vials for testing. 2. Return unused reagents to the appropriate storage conditions. 3. Open Master Mix carefully to avoid disruption of the pellet. 4. Add 135 µl of Rehydration Solution to the Master Mix. 5. Place vial at room temperature for 1 to 2 minutes to allow rehydration of pellet. 6. Gently pipette up and down 3 to 5 times (avoiding the formation of bubbles) prior to dispensing into the first PCR tube or plate well. Note: The rehydrated Master Mix is sufficient for eight reactions. The rehydrated Master Mix may be kept at room temperature (20 C to 25 C) for up to 1 hour, or at 2 C to 8 C for up to 9 hours or at 20 C for up to 3 days. The rehydrated Master Mix should be recapped, sealed with parafilm, and stored in an upright position. Protect the Master Mix from light during storage. HSV 1+2/VZV PCR Set-up Procedure: 1. Add 15 µl of the rehydrated Master Mix to each reaction tube or plate well. 2. Add 5 µl of prepared sample (heated specimen with the process buffer added) into the reaction tubes or plate wells. Mixing of reagents is not required. Note: Use a micropipettor with a new non-aerosol tip with each prepared sample. 3. Close the reaction tubes or seal the plate. Note: Quidel suggests each thermocycler run should include a reaction tube or well with HSV-1, HSV-2 and VZV External Control and Negative Control. Run controls in keeping with your lab practices and policies. 4. Centrifuge the reaction tubes or plate for a minimum of 15 seconds. Ensure that all liquid is at the bottom of the tube. 5. Insert tubes or plate into the thermocycler. AMPLIFICATION PROTOCOL FOR HSV 1+2/VZV DETECTION AND IDENTIFICATION Amplification Protocol on the Life Technologies QuantStudio Dx 1. Switch on Life Technologies QuantStudio Dx. 2. Choose IVD mode on the instrument. 3. Launch the Life Technologies QuantStudio Dx IVD software package. 4. Enter the system Username and Password when prompted. 5. The Home screen window will open. 6. In the Setup box, highlight the previously loaded test name Lyra Direct HSV 1+2/VZV Assay. 7. Click the Setup button to begin a run. 8. The Setup, Test Properties screen will be displayed. Enter run information accordingly. a. Enter the Experiment Name (default setting launches the run with a date and time stamp in the plate name field). b. Enter the Plate Barcode information. c. Record material lot numbers under Reagent Information. d. Save the run as YYMMDD-Lyra HSV-VZV.eds. e. A window will open asking for the Reason for change of entry. Enter Setup and any other comments relevant to the run. 9. In the left menu bar, select Define. 10. Edit sample information. a. Enter specific sample information for each well by deleting the default identifier (Patient 1, Patient 2, etc.) and entering new information, OR b. Select Import from File across the top of the display to upload a predefined plate map from a Text (tab delimited) file. 11. In the left menu bar, select Assign to verify proper plate setup. 12. Loading the sample plate. a. Eject the instrument tray. b. Insert the 96-well PCR plate into the machine with the A1 well positioned in the top, left corner. Lyra Direct HSV 1+2/VZV Assay Page 21 of 50

22 c. Retract the instrument tray. 13. Starting the run. a. In the left menu bar, select Run. b. Click the green Start Run button at the top of the screen. i. If prompted, select the serial number specific to the instrument being used. 14. When the run is complete, select Analysis in the left menu bar. a. Save the file by pressing Save in the task bar. A window will open asking for the Reason for change of entry. Enter Data analysis post run and any other comments relevant to the run. b. The Amplification Plot will show by default. To view other plot types, select them from the left menu bar. c. To view run information with Ct values, select the Well Table tab in the right side of the screen. 15. Printing a report. a. In the top menu bar, select Print Report. Customize the report contents by selecting or deselecting boxes from the report window. b. Select the Print Report button at the bottom of the dialogue box. 16. Exporting data files. a. In the left menu bar, select Export. b. Enter the Export File Location OR click Browse to locate the desired path. c. The Export File Name will default to that of the saved run. d. Select Excel as the file type. e. Customize the exported data report by toggling across the provided tabs and selecting or deselecting options. f. Select Start Export along the bottom of the screen. Amplification Protocol on the Applied Biosystems 7500 Fast and Fast Dx 1. Switch on Applied Biosystems 7500 Fast or Fast Dx. 2. Launch the Applied Biosystems 7500 Fast or Fast Dx software package. 3. The Quick Startup document dialog window will open. 4. Click on Create a new document. 5. Most of the following should be the default setting. If not, change accordingly. Assay: Standard Curve (Absolute Quantitation) Container: 96-Well Clear Template: Lyra Direct HSV+VZV Run Mode: Fast 7500 Operator: your operator name Comments: SDS v1.4.1 (add more if needed) Plate Name: YYMMDD-Lyra Direct HSV+VZV 6. Click Finish once settings have been entered. 7. Set Up Sample Plate a. Under the Setup and Plate tabs the plate setup will appear. b. Select all wells that will contain sample, right-click and select the Well Inspector from the drop-down menu. When the Well Inspector pop-up window opens, select the detectors for HSV-1, HSV-2, VZV, and PRC. c. Use the Well Inspector to enter the sample names. Patient IDs may be entered in the Well Inspector window; however, it is recommended that this is done prior to re-suspending the lyophilized master mix, post run, or using the import function to minimize the time the PCR reactions will sit at room temperature prior to starting the run. d. Save the run as YYMMDD-Lyra Direct HSV+VZV.sds. e. A window will open asking for the Reason for change of entry. Enter Setup and any other comments relevant to the run. 8. Starting the PCR a. Select the Instrument tab. b. Insert the 96 well PCR plate into the machine. c. Under Instrument Control, select the Start button to initiate the run. Lyra Direct HSV 1+2/VZV Assay Page 22 of 50

23 9. Post PCR a. IMPORTANT: When the run is finished, press OK. Analyze the data by pressing the Analyze button in the top menu, and save the file. b. Save the file by pressing Save Document in the task bar. A window will open asking for the Reason for change of entry. Enter Data analysis post run and any other comments relevant to the run. Amplification Protocol on the Cepheid SmartCycler II System 1. Switch on SmartCycler Block(s). 2. Launch the Cepheid SmartCycler II System Version 3.0b software package. 3. Select the Create Run button from the top of the screen to set up the run. 4. Under Run Name, enter a name for the current run (e.g. YYMMDD-Lyra Direct HSV+VZV). 5. Under Notes, enter any notes about the run for future reference. 6. Under Assay, select the Lyra Direct HSV+VZV assay from the drop-down menu. 7. Under Assay Information, enter lot number and expiration date of the kit. 8. To select the wells that will be used, do one of the following: a. To automatically assign wells do the following: i. Under Number of specimens, enter the number of samples in the provided text box. ii. Select the Apply button. The entered number of rows will appear in the Site Table. b. To manually choose wells on the SmartCycler blocks, do the following: i. Select the Add/Remove Sites button towards the bottom of the screen. ii. This will open the Select Sites pop-up window with two columns. The column on the left (Sites) lists all available sites and the column on the right (Selections) holds all selected sites. iii. To select all sites, click the Select All Sites button. iv. To select specific sites, highlight one or more sites, and select the right arrow to add the site(s) to the Selections column. v. Select the OK button to close the window. The selected sites will appear in the Site Table. 9. Enter the sample identifiers under the Sample ID column within the Site Table (this can also be done after the run is started). 10. Enter any notes under the Notes column, and leave the Sample Type column entries as SPEC. 11. Select the Start Run button at the bottom of the screen. 12. Select the View Results button to view the progress of the run. 13. Save the run after it is finished and prior to exiting the software. AMPLIFICATION PROTOCOL FOR HSV 1+2 DETECTION AND IDENTIFICATION Amplification Protocol on the Life Technologies QuantStudio Dx 1. Switch on Life Technologies QuantStudio Dx. 2. Choose IVD mode on the instrument. 3. Launch the Life Technologies QuantStudio Dx IVD software package. 4. Enter the system Username and Password when prompted. 5. The Home screen window will open. 6. In the Setup box, highlight the previously loaded test name Lyra HSV 1+2 Assay. 7. Click the Setup button to begin a run. 8. The Setup, Test Properties screen will be displayed. Enter run information accordingly. a. Enter the Experiment Name (default setting launches the run with a date and time stamp in the plate name field). b. Enter the Plate Barcode information. c. Record material lot numbers under Reagent Information. d. Save the run as YYMMDD-Lyra HSV.eds. e. A window will open asking for the Reason for change of entry. Enter Setup and any other comments relevant to the run. 9. In the left menu bar, select Define. 10. Edit sample information. a. Enter specific sample information for each well by deleting the default identifier (Patient 1, Patient 2, etc.) and entering new information, OR Lyra Direct HSV 1+2/VZV Assay Page 23 of 50

24 b. Select Import from File across the top of the display to upload a predefined plate map from a Text (tab delimited) file. 11. In the left menu bar, select Assign to verify proper plate setup. 12. Loading the sample plate. a. Eject the instrument tray. b. Insert the 96-well PCR plate into the machine with the A1 well positioned in the top, left corner. c. Retract the instrument tray. 13. Starting the run. a. In the left menu bar, select Run. b. Click the green Start Run button at the top of the screen. i. If prompted, select the serial number specific to the instrument being used. 14. When the run is complete, select Analysis in the left menu bar. a. Save the file by pressing Save in the task bar. A window will open asking for the Reason for change of entry. Enter Data analysis post run and any other comments relevant to the run. b. The Amplification Plot will show by default. To view other plot types, select them from the left menu bar. c. To view run information with Ct values, select the Well Table tab in the right side of the screen. 15. Printing a report. a. In the top menu bar, select Print Report. Customize the report contents by selecting or deselecting boxes from the report window. b. Select the Print Report button at the bottom of the dialogue box. 16. Exporting data files. a. In the left menu bar, select Export. b. Enter the Export File Location OR click Browse to locate the desired path. c. The Export File Name will default to that of the saved run. d. Select Excel as the file type. e. Customize the exported data report by toggling across the provided tabs and selecting or deselecting options. f. Select Start Export along the bottom of the screen. Amplification Protocol on the Applied Biosystems 7500 Fast and Fast Dx 1. Switch on Applied Biosystems 7500 Fast or Fast Dx. 2. Launch the Applied Biosystems 7500 Fast or Fast Dx software package. 3. The Quick Startup document dialog window will open. 4. Click on Create a new document. 5. Most of the following should be the default setting. If not, change accordingly. Assay: Standard Curve (Absolute Quantitation) Container: 96-Well Clear Template: Lyra Direct HSV Run Mode: Fast 7500 Operator: your operator name Comments: SDS v1.4.1 (add more if needed) Plate Name: YYMMDD-Lyra Direct HSV 6. Click Finish once settings have been entered. 7. Set Up Sample Plate a. Under the Setup and Plate tabs the plate setup will appear. b. Select all wells that will contain sample, right-click and select the Well Inspector from the drop-down menu. When the Well Inspector pop-up window opens, select the detectors for HSV-1, HSV-2, and PRC. c. Use the Well Inspector to enter the sample names. Patient IDs may be entered in the Well Inspector window; however, it is recommended that this is done prior to re-suspending the lyophilized master mix, post run, or using the import function to minimize the time the PCR reactions will sit at room temperature prior to starting the run. d. Save the run as YYMMDD-Lyra Direct HSV.sds. Lyra Direct HSV 1+2/VZV Assay Page 24 of 50

25 e. A window will open asking for the Reason for change of entry. Enter Setup and any other comments relevant to the run. 8. Starting the PCR a. Select the Instrument tab. b. Insert the 96 well PCR plate into the machine. c. Under Instrument Control, select the Start button to initiate the run. 9. Post PCR a. IMPORTANT: When the run is finished, press OK. Analyze the data by pressing the Analyze button in the top menu, and save the file. b. Save the file by pressing Save Document in the task bar. A window will open asking for the Reason for change of entry. Enter Data analysis post run and any other comments relevant to the run. Amplification Protocol on the Cepheid SmartCycler II System 1. Switch on SmartCycler Block(s). 2. Launch the Cepheid SmartCycler II System Version 3.0b software package. 3. Select the Create Run button from the top of the screen to set up the run. 4. Under Run Name, enter a name for the current run (e.g. YYMMDD-Lyra Direct HSV). 5. Under Notes, enter any notes about the run for future reference. 6. Under Assay, select the Lyra Direct HSV assay from the drop-down menu. 7. Under Assay Information, enter lot number and expiration date of the kit. 8. To select the wells that will be used, do one of the following: a. To automatically assign wells do the following: i. Under Number of specimens, enter the number of samples in the provided text box. ii. Select the Apply button. The entered number of rows will appear in the Site Table. b. To manually choose wells on the SmartCycler blocks, do the following: i. Select the Add/Remove Sites button towards the bottom of the screen. ii. This will open the Select Sites pop-up window with two columns. The column on the left (Sites) lists all available sites and the column on the right (Selections) holds all selected sites. iii. To select all sites, click the Select All Sites button. iv. To select specific sites, highlight one or more sites, and select the right arrow to add the site(s) to the Selections column. v. Select the OK button to close the window. The selected sites will appear in the Site Table. 9. Enter the sample identifiers under the Sample ID column within the Site Table (this can also be done after the run is started). 10. Enter any notes under the Notes column, and leave the Sample Type column entries as SPEC. 11. Select the Start Run button at the bottom of the screen. 12. Select the View Results button to view the progress of the run. 13. Save the run after it is finished and prior to exiting the software. AMPLIFICATION PROTOCOL FOR VZV DETECTION Amplification Protocol on the Life Technologies QuantStudio Dx 1. Switch on Life Technologies QuantStudio Dx. 2. Choose IVD mode on the instrument. 3. Launch the Life Technologies QuantStudio Dx IVD software package. 4. Enter the system Username and Password when prompted. 5. The Home screen window will open. 6. In the Setup box, highlight the previously loaded test name Lyra Direct VZV Assay. 7. Click the Setup button to begin a run. 8. The Setup, Test Properties screen will be displayed. Enter run information accordingly. a. Enter the Experiment Name (default setting launches the run with a date and time stamp in the plate name field). b. Enter the Plate Barcode information. c. Record material lot numbers under Reagent Information. d. Save the run as YYMMDD-Lyra VZV.eds. Lyra Direct HSV 1+2/VZV Assay Page 25 of 50

26 e. A window will open asking for the Reason for change of entry. Enter Setup and any other comments relevant to the run. 9. In the left menu bar, select Define. 10. Edit sample information. a. Enter specific sample information for each well by deleting the default identifier (Patient 1, Patient 2, etc.) and entering new information, OR b. Select Import from File across the top of the display to upload a predefined plate map from a Text (tab delimited) file. 11. In the left menu bar, select Assign to verify proper plate setup. 12. Loading the sample plate. a. Eject the instrument tray. b. Insert the 96-well PCR plate into the machine with the A1 well positioned in the top, left corner. c. Retract the instrument tray. 13. Starting the run. a. In the left menu bar, select Run. b. Click the green Start Run button at the top of the screen. i. If prompted, select the serial number specific to the instrument being used. 14. When the run is complete, select Analysis in the left menu bar. a. Save the file by pressing Save in the task bar. A window will open asking for the Reason for change of entry. Enter Data analysis post run and any other comments relevant to the run. b. The Amplification Plot will show by default. To view other plot types, select them from the left menu bar. c. To view run information with Ct values, select the Well Table tab in the right side of the screen. 15. Printing a report. a. In the top menu bar, select Print Report. Customize the report contents by selecting or deselecting boxes from the report window. b. Select the Print Report button at the bottom of the dialogue box. 16. Exporting data files. a. In the left menu bar, select Export. b. Enter the Export File Location OR click Browse to locate the desired path. c. The Export File Name will default to that of the saved run. d. Select Excel as the file type. e. Customize the exported data report by toggling across the provided tabs and selecting or deselecting options. f. Select Start Export along the bottom of the screen. Amplification Protocol on the Applied Biosystems 7500 Fast and Fast Dx 1. Switch on Applied Biosystems 7500 Fast or Fast Dx. 2. Launch the Applied Biosystems 7500 Fast or Fast Dx software package. 3. The Quick Startup document dialog window will open. 4. Click on Create a new document. 5. Most of the following should be the default setting. If not, change accordingly. Assay: Standard Curve (Absolute Quantitation) Container: 96-Well Clear Template: Lyra Direct VZV Run Mode: Fast 7500 Operator: your operator name Comments: SDS v1.4.1 (add more if needed) Plate Name: YYMMDD-Lyra Direct VZV 6. Click Finish once settings have been entered. 7. Set Up Sample Plate a. Under the Setup and Plate tabs the plate setup will appear. b. Select all wells that will contain sample, right-click and select the Well Inspector from the drop-down menu. When the Well Inspector pop-up window opens, select the detectors for VZV and PRC. Lyra Direct HSV 1+2/VZV Assay Page 26 of 50

27 c. Use the Well Inspector to enter the sample names. Patient IDs may be entered in the Well Inspector window; however, it is recommended that this is done prior to re-suspending the lyophilized master mix, post run, or using the import function to minimize the time the PCR reactions will sit at room temperature prior to starting the run. d. Save the run as YYMMDD-Lyra Direct VZV.sds. e. A window will open asking for the Reason for change of entry. Enter Setup and any other comments relevant to the run. 8. Starting the PCR a. Select the Instrument tab. b. Insert the 96 well PCR plate into the machine. c. Under Instrument Control, select the Start button to initiate the run. 9. Post PCR a. IMPORTANT: When the run is finished, press OK. Analyze the data by pressing the Analyze button in the top menu, and save the file. b. Save the file by pressing Save Document in the task bar. A window will open asking for the Reason for change of entry. Enter Data analysis post run and any other comments relevant to the run. Amplification Protocol on the Cepheid SmartCycler II System 1. Switch on SmartCycler Block(s). 2. Launch the Cepheid SmartCycler II System Version 3.0b software package. 3. Select the Create Run button from the top of the screen to set up the run. 4. Under Run Name, enter a name for the current run (e.g. YYMMDD-Lyra Direct VZV). 5. Under Notes, enter any notes about the run for future reference. 6. Under Assay, select the Lyra Direct VZV assay from the drop-down menu. 7. Under Assay Information, enter lot number and expiration date of the kit. 8. To select the wells that will be used, do one of the following: a. To automatically assign wells do the following: i. Under Number of specimens, enter the number of samples in the provided text box. ii. Select the Apply button. The entered number of rows will appear in the Site Table. b. To manually choose wells on the SmartCycler blocks, do the following: i. Select the Add/Remove Sites button towards the bottom of the screen. ii. This will open the Select Sites pop-up window with two columns. The column on the left (Sites) lists all available sites and the column on the right (Selections) holds all selected sites. iii. To select all sites, click the Select All Sites button. iv. To select specific sites, highlight one or more sites, and select the right arrow to add the site(s) to the Selections column. v. Select the OK button to close the window. The selected sites will appear in the Site Table. 9. Enter the sample identifiers under the Sample ID column within the Site Table (this can also be done after the run is started). 10. Enter any notes under the Notes column, and leave the Sample Type column entries as SPEC. 11. Select the Start Run button at the bottom of the screen. 12. Select the View Results button to view the progress of the run. 13. Save the run after it is finished and prior to exiting the software. Lyra Direct HSV 1+2/VZV Assay Page 27 of 50

28 INTERPRETATION OF RESULTS Interpretation of Results using the Life Technologies QuantStudio Dx The printed results from the instrument are reviewed and interpreted based on the table below. Interpretation of the Lyra Direct HSV 1+2/VZV Assay Results on the Life Technologies QuantStudio Dx Detector: Detector: Detector: Detector: HSV-1 HSV-2 VZV Process Control Interpretation of Results Ct <1.0 or Ct >40.0 Ct <1.0 or Ct >40.0 Ct <1.0 or Ct >40.0 Ct 1.0 or Ct 40.0 Negative HSV-1, HSV-2, and VZV viral DNA not detected; PRC Detected Ct 1.0 or Ct >1.0 or Ct <1.0 or Ct 40.0 Ct <40.0 Ct >40.0 NA* HSV-1 HSV-1 viral DNA detected Ct <1.0 or Ct 1.0 or Ct >1.0 or Ct >40.0 Ct 40.0 Ct <40.0 NA* HSV-2 HSV-2 viral DNA detected Ct <1.0 or Ct <1.0 or Ct 1.0 or Ct >40.0 Ct >40.0 Ct 40.0 NA* VZV VZV viral DNA detected Ct 1.0 or Ct 1.0 or Ct <1.0 or HSV-1 and HSV-2 HSV-1 and HSV-2 viral NA* Ct 40.0 Ct 40.0 Ct >40.0 DNA detected Ct 1.0 or Ct <1.0 or Ct 1.0 or HSV-1 and VZV HSV-1 and VZV viral DNA NA* Ct 40.0 Ct >40.0 Ct 40.0 detected Ct <1.0 or Ct 1.0 or Ct 1.0 or HSV-2 and VZV HSV-2 and VZV viral DNA NA* Ct >40.0 Ct 40.0 Ct 40.0 detected Ct 1.0 or Ct 1.0 or Ct 1.0 or HSV-1, HSV-2 and VZV HSV-1, HSV-2, NA* Ct 40.0 Ct 40.0 Ct 40.0 and VZV viral DNA detected Invalid PCR inhibition or reagent failure. Retest Ct <1.0 or Ct >40.0 Ct <1.0 or Ct >40.0 Ct <1.0 or Ct >40.0 Ct <1.0 or Ct >40.0 the same purified sample. If the test is also invalid, re prepare and retest another aliquot of the same sample or obtain a new sample and retest. *No Ct value is required for the Process Control to make a positive call. Interpretation of Results using the Applied Biosystems 7500 Fast and Fast Dx The printed results from the instrument are reviewed and interpreted based on the table below. Interpretation of the Lyra Direct HSV 1+2/VZV Assay Results on the Applied Biosystems 7500 Fast and Fast Dx Detector: Detector: Detector: Detector: HSV-1 HSV-2 VZV Process Control Interpretation of Results Ct <1.0 or Ct >40.0 Ct <1.0 or Ct >40.0 Ct <1.0 or Ct >40.0 Ct 1.0 or Ct 40.0 Negative HSV-1, HSV-2, and VZV viral DNA not detected; PRC Detected Ct 1.0 or Ct <1.0 or Ct <1.0 or Ct 40.0 Ct >40.0 Ct >40.0 NA* HSV-1 HSV-1 viral DNA detected Ct <1.0 or Ct 1.0 or Ct <1.0 or Ct >40.0 Ct 40.0 Ct >40.0 NA* HSV-2 HSV-2 viral DNA detected Ct <1.0 or Ct <1.0 or Ct 1.0 or Ct >40.0 Ct >40.0 Ct 40.0 NA* VZV VZV viral DNA detected Ct 1.0 or Ct 1.0 or Ct < 1.0 or HSV-1 and HSV-2 HSV-1 and HSV-2 viral NA* Ct 40.0 Ct 40.0 Ct > 40.0 DNA detected Ct 1.0 or Ct <1.0 or Ct 1.0 or HSV-1 and VZV HSV-1 and VZV viral DNA NA* Ct 40.0 Ct >40.0 Ct 40.0 detected Ct <1.0 or Ct 1.0 or Ct 1.0 or HSV-2 and VZV HSV-2 and VZV viral DNA NA* Ct >40.0 Ct 40.0 Ct 40.0 detected Ct 1.0 or Ct 1.0 or Ct 1.0 or HSV-1, HSV-2 and VZV HSV-1, HSV-2, and NA* Ct 40.0 Ct 40.0 Ct 40.0 VZV viral DNA detected Ct <1.0 or Ct <1.0 or Ct <1.0 or Ct <1.0 or Invalid PCR inhibition or reagent failure. Retest Lyra Direct HSV 1+2/VZV Assay Page 28 of 50

29 Interpretation of the Lyra Direct HSV 1+2/VZV Assay Results on the Applied Biosystems 7500 Fast and Fast Dx Detector: Detector: Detector: Detector: HSV-1 HSV-2 VZV Process Control Interpretation of Results Ct >40.0 Ct >40.0 Ct >40.0 Ct >40.0 the same purified sample. If the test is also invalid, re prepare and retest another aliquot of the same sample or obtain a new sample and retest. *No Ct value is required for the Process Control to make a positive call. Interpretation of Results using the Cepheid SmartCycler II System 1. Select View Results tab after the run is finished. 2. Select Sample Results tab. 3. The Cepheid SmartCycler II System software will automatically report whether HSV-1, HSV-2 and/or VZV viral DNA has been detected in the samples or whether the run was invalid (unresolved). 4. More detailed information may be found under the respective tabs of each analyte in the same window. If there is a positive call for HSV-1, HSV-2, or VZV (or any combination thereof), the PRC result is not applicable. The PRC is only required for negative calls. Interpretation of the Lyra Direct HSV 1+2/VZV Assay Results on Cepheid SmartCycler II System Assay Result HSV-1 HSV-2 VZV Process Control Result Interpretation of Results Negative NEG NEG NEG Pass Negative HSV-1, HSV-2, and VZV viral DNA not detected; PRC Detected HSV-1 POS NEG NEG NA* HSV-1 HSV-1 viral DNA detected HSV-2 NEG POS NEG NA* HSV-2 HSV-2 viral DNA detected VZV NEG NEG POS NA* VZV VZV viral DNA detected HSV-1 and HSV-2 POS POS NEG NA* HSV-1 and HSV-2 HSV-1 and HSV-2 viral DNA detected HSV-1 and VZV POS NEG POS NA* HSV-1 and VZV HSV-1 and VZV viral DNA detected HSV-2 and VZV NEG POS POS NA* HSV-2 and VZV HSV-2 and VZV viral DNA detected HSV-1, HSV-2, and VZV HSV-1, HSV-2 and VZV HSV-1, HSV-2, and POS POS POS NA* VZV viral DNA detected Invalid PCR inhibition or reagent failure. Retest Invalid NEG NEG NEG Fail the same purified sample. If the test is also invalid, re prepare and retest another aliquot of the same sample or obtain a new sample and retest. *No value is required for the Process Control to make a positive call. QUALITY CONTROL The Lyra Direct HSV 1+2/VZV Assay incorporates several controls to monitor assay performance. 1. The Process Control should be used during sample preparation and amplification in the assay. This control should be added to each sample aliquot prior to PCR. 2. Commercially available external positive HSV-1, HSV-2, and VZV controls may be treated as a patient specimen and should be used in accordance with your lab standards. Previously characterized positive HSV-1, HSV-2, and VZV specimens may be used in lieu of commercial HSV-1, HSV-2, and VZV controls. 3. Viral transport media or previously characterized negative specimen may be used as an external negative control. This must be treated as a patient specimen and should be performed in accordance with current lab standards. Lyra Direct HSV 1+2/VZV Assay Page 29 of 50

30 LIMITATIONS Specimens should be tested with Lyra Direct HSV 1+2/VZV for only those viruses requested by the physician. Testing and reporting the additional, unrequested virus(es) may lead to confusion and delayed diagnosis due to an unexpected positive result. The samples used for the device should be limited to samples from lesions, indicating an active infection. Negative results do not preclude infection with HSV-1, HSV-2, or VZV and should not be the sole basis of a treatment decision. As with other assays of this type, there is a risk of false negative results due to the presence of sequence variants in the viral target. Improper collection, storage, or transport may lead to false negative results. Inhibitors present in the sample and/or errors in following the assay procedure may lead to false negative results. The programming instructions provided for each instrument are for use with the Lyra Direct HSV 1+2/VZV Assay. There use for other instruments or assays has not been established. The performance of the Lyra Direct HSV 1+2/VZV Assay from ocular or nares lesion specimens has not been adequately established in the clinical study due to low number of samples tested. EXPECTED VALUES The observed expected values in the clinical study are presented below for the Life Technologies QuantStudio Dx, the Applied Biosystems 7500 Fast Dx, and the Cepheid SmartCycler II System. The tables below provide the expected value for each viruses detected on the three instruments based patient age and the specific lesion categories. The study design did not differentiate samples in which an HSV-1, HSV-2, VZV test was requested or if all of the tests were requested in each of the samples tests. The samples included in the study consisted of any sample which was sent for HSV or VZV testing which may have included samples in which both the HSV and VZV testing was ordered. The expected values shown below, therefore, only reflect the expected values for this study design only (samples submitted for HSV-1, or HSV-2, or VZV testing) and are not reflective of the prevalence values of VZV DNA in patients suspected of VZV cutaneous and mucocutaneous infections, or the prevalence of HSV-1 and HSV-2 DNA in patients suspected of HSV cutaneous and mucocutaneous infections. Life Technologies QuantStudio Dx Expected Values (Cutaneous) (N=279)* HSV-1 HSV-2 VZV Age # Prevalence # Prevalence # Prevalence <5 years % % % 6 to 21 years % % % 22 to 59 years % % % >60 years* % % % *1 Specimen was invalid when tested on the Life Technologies QuantStudio Dx. It has been removed from analysis. Expected Values (Cutaneous) (N=279)* HSV-1 HSV-2 VZV Specimen Source # Prevalence # Prevalence # Prevalence Skin lesion 213* % 213* % 213* % Genital penis % % % *1 Specimen was invalid when tested on the Life Technologies QuantStudio Dx. It has been removed from analysis. Lyra Direct HSV 1+2/VZV Assay Page 30 of 50

31 Expected Values (Mucocutaneous) (N=650)* HSV-1 HSV-2 VZV Age # Prevalence # Prevalence # Prevalence <5 years % 18 0 N/A 18 0 N/A 6 to 21 years % % % 22 to 59 years* % % % >60 years % % % *1 Specimen was invalid when tested on the Life Technologies QuantStudio Dx. It has been removed from analysis. Expected Values (Mucocutaneous) (N=650)* HSV-1 HSV-2 VZV Specimen Source # Prevalence # Prevalence # Prevalence Anorectal % % % Genital vaginal/cervical 473* % 473* % 473* 4 0.8% Nares % 9 0 N/A 9 0 N/A Ocular 6 0 N/A 6 0 N/A % Oral lesion % % % *1 Specimen was invalid when tested on the Life Technologies QuantStudio Dx. It has been removed from analysis. Applied Biosystems 7500 Fast Dx Expected Values (Cutaneous) (N=279) HSV-1 HSV-2 VZV Age Prevalence Prevalence # # # Prevalence <5 years % % % 6 to 21 years % % % 22 to 59 years % % % >60 years % % % Expected Values (Cutaneous) (N=279) HSV-1 HSV-2 VZV Specimen Source Prevalence Prevalence # # # Prevalence Skin lesion % % % Genital penis % % 65 0 N/A Expected Values (Mucocutaneous) (N=650)* HSV-1 HSV-2 VZV Age # Prevalence # Prevalence # Prevalence <5 years % 18 0 N/A 18 0 N/A 6 to 21 years % % % 22 to 59 years* % % % >60 years % % % *Three (3) specimens were invalid when tested on the Applied Biosystems 7500 Fast Dx. They have been removed from analysis. Lyra Direct HSV 1+2/VZV Assay Page 31 of 50

32 Expected Values (Mucocutaneous) (N=650)* HSV-1 HSV-2 VZV Specimen Source Prevalence # Prevalence # # Prevalence Anorectal % % % Genital vaginal/cervical 471* % 471* % 471* 3 0.6% Nares % 9 0 N/A 9 0 N/A Ocular 6 0 N/A 6 0 N/A % Oral lesion % % % *Three (3) specimens were invalid when tested on the Applied Biosystems 7500 Fast Dx. They have been removed from analysis. Cepheid SmartCycler II System Expected Values (Cutaneous) (N=279)* HSV-1 HSV-2 VZV Age # Prevalence # Prevalence # Prevalence <5 years % % % 6 to 21 years % % % 22 to 59 years* % % % >60 years % % % *One (1) specimen was invalid when tested on the Cepheid SmartCycler II. It has been removed from analysis. Expected Values (Cutaneous) (N=279)* HSV-1 HSV-2 VZV Specimen Source # Prevalence # Prevalence # Prevalence Skin lesion 213* % 213* % 213* % Genital penis % % 65 0 N/A *One (1) specimen was invalid when tested on the Cepheid SmartCycler II. It has been removed from analysis. Expected Values (Mucocutaneous) (N=650)* HSV-1 HSV-2 VZV Age # Prevalence # Prevalence # Prevalence <5 years % 18 0 N/A 18 0 N/A 6 to 21 years % % % 22 to 59 years* % % % >60 years % % % *Four (4) specimens were invalid when tested on the Cepheid SmartCycler II. They have been removed from analysis. Expected Values (Mucocutaneous) (N=650)* HSV-1 HSV-2 VZV Specimen Source # Prevalence Prevalence # # Prevalence Anorectal % % % Genital vaginal/cervical 470* % 470* % 470* 4 0.9% Nares % 9 0 N/A 9 0 N/A Ocular 6 0 N/A 6 0 N/A % Oral lesion % % % *Four (4) specimens were invalid when tested on the Cepheid SmartCycler II. They have been removed from analysis. Lyra Direct HSV 1+2/VZV Assay Page 32 of 50

33 CLINICAL PERFORMANCE A multi-center study was performed between April 2013 and October 2013 to evaluate the Lyra Direct HSV 1+2/VZV Assay using lesion swab specimens obtained from cutaneous or mucocutaneous lesions and submitted for HSV and/or VZV culture. These specimens were processed with the Lyra Direct HSV 1+2/VZV Assay kit and tested on the Life Technologies QuantStudio Dx, the Applied Biosystems 7500 Fast Dx, and the Cepheid SmartCycler II at three (3) locations. Each specimen was also processed and inoculated into two (2) different cell culture systems within 72 hours of collection. The isolation and identification of HSV-1 and HSV-2 was performed using the FDA-cleared ELVIS HSV ID and D³ Typing Test. This testing was performed according to the manufacturer s product insert. The detection and isolation of VZV was performed by staining cells present in the specimen with a FDA-cleared VZV detection reagent (DSFA) and by culturing the specimen for 96-hours using commercially available mixed cell culture (H&V mixed cells 3,4 ) consisting of MRC-5 cells (human diploid fibroblast) and CV-1 cells (African green monkey kidney), and staining the cultures with the same FDAcleared reagent used for DSFA. A specimen was considered positive for VZV if either the DSFA or the culture with DFA were positive. The specimens have been categorized as cutaneous (skin lesion, penis), or mucocutaneous (anorectal, vaginal/cervical, and oral lesion). The gender and age demographics for each category are listed below. Age and Gender Distribution (Cutaneous) Gender Female Male Age <5 years to 21 years to 59 years >60 years Age and Gender Distribution (Mucocutaneous) Gender Female Male Age <5 years to 21 years to 59 years >60 years 47 6 Life Technologies QuantStudio Dx Cutaneous Lesions Two-hundred and seventy-nine (279) active cutaneous lesion specimens were cultured for HSV-1, using the FDA-cleared ELVIS cell culture system and were also tested with the subject device for HSV-1 viral DNA. One (1) specimen was invalid in Lyra Direct HSV 1+2/VZV Assay. This specimen has been excluded from further analysis. The table below details the HSV-1 results for the remaining Two-hundred and seventy-eight (278) specimens. HSV-1 Results Lyra Direct Comparator: ELVIS HSV ID and D³ Typing Test HSV 1+2/VZV Assay Negative 24 4* 28 Negative % CI Sensitivity 24/24 100% 86.2% to 100% Specificity 250/ % 96.0% to 99.4% *One (1) of the four (4) positives was positive by an additional RT-PCR assay. Lyra Direct HSV 1+2/VZV Assay Page 33 of 50

34 Two-hundred and seventy-nine (279) active cutaneous lesion specimens were cultured for HSV-2, using the FDA-cleared ELVIS cell culture system and were also tested with the subject device for HSV-2 viral DNA. One (1) specimen was invalid in Lyra Direct HSV 1+2/VZV Assay. This specimen has been excluded from further analysis. The table below details the HSV-2 results for the remaining Two-hundred and seventy-eight (278) specimens. HSV-2 Results Lyra Direct Comparator: ELVIS HSV ID and D³ Typing Test HSV 1+2/VZV Assay Negative 35 9* 44 Negative % CI Sensitivity 35/35 100% 90.1% to 100% Specificity 234/ % 93.1% to 98.0% *Nine (9) of the nine (9) positives were positive by an additional RT-PCR assay. Two-hundred and seventy-nine (279) active cutaneous lesion specimens were cultured for VZV using the H&V mixed cells with DFA cell culture systems and were also tested with the subject device for VZV viral DNA. Due the presence of either HSV-1 or HSV-2, fifty-six (56) specimens have been excluded from analysis. One (1) specimen was invalid in Lyra Direct HSV 1+2/VZV Assay. These fifty-seven (57) specimens have been excluded from analysis. The table below details the VZV results for the remaining Two-hundred and twenty-two (222) specimens. VZV Results Lyra Direct Comparator: DSFA and Culture with DFA HSV 1+2/VZV Assay Negative 27 8* 35 Negative % CI Sensitivity 27/27 100% 87.5% to 100% Specificity 187/ % 92.1% to 97.9% *Seven (7) of the eight (8) positives were positive by an additional RT-PCR assay. Mucocutaneous Lesions Six-hundred and fifty (650) active mucocutaneous lesion specimens were cultured for HSV-1, using the FDA-cleared ELVIS cell culture system and were also tested with the subject device for HSV-1 viral DNA. Three (3) specimens were contaminated in the ELVIS cell culture, and one (1) specimen was invalid in Lyra Direct HSV 1+2/VZV Assay. These four (4) specimens have been excluded from further analysis. The table below details the HSV-1 results for the remaining sixhundred forty-six (646) specimens. HSV-1 Results Lyra Direct Comparator: ELVIS HSV ID and D³ Typing Test HSV 1+2/VZV Assay Negative * 116 Negative 3** % CI Sensitivity 100/ % 91.8% to 99.0% Specificity 527/ % 95.3% to 98.2% *Thirteen (13) of the sixteen (16) positives were positive by an additional RT-PCR assay. **Three (3) of the three (3) negatives were positive by an additional RT-PCR assay. Lyra Direct HSV 1+2/VZV Assay Page 34 of 50

35 Six-hundred and fifty (650) active mucocutaneous lesion specimens were cultured for HSV-2, using the FDA-cleared ELVIS cell culture system and were also tested with the subject device for HSV-2 viral DNA. Three (3) specimens were contaminated in the ELVIS cell culture, and one (1) specimen was invalid in Lyra Direct HSV 1+2/VZV Assay. These four (4) specimens have been excluded from further analysis. The table below details the HSV-2 results for the remaining sixhundred forty-six (646) specimens. Lyra Direct HSV 1+2/VZV Assay HSV-2 Results Comparator: ELVIS HSV ID and D³ Typing Test Negative 95 21* 116 Negative % CI Sensitivity 95/95 100% 96.1% to 100% Specificity 530/ % 94.2% to 97.5% *Eighteen (18) of the twenty-one (21) positives were positive by an additional RT-PCR assay. Six-hundred and fifty (650) active mucocutaneous lesion specimens were cultured for VZV using the H&V mixed cells with DFA cell culture systems and were also tested with the subject device for VZV viral DNA. Due the presence of either HSV-1 or HSV-2, two hundred seventeen (217) specimens have been excluded from analysis. One (1) specimen was invalid in Lyra Direct HSV 1+2/VZV Assay. These two hundred eighteen (218) specimens have been excluded from analysis. The table below details the VZV results for the remaining four-hundred and thirty-two (432) specimens. Lyra Direct HSV 1+2/VZV Assay VZV Results Comparator: DSFA and Culture with DFA Negative 4 5* 9 Negative % CI Sensitivity 4/4 100% 51.0% to 100% Specificity 423/ % 97.3% to 99.5% *Five (5) of the five (5) positives were positive by an additional RT-PCR assay. Note: The data presented for the detection of VZV is consistent with limited presence of VZV in mucocutaneous lesions. The use of mucocutaneous lesions has no discernible impact on the performance characteristics of Lyra Direct HSV 1+2/VZV Assay. Applied Biosystems 7500 Fast Dx Cutaneous Lesions Two-hundred and seventy-nine (279) active cutaneous lesion specimens were cultured for HSV-1, using the FDA-cleared ELVIS cell culture system and were also tested with the subject device for HSV-1 viral DNA. The table below details the HSV-1 results for the two-hundred and seventy-nine (279) specimens. Lyra Direct HSV 1+2/VZV Assay Page 35 of 50

36 Lyra Direct HSV 1+2/VZV Assay HSV-1 Results Comparator: ELVIS HSV ID and D³ Typing Test Negative 24 3* 27 Negative % CI Sensitivity 24/24 100% 86.2% to 100% Specificity 252/ % 96.6% to 99.6% *One (1) of the three (3) positives was positive by an additional RT-PCR assay. Two-hundred and seventy-nine (279) active cutaneous lesion specimens were cultured for HSV-2, using the FDA-cleared ELVIS cell culture system and were also tested with the subject device for HSV-2 viral DNA. The table below details the HSV-2 results for the two-hundred and seventy-nine (279) specimens. Lyra Direct HSV 1 + 2/VZV Assay HSV-2 Results Comparator: ELVIS HSV ID and D³ Typing Test Negative 34 8* 42 Negative 1** % CI Sensitivity 34/ % 85.5% to 99.5% Specificity 236/ % 93.7% to 98.3% *Seven (7) of the eight (8) positives were positive by an additional RT-PCR assay. **One (1) of the one (1) negative was positive by an additional RT-PCR assay. Two-hundred and seventy-nine (279) active cutaneous lesion specimens were cultured for VZV using the H&V mixed cells with DFA cell culture systems and were also tested with the subject device for VZV viral DNA. Due the presence of either HSV-1 or HSV-2, fifty-six (56) specimens have been excluded from analysis. The table below details the VZV results for the two-hundred and twenty-three (223) specimens. VZV Results Lyra Direct Comparator: DSFA and Culture with DFA HSV 1+2/VZV Assay Negative 26 7* 33 Negative 1** % CI Sensitivity 26/ % 87.5% to 99.3% Specificity 189/ % 92.8% to 98.3% *Seven (7) of the seven (7) positives were positive by an additional RT-PCR assay. **One (1) of the one (1) negative was positive by an additional RT-PCR assay. Mucocutaneous Lesions Six-hundred and fifty (650) active mucocutaneous lesion specimens were cultured for HSV-1, using the FDA-cleared ELVIS cell culture system and were also tested with the subject device for HSV-1 viral DNA. Three (3) specimens were contaminated in the ELVIS cell culture, and three (3) specimens were invalid in Lyra Direct HSV 1+2/VZV Assay. These six (6) specimens have been excluded from further analysis. The table below details the HSV-1 results for the remaining sixhundred forty-four (644) specimens. Lyra Direct HSV 1+2/VZV Assay Page 36 of 50

37 HSV-1 Results Lyra Direct Comparator: ELVIS HSV ID and D³ Typing Test HSV 1+2/VZV Assay Negative 98 10* 108 Negative 5** % CI Sensitivity 98/ % 89.1% to 97.9% Specificity 531/ % 96.6% to 99.0% *Ten (10) of the ten (10) positives were positive by an additional RT-PCR assay. **Five (5) of the five (5) negatives were positive by an additional RT-PCR assay. Six-hundred and fifty (650) active mucocutaneous lesion specimens were cultured for HSV-2, using the FDA-cleared ELVIS cell culture system and were also tested with the subject device for HSV-2 viral DNA. Three (3) specimens were contaminated in the ELVIS cell culture, and three (3) specimens were invalid in Lyra Direct HSV 1+2/VZV Assay. These six (6) specimens have been excluded from further analysis. The table below details the HSV-2 results for the remaining sixhundred forty-four (644) specimens. HSV-2 Results Lyra Direct Comparator: ELVIS HSV ID and D³ Typing Test HSV 1+2/VZV Assay Negative 93 16* 109 Negative 2** % CI Sensitivity 93/ % 92.6% to 99.4% Specificity 533/ % 95.3% to 98.2% *Sixteen (16) of the sixteen (16) positives were positive by an additional RT-PCR assay. **Two (2) of the two (2) negatives were positive by an additional RT-PCR assay. Six-hundred and fifty (650) active mucocutaneous lesion specimens were cultured for VZV using the H&V mixed cells with DFA cell culture systems and were also tested with the subject device for VZV viral DNA. Due the presence of either HSV-1 or HSV-2, two hundred seventeen (217) specimens have been excluded from analysis. Three (3) specimens were invalid in Lyra Direct HSV 1+2/VZV Assay. These two hundred twenty (220) specimens have been excluded from analysis. The table below details the VZV results for the remaining four-hundred and thirty (430) specimens. VZV Results Lyra Direct Comparator: DSFA and Culture with DFA HSV 1+2/VZV Assay Negative 4 3* 7 Negative % CI Sensitivity 4/4 100% 51.0% to 100% Specificity 423/ % 98.0% to 99.8% *Three (3) of the three (3) positives were positive by an additional RT-PCR assay. Note: The data presented for the detection of VZV is consistent with limited presence of VZV in mucocutaneous lesions. The use of mucocutaneous lesions has no discernible impact on the performance characteristics of Lyra Direct HSV 1+2/VZV Assay. Lyra Direct HSV 1+2/VZV Assay Page 37 of 50

38 Cepheid SmartCycler II System Cutaneous Lesions Two-hundred and seventy-nine (279) active cutaneous lesion specimens were cultured for HSV-1, using the FDA-cleared ELVIS cell culture system and were also tested with the subject device for HSV-1 viral DNA. One (1) specimen was invalid in Lyra Direct HSV 1+2/VZV Assay. This specimen has been excluded from further analysis. The table below details the HSV-1 results for the two-hundred and seventy-eight (278) specimens. HSV-1 Results Lyra Direct Comparator: ELVIS HSV ID and D³ Typing Test HSV 1+2/VZV Assay Negative 24 3* 27 Negative % CI Sensitivity 24/24 100% 86.2% to 100% Specificity 251/ % 96.6% to 99.6% *One (1) of the three (3) positives was positive by an additional RT-PCR assay. Two-hundred and seventy-nine (279) active cutaneous lesion specimens were cultured for HSV-2, using the FDA-cleared ELVIS cell culture system and were also tested with the subject device for HSV-2 viral DNA. One (1) specimen was invalid in Lyra Direct HSV 1+2/VZV Assay. This specimen has been excluded from further analysis. The table below details the HSV-1 results for the two-hundred and seventy-eight (278) specimens. HSV-2 Results Lyra Direct Comparator: ELVIS HSV ID and D³ Typing Test HSV 1+2/VZV Assay Negative 35 8* 43 Negative % CI Sensitivity 35/35 100% 90.1% to 100% Specificity 235/ % 93.6% to 98.3% *Seven (7) of the eight (8) positives were positive by an additional RT-PCR assay. Two-hundred and seventy-nine (279) active cutaneous lesion specimens were cultured for VZV using the H&V mixed cells with DFA cell culture systems and were also tested with the subject device for VZV viral DNA. Due the presence of either HSV-1 or HSV-2, fifty-six (56) specimens have been excluded from analysis. One (1) specimen was invalid in Lyra Direct HSV 1+2/VZV Assay. The table below details the VZV results for the two-hundred and twenty-two (222) specimens. VZV Results Lyra Direct Comparator: DSFA and Culture with DFA HSV 1+2/VZV Assay Negative 27 10* 37 Negative % CI Sensitivity 27/27 100% 87.5% to 100% Specificity 185/ % 90.8% to 97.2% *Six (6) of the ten (10) positives were positive by an additional RT-PCR assay. Lyra Direct HSV 1+2/VZV Assay Page 38 of 50

39 Mucocutaneous Lesions Six-hundred and fifty (650) active mucocutaneous lesion specimens were cultured for HSV-1, using the FDA-cleared ELVIS cell culture system and were also tested with the subject device for HSV-1 viral DNA. Three (3) specimens were contaminated in the ELVIS cell culture, and four (4) specimens were invalid in Lyra Direct HSV 1+2/VZV Assay. These seven (7) specimens have been excluded from further analysis. The table below details the HSV-1 results for the remaining sixhundred forty-three (643) specimens. HSV-1 Results Lyra Direct Comparator: ELVIS HSV ID and D³ Typing Test HSV 1+2/VZV Assay Negative * 116 Negative 2** % CI Sensitivity 101/ % 93.2% to 99.5% Specificity 525/ % 95.5% to 98.3% *Fourteen (14) of the fifteen (15) positives were positive by an additional RT-PCR assay. **Two (2) of the two (2) negatives were positive by an additional RT-PCR assay. Six-hundred and fifty (650) active mucocutaneous lesion specimens were cultured for HSV-2, using the FDA-cleared ELVIS cell culture system and were also tested with the subject device for HSV-2 viral DNA. Three (3) specimens were contaminated in the ELVIS cell culture, and four (4) specimens were invalid in Lyra Direct HSV 1+2/VZV Assay. These seven (7) specimens have been excluded from further analysis. The table below details the HSV-2 results for the remaining sixhundred forty-three (643) specimens. HSV-2 Results Lyra Direct Comparator: ELVIS HSV ID and D³ Typing Test HSV 1+2/VZV Assay Negative 94 16* 110 Negative 1** % CI Sensitivity 94/ % 94.3% to 99.8% Specificity 532/ % 95.3% to 98.2% *Sixteen (16) of the sixteen (16) positives were positive by an additional RT-PCR assay. **The one (1) negative was positive by an additional RT-PCR assay. Six-hundred and fifty (650) active mucocutaneous lesion specimens were cultured for VZV using the H&V mixed cells with DFA cell culture system and were also tested with the subject device for VZV viral DNA. Due the presence of either HSV-1 or HSV-2, two hundred seventeen (217) specimens have been excluded from analysis. Four (4) specimens were invalid in Lyra Direct HSV 1+2/VZV Assay. These two hundred twenty-one (221) specimens have been excluded from analysis. The table below details the VZV results for the remaining four-hundred and twenty-nine (429) specimens. Lyra Direct HSV 1+2/VZV Assay VZV Results Comparator: DSFA and Culture with DFA Negative 4 5* 9 Negative % CI Sensitivity 4/4 100% 51.0% to 100% Specificity 420/ % 97.3% to 99.5% *Five (5) of the five (5) positives were positive by an additional RT-PCR assay. Lyra Direct HSV 1+2/VZV Assay Page 39 of 50

40 Note: The data presented for the detection of VZV is consistent with limited presence of VZV in mucocutaneous lesions. The use of mucocutaneous lesions has no discernible impact on the performance characteristics of Lyra Direct HSV 1+2/VZV Assay. ANALYTICAL PERFORMANCE Level of Detection The analytical sensitivity (limit of detection or LOD) of the Lyra Direct HSV 1+2/VZV Assay was determined on each instrument in three separate studies using quantified (TCID50/mL) stocks of 2-strains of HSV-1, 2-strains of HSV-2 and 2- strains of VZV serially diluted in a negative matrix. Each dilution was processed in replicates of 20 per concentration of virus and tested on Life Technologies QuantStudio Dx, the Applied Biosystems 7500 Fast Dx, or the Cepheid SmartCycler II System. Testing was performed concurrently using the same serial dilutions on each instrument. Analytical sensitivity is defined as the lowest concentration at which 95% of all replicates tested positive. Instrument Life Technologies QuantStudio Dx Applied Biosystems 7500 Fast Dx Cepheid SmartCycler II System HSV-1 Macintyre HSV-1 QC 316 HSV-2 Strain G Virus TCID50/mL HSV-2 QC Comp VZV Ellen VZV x x x x x x x x x x x x x x x x x x10 1 Precision Repeatability For the Precision/Within Laboratory Repeatability study, a blinded four-member panel consisting of HSV-1, HSV-2, and VZV positive and negative sample was tested by two (2) operators, twice a day (2x) for twelve (12) days on all three instruments. HSV-1 HSV-2 VZV Life Technologies QuantStudio Dx Results Summary Ct Values and Percent (%) Target Negative 5x LOD 2x LOD 0.25x LOD Control Matrix Operator 1 Avg Ct Neg Operator 2 Avg Ct Neg Positivity Operator 1 Avg Ct Neg Operator 2 Avg Ct Neg Positivity Operator 1 Avg Ct Neg Operator 2 Avg Ct Neg Positivity Lyra Direct HSV 1+2/VZV Assay Page 40 of 50

41 HSV-1 HSV-2 VZV Applied Biosystems 7500 Fast Dx Results Summary Ct Values and Percent (%) Target Negative 5x LOD 2x LOD 0.25x LOD Control Matrix Operator 1 Avg Ct Neg Operator 2 Avg Ct Neg Positivity % Operator 1 Avg Ct Neg Operator 2 Avg Ct Neg Positivity % Operator 1 Avg Ct Neg Operator 2 Avg Ct Neg Positivity % Cepheid SmartCycler II System Results Summary Ct Values and Percent (%) Target Negative 5x LOD 2x LOD 0.25x LOD Control Matrix Operator 1 Avg Ct Neg HSV-1 Operator 2 Avg Ct Neg Positivity % Operator 1 Avg Ct Neg HSV-2 Operator 2 Avg Ct Neg Positivity % Operator 1 Avg Ct Neg VZV Operator 2 Avg Ct Neg Positivity % The Lyra Direct HSV 1+2/VZV Assay produces results that are highly reproducible on all three (3) instruments. Analytical Reactivity (Inclusivity) A study was performed on the Applied Biosystems 7500 Fast Dx to show that the Lyra Direct HSV 1+2/VZV Assay detects multiple strains of HSV-1, HSV-2 and VZV at concentrations near the LOD. Sixteen (16) viruses (four HSV-1, five HSV-2, and seven VZV) were tested and detected by the Lyra Direct HSV 1+2/VZV Assay. Lyra Direct HSV 1+2/VZV Assay Page 41 of 50

42 HSV-1 Results HSV-1 Strain and Test Concentration (TCID50/mL) Replicate # MacIntyre (Control) QC 316 SFG-029 Clinical Isolate x x x x10 3 HSV-1 PRC HSV-1 PRC HSV-1 PRC HSV-1 PRC Avg. Ct SD CV % 0.00% 0.90% 2.40% 1.90% 0.40% 1.70% 1.00% 0.40% HSV-2 Results HSV-2 Strain and Test Concentration (TCID50/mL) Replicate # Strain G (control) Clinical Isolate 6 Clinical Isolate 9 Clinical Isolate 10 Strain MS 4.88x x x x x10 3 HSV-2 PRC HSV-2 PRC HSV-2 PRC HSV-2 PRC HSV-2 PRC Avg. Ct SD CV % 0.4% 0.7% 0.8% 0.4% 0.7% 0.3% 1.1% 1.6% 3.6% 0.2% VZV Results Replicate # 130 (Control) AV-92-3 VZV Strain and Test Concentration (TCID50/mL) Ellen Clinical Isolate B Clinical Isolate D Strain 82 Strain x x x x x x x10 1 VZV PRC VZV PRC VZV PRC VZV PRC VZV PRC VZV PRC VZV PRC Avg. Ct SD CV % 1.1% 2.8% 1.4% 0.9% 0.2% 0.3% 4.3% 0.7% 2.2% 0.8% 1.2% 1.0% 0.8% 0.4% Reproducibility Study The reproducibility of the Lyra Direct HSV 1+2/VZV Assay was evaluated at three (3) laboratory sites (two external, one in-house). Reproducibility was assessed using a panel of six (6) simulated samples that include moderate positive and low positive, high negative and negative HSV-1, HSV-2, and VZV samples. The LOD values for each sample were based on the values obtained in the LOD study and are presented in the table below. Instrument Platform Medium Concentration Low High Negative Virus: HSV-1 HSV-2 VZV HSV-1 HSV-2 VZV HSV-1 HSV-2 VZV Life Technologies QuantStudio Dx 5x LOD 5x LOD 5x LOD 2x LOD 2x LOD 2x LOD 0.25x LOD 0.25x LOD 0.25x LOD Applied Biosystems 7500 Fast Dx 5x LOD 5x LOD 5x LOD 2x LOD 2x LOD 2x LOD 0.3x LOD 0.3x LOD 0.3x LOD Cepheid SmartCycler II System 5x LOD 5x LOD 5x LOD 2x LOD 2x LOD 2x LOD 0.3x LOD 0.3x LOD 0.3x LOD Lyra Direct HSV 1+2/VZV Assay Page 42 of 50

43 The panels and controls were processed and tested on the Life Technologies QuantStudio Dx, the Applied Biosystems 7500 Fast Dx, or the Cepheid SmartCycler II System. Panels and controls were tested at each site by 2 operators for 5 days (triplicate testing x 2 operators x 5 days x 3 sites = 90 results per level for each virus). Panel Member ID Reproducibility Results Life Technologies QuantStudio Dx Site 1 Site 2 Site 3 Combined Site Data Rate of AVE % Rate of AVE % Rate of AVE % Rate of AVE Ct %CV Detection Ct CV Detection Ct CV Detection Ct CV Detection HSV-1 High Negative 10/ / / / HSV-1 Low 30/ / / / HSV-1 Moderate 30/ / / / HSV-1 Negative 0/30 N/A N/A 0/29* N/A N/A 0/30 N/A N/A 0/89* N/A N/A HSV-2 High Negative 30/ / / / HSV-2 Low 30/ / / / HSV-2 Moderate 30/ / / / HSV-2 Negative 0/30 N/A N/A 0/29* N/A N/A 0/30 N/A N/A 0/89* N/A N/A VZV High Negative 9/ / / / VZV Low 30/ / / / VZV Moderate 30/ / / / VZV Negative 0/30 N/A N/A 0/29* N/A N/A 0/30 N/A N/A 0/89* N/A N/A HSV-1 30/ / / / Control HSV-2 30/ / / / Control VZV 30/ / / / Control Negative Control 0/30 N/A N/A 0/30 N/A N/A 0/30 N/A N/A 0/90 N/A N/A *One replicate s PRC was not detected. The replicate was reported as invalid. Lyra Direct HSV 1+2/VZV Assay Page 43 of 50

44 Panel Member ID Reproducibility Results Applied Biosystems 7500 Fast Dx Site 1 Site 2 Site 3 Combined Site Data Rate of AVE % Rate of AVE % Rate of AVE % Rate of AVE Ct %CV Detection Ct CV Detection Ct CV Detection Ct CV Detection HSV-1 High Negative 9/ / /29* /89* HSV-1 Low 30/ / / / HSV-1 Moderate 30/ / / / HSV-1 Negative 0/30 N/A N/A 0/30 N/A N/A 0/30 N/A N/A 0/90 N/A N/A HSV-2 High Negative 30/ / / / HSV-2 Low 30/ / / / HSV-2 Moderate 30/ / / / HSV-2 Negative 0/30 N/A N/A 0/30 N/A N/A 0/30 N/A N/A 0/90 N/A N/A VZV High Negative 0/30 N/A N/A 3/ /30 N/A N/A 3/ VZV Low 29/ / / / VZV Moderate 30/ / / / VZV Negative 0/30 N/A N/A 0/30 N/A N/A 0/30 N/A N/A 0/90 N/A N/A HSV-1 30/ / / / Control HSV-2 30/ / / / Control VZV 30/ / / / Control Negative Control 0/30 N/A N/A 0/30 N/A N/A 0/30 N/A N/A 0/90 N/A N/A *One replicate s PRC was not detected. The replicate was reported as invalid. Lyra Direct HSV 1+2/VZV Assay Page 44 of 50

45 Panel Member ID Reproducibility Results Cepheid SmartCycler II System Site 1 Site 2 Site 3 Combined Site Data Rate of AVE % Rate of AVE % Rate of AVE % Rate of AVE Ct %CV Detection Ct CV Detection Ct CV Detection Ct CV Detection HSV-1 High Negative 5/ / / / HSV-1 Low 30/ / / / HSV-1 Moderate 29/29* / / /89* HSV-1 Negative 0/30 N/A N/A 0/30 N/A N/A 0/30 N/A N/A 0/90 N/A N/A HSV-2 High Negative 24/ / / / HSV-2 Low 30/ / / / HSV-2 Moderate 29/29* / / /89* HSV-2 Negative 0/30 N/A N/A 0/30 N/A N/A 0/30 N/A N/A 0/90 N/A N/A VZV High Negative 0/30 N/A N/A 1/30 N/A N/A 0/30 N/A N/A 1/90 N/A N/A VZV Low 29/ / / / VZV Moderate 29/29* / / /89* VZV Negative 0/30 N/A N/A 0/30 N/A N/A 0/30 N/A N/A 0/90 N/A N/A HSV-1 30/ / / / Control HSV-2 Control 30/ / / / VZV / Control 30/ / / Negative Control 0/30 N/A N/A 0/30 N/A N/A 0/30 N/A N/A 0/90 N/A N/A *One (1) replicate s PRC was not detected. The replicate was reported as invalid. Analytical Specificity Interfering or Cross-Reactive Microorganism A study was performed on the Applied Biosystems 7500 Fast Dx to evaluate the performance of the Lyra Direct HSV 1+2/VZV Assay in the presence of seventy (70) other microorganisms that might be found in lesion specimens. Each potentially interfering or cross-reactive microorganism was tested in the presence of 2x LOD HSV-1, HSV-2 and VZV viruses, or negative matrix. Clinically relevant levels of viruses and bacteria are typically 10 6 cfu/ml or higher for bacteria and 10 5 pfu/ml or higher for viruses. Lyra Direct HSV 1+2/VZV Assay Page 45 of 50

46 Organism Test Concentration Lyra Direct HSV 1+2/VZV Assay Result Negative Matrix HSV-1 HSV-2 VZV Adenovirus x10 5 TCID50/mL Neg Pos Pos Pos Coronavirus OC x10 5 TCID50/mL Neg Pos Pos Pos Coxsackievirus B4 2.60x10 6 TCID50/mL Neg Pos Pos Pos Cytomegalovirus Towne VR x10 5 TCID50/mL Neg Pos Pos Pos Echovirus x10 5 TCID50/mL Neg Pos Pos Pos HHV x10 6 TCID50/mL Neg Pos Pos Pos HHV x10 5 TCID50/mL Neg Pos Pos Pos HIV Virus 1.05x10 5 TCID50/mL Neg Pos Pos Pos hmpv A1 1.24x10 5 TCID50/mL Neg Pos Pos Pos HSV-1 SFG x10 5 TCID50/mL Neg Pos Pos Pos HSV-1 Macintyre 2.14x10 5 TCID50/mL Neg Pos Pos Pos HSV-2 MS 6.44x10 6 TCID50/mL Neg Pos Pos Pos HSV-2 Strain G 1.38x10 5 TCID50/mL Neg Pos Pos Pos Influenza A/Mexico/4108/ x10 6 TCID50/mL Neg Pos Pos Pos Influenza B Hong Kong VR x10 5 TCID50/mL Neg Pos Pos Pos Measles virus 1.46x10 5 TCID50/mL Neg Pos Pos Pos Mumps virus 2.07x10 7 TCID50/mL Neg Pos Pos Pos Parainfluenza Type x10 5 TCID50/mL Neg Pos Pos Pos Parainfluenza Type x10 5 TCID50/mL Neg Pos Pos Pos Parainfluenza Type x10 5 TCID50/mL Neg Pos Pos Pos RSV A Long 7.13x10 5 TCID50/mL Neg Pos Pos Pos RSV B Washington 1.01x10 6 TCID50/mL Neg Pos Pos Pos Rubella Virus 9.45x10 5 TCID50/mL Neg Pos Pos Pos Acholeplasma laidlawi 5.33x10 6 cfu/ml Neg Pos Pos Pos Bordetella bronchiseptica 8.78x10 7 cfu/ml Neg Pos Pos Pos Bordetella pertussis 1.41x10 7 cfu/ml Neg Pos Pos Pos Clostridium perfringens 1.18x10 6 cfu/ml Neg Pos Pos Pos Mycoplasma hominis 9.75x10 6 cfu/ml Neg Pos Pos Pos Mycoplasma hyorhinis 4.95x10 6 cfu/ml Neg Pos Pos Pos Mycoplasma orale 1.16x10 7 cfu/ml Neg Pos Pos Pos Mycoplasma pneumoniae 7.50x10 6 ccu/ml Neg Pos Pos Pos Mycoplasma salivarium 1.25x10 7 cfu/ml Neg Pos Pos Pos Enterovirus x10 4 TCID50/mL Neg Pos Pos Pos Epstein Barr Virus 1.67x10 4 TCID50/mL Neg Pos Pos Pos HBV 1.67x10 3 TCID50/mL Neg Pos Pos Pos HCV 1.67x10 4 TCID50/mL Neg Pos Pos Pos HHV x10 4 TCID50/mL Neg Pos Pos Pos HPV Not Available Neg Pos Pos Pos Parainfluenza Type x10 4 TCID50/mL Neg Pos Pos Pos VZV Ellen 8.75x10 2 TCID50/mL Neg Pos Pos Pos VZV Webster 1.85x10 3 TCID50/mL Neg Pos Pos Pos Chlamydia trachomatis 5.00x10 5 cfu/ml Neg Pos Pos Pos Chlamydophila pneumoniae 2.67x10 3 cp/ml Neg Pos Pos Pos Toxoplasma gondii 1.10x10 6 tachyzoites/ml Neg Pos Pos Pos Trichomonas vaginalis 2.75x10 5 trophozoites/ml Neg Pos Pos Pos Acinetobacter calcoaceticus 7.35x10 8 cfu/ml Neg Pos Pos Pos Bacteroides fragilis 5.67x10 5 cfu/ml Neg Pos Pos Pos Candida albicans 1.50x10 6 cfu/ml Neg Pos Pos Pos Candida glabrata 1.50x10 6 cfu/ml Neg Pos Pos Pos Corynebacterium diphtheriae 2.40x10 8 cfu/ml Neg Pos Pos Pos Enterococcus faecalis 1.65x10 8 cfu/ml Neg Pos Pos Pos Lyra Direct HSV 1+2/VZV Assay Page 46 of 50

47 Organism Test Concentration Lyra Direct HSV 1+2/VZV Assay Result Negative Matrix HSV-1 HSV-2 VZV Escherichia coli 4.35x10 7 cfu/ml Neg Pos Pos Pos Gardnerella vaginalis 9.00x10 6 cfu/ml Neg Pos Pos Pos Haemophilis influenzae type A 6.30x10 8 cfu/ml Neg Pos Pos Pos Klebsiella pneumoniae 3.90x10 7 cfu/ml Neg Pos Pos Pos Lactobacillus acidophilus 1.35x10 6 cfu/ml Neg Pos Pos Pos Legionella pneumophila 2.85x10 7 cfu/ml Neg Pos Pos Pos Mobiluncus mulieris 1.91x10 8 cfu/ml Neg Pos Pos Pos Moraxella cartarrhalis 2.25x10 7 cfu/ml Neg Pos Pos Pos Neisseria gonorrhoeae 2.40x10 7 cfu/ml Neg Pos Pos Pos Proteus mirabilis 9.60x10 7 cfu/ml Neg Pos Pos Pos Pseudomonas aeruginosa 5.25x10 7 cfu/ml Neg Pos Pos Pos Salmonella enteriditis 4.05x10 7 cfu/ml Neg Pos Pos Pos Salmonella typhimurium 3.45x10 7 cfu/ml Neg Pos Pos Pos Staphylococcus aureus 1.38x10 9 cfu/ml Neg Pos Pos Pos Staphylococcus saprophyticus 2.25x10 8 cfu/ml Neg Pos Pos Pos Streptococcus agalactiae 1.65x10 8 cfu/ml Neg Pos Pos Pos Streptococcus pneumoniae 2.10x10 6 cfu/ml Neg Pos Pos Pos Streptococcus pyogenes 2.85x10 8 cfu/ml Neg Pos Pos Pos Ureaplasma uralyticum Unable to titer Neg Pos Pos Pos The organism, Treponema pallidum, could not be sourced for the Lyra Direct HSV 1+2/VZV Assay study and therefore an in silico analysis was required to confirm no cross-reactivity of the HSV-1, HSV-2, or VZV primers. The in silico analysis determined that the three primer pairs will not cross-react with T. pallidum under the Lyra assay conditions. Therefore, the presence of the organism should not interfere with the assay. None of the seventy (70) other microorganisms that might be found in lesion specimens interfere with the assay. Analytical Specificity Interfering or Cross-Reactive Substances A study was performed on the Applied Biosystems 7500 Fast Dx to evaluate the performance of the Lyra Direct HSV 1+2/VZV Assay in the presence of twenty-six (26) clinically relevant levels of potentially interfering/cross-reactive substances that might be present in lesion specimens. Substance Name Substance Final Lyra Direct HSV 1+2/VZV Assay Result Concentration Negative Matrix HSV-1 HSV-2 VZV Seminal Fluid 7% Negative Cornstarch 2.5 mg/ml Negative Acetamidophenol 10 mg/ml Negative Feces 0.219% Negative Chlorpheniramine 5 mg/ml Negative Dextromethorphan 5 mg/ml Negative Blood/EDTA 0.625% Negative Female Urine 7% Negative Male Urine 7% Negative Acyclovir 7 mg/ml Negative Albumin 3.3 mg/ml Negative Casein 7 mg/ml Negative KY Jelly 7% Negative Douche 7% Negative Miconazole 1 7% Negative Miconazole 3 7% Negative Tioconazole 1 7% Negative Lyra Direct HSV 1+2/VZV Assay Page 47 of 50

48 Substance Name Substance Final Lyra Direct HSV 1+2/VZV Assay Result Preparation H 7% Negative Lanacane 3.5% Negative Listerine 7% Negative Abreva 7% Negative Carmex 7% Negative Releev 7% Negative Colgate 7% Negative Mucin 60 µg/ml Negative Leukocytes 2.5e5 cells/ml Negative None of the twenty-six (26) substances interfered with the detection of 2x LOD HSV-1, HSV-2, or VZV, or were cross-reactive with the Lyra Direct HSV 1+2/VZV Assay. Carryover and Cross-Contamination Studies Studies were performed on each instrument using a 96-sample panel consisting of 48 high positives and 48 negative specimens. Each high positive specimen contained 1.00 x10 5 TCID50/mL of each analyte (combined into one specimen). The negative specimen was negative matrix. The high positive samples were analyzed in series alternating with the negative samples. The testing was repeated over a 5-day period. Over the course of 5-days, cross-contamination and amplicon carry-over did not occur with the Lyra Direct HSV 1+2/VZV Assay on any of the three instruments. CUSTOMER AND TECHNICAL ASSISTANCE To place an order or for technical support, please contact a Quidel Representative at (in the U.S.) or (outside the U.S.), Monday through Friday, from 8:00 a.m. to 5:00 p.m., Eastern Time. Orders may also be placed by fax at For support contact customerservice@quidel.com or technicalsupport@quidel.com. For services outside the U.S., please contact your local distributor. Additional information about Quidel, our products, and our distributors can be found on our website at quidel.com. INTELLECTUAL PROPERTY Dye compounds in this product are sold under license from BioSearch Technologies, Inc. and protected by U.S. and worldwide patents either issued or under application. The purchase of this product grants the purchaser rights under certain Roche patents to use it solely for providing human in vitro diagnostic services. No general patent or other license of any kind other than this specific right of use from purchase is granted hereby. REFERENCES 1. National Health and Nutrition Examination Survey Data Documentation, Codebook, and Frequencies Herpes Simplex Virus Type-1 and Type-2 (HSV_E). Revised May, Clinical and Laboratory Standards Institute. Viral Culture; Approved Guidelines. CLSI document M41-A [ISBN ] Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania , USA Huang Y.T., Hite S., Duane V., Yan H. CV-1 and MRC-5 mixed cells for simultaneous detection of herpes simplex viruses and varicella zoster virus in skin lesions. J Clin Virol Feb; 24(1-2): Leland D., Ginocchio C. Role of Cell Culture for Virus Detection in the Age of Technology, Clin Microbiol Rev. Jan 2007; 20(1): Lyra Direct HSV 1+2/VZV Assay Page 48 of 50

49 M102 Lyra Direct HSV 1+2/VZV Assay kit MDSS GmBH Schiffgraben Hannover, Germany Quidel Corporation 2005 East State Street, Suite 100 Athens, OH USA quidel.com PIM102003EN00 (09/18) Lyra Direct HSV 1+2/VZV Assay Page 49 of 50

50 GLOSSARY Lyra Direct HSV 1+2/VZV Assay Page 50 of 50