QUIDEL MOLECULAR DIRECT HSV 1 +2/VZV ASSAY
Device Facts
| Record ID | DEN140004 |
|---|---|
| Device Name | QUIDEL MOLECULAR DIRECT HSV 1 +2/VZV ASSAY |
| Applicant | Diagnostic Hybrids, Inc. |
| Product Code | PGI · Microbiology |
| Decision Date | May 13, 2014 |
| Decision | DENG |
| Submission Type | Post-NSE |
| Regulation | 21 CFR 866.3309 |
| Device Class | Class 2 |
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 varicella-zoster 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.
Device Story
The Lyra™ Direct HSV 1 + 2/VZV Assay is a multiplex real-time PCR test for qualitative detection and differentiation of HSV-1, HSV-2, and VZV DNA. Input consists of cutaneous or mucocutaneous lesion swabs. Samples are heated at 60°C for 5 minutes, then combined with Process Buffer (containing a Process Control) and rehydrated Master Mix. The assay uses TaqMan® chemistry; DNA polymerase with 5'-3' exonuclease activity cleaves fluorescent-labeled probes upon hybridization to target DNA, generating signal. The device is used in clinical laboratories by trained personnel on the Life Technologies QuantStudio™ Dx, Applied Biosystems® 7500 Fast Dx, or Cepheid SmartCycler® II. Output is a qualitative report of HSV-1, HSV-2, and VZV presence. Results aid clinicians in diagnosing active infections and initiating appropriate antiviral therapy. Benefits include accurate, rapid diagnosis of potentially severe infections.
Clinical Evidence
Multi-center clinical study (April-October 2013) compared the assay to ELVIS® cell culture (HSV) and DSFA/culture (VZV). Evaluated 279 cutaneous and 650 mucocutaneous lesion specimens across three instruments. Sensitivity for HSV-1/2 ranged from 95.1% to 100%; specificity ranged from 94.9% to 99.3%. VZV sensitivity was 100% (95% CI 51.0-100%) with specificity 94.9-99.3%. Invalid rates were low. Additional RT-PCR was used to resolve discrepancies.
Technological Characteristics
Multiplex real-time PCR assay using TaqMan® chemistry. Reagents: DNA polymerase, primers/probes, dNTPs, Process Control (PRC). Detection: FAM (HSV-1), CAL Fluor® Orange 560 (HSV-2), CAL Fluor® Red 610 (VZV), Quasar® 670 (PRC). Platforms: QuantStudio™ Dx, 7500 Fast Dx, SmartCycler® II. Software: Instrument-specific control/analysis software (Moderate Level of Concern).
Indications for Use
Indicated for symptomatic patients suspected of active HSV-1, HSV-2, or VZV infection. For use with cutaneous or mucocutaneous lesion swab specimens. Not for use with cerebrospinal fluid, CNS infection diagnosis, or prenatal screening.
Regulatory Classification
Identification
A herpes virus nucleic acid-based cutaneous and mucocutaneous lesion panel is a qualitative in vitro diagnostic device intended for the simultaneous detection and differentiation of different herpes viruses in cutaneous and mucocutaneous lesion samples from symptomatic patients suspected of Herpetic infections. Negative results do not preclude infection and should not be used as the sole basis for treatment or other patient management decisions. The assay is not intended for use in cerebrospinal fluid samples.
Special Controls
*Classification.* Class II (special controls). The special controls for this device are:(1) Premarket notification submissions must include detailed documentation for the device description, including the device components, ancillary reagents required but not provided, and a detailed explanation of the methodology including primer design and selection. (2) Premarket notification submissions must include detailed documentation from the following analytical and clinical performance studies: Analytical sensitivity (Limit of Detection), reactivity, inclusivity, precision, reproducibility, interference, cross reactivity, carry-over, and cross contamination. (3) Premarket notification submissions must include detailed documentation of a clinical study using lesion samples in which Herpes Simplex Virus 1, Herpes Simplex Virus 2, or Varicella Zoster Virus DNA detection was requested. The study must compare the device performance to an appropriate well established reference method. (4) A detailed explanation of the interpretation of results and acceptance criteria must be included in the device's 21 CFR 809.10(b)(9) compliant labeling. (5) The device labeling must include a limitation statement that reads: “The device 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).” (6) Premarket notification submissions must include quality assurance protocols and a detailed documentation for device software, including, but not limited to, standalone software applications and hardware-based devices that incorporate software. (7) The risk management activities performed as part of the manufacturer's 21 CFR 820.10(c) design and development activities must document an appropriate end user device training program that will be offered as part of efforts to mitigate the risk of failure to correctly operate the instrument.
Reference Devices
- ELVIS® HSV ID and D3 Typing Test
- DSFA (VZV detection reagent)
Related Devices
- K173798 — Simplexa HSV 1 & 2 Direct, Simplexa HSV 1 & 2 Positive Control Pack · Diasorin Molecular, LLC · Mar 14, 2018
- K232286 — Savanna HSV 1+2/VZV Assay, Savanna HSV 1+2/VZV Control Set, Savanna Instrument · Quidel Corporation · Dec 20, 2023
Submission Summary (Full Text)
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# EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR Lyra™ Direct HSV 1 + 2/VZV Assay
# DECISION SUMMARY
# A. 510(k) Number:
K133448
# B. Purpose for Submission:
De novo request for evaluation of automatic class III designation for the Lyra™ Direct HSV 1 + 2/VZV Assay
### C. Measurand:
Target DNA sequences from conserved regions of the herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2) and varicella-zoster virus (VZV) genes.
# D. Type of Test:
A real-time Polymerase Chain Reaction (PCR) test for qualitative detection and differentiation of HSV-1, HSV- 2, and VZV DNA isolated and purified from cutaneous or mucocutaneous lesion samples.
# E. Applicant:
Quidel Corporation.
# F. Proprietary and Established Names:
Lyra™ Direct HSV 1 + 2/VZV Assay
### G. Regulatory Information:
- 21 CFR 866.3309 1. Regulation:
- 2. Classification: Class II (special controls)
- 3. Product code: PGI
- 4. Panel: Microbiology (83)
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### H. Intended Use:
### 1. Intended use(s):
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 varicella-zoster 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.
- 2. Indication(s) for use:
Same as intended use
- 3. Special conditions for use statement(s):
For prescription use only in accordance with 21 CFR 801.109.
- 4. Special instrument requirements:
To be used with: Life Technologies QuantStudio™ Dx (software version 1.0) Applied Biosystems® 7500 Fast Dx (software version 1.4) Cepheid SmartCycler® II System (software version 3.0b)
### I. Device Description:
The Lyra™ Direct HSV 1 + 2/VZV 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 amplicons for 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, and VZV and to the PRC, respectively.
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| Target | Dye |
|--------|-----------------------|
| HSV-1 | FAM |
| HSV-2 | CAL Fluor® Orange 560 |
| VZV | CAL Fluor® Red 610 |
| PRC | Quasar® 670 |
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The following table and list describe: the reagents provided in the kit, the materials which are required but not provided with the kit and the optional reagents and materials.
# Materials Provided With The Kit:
The Lyra™ Direct HSV 1 + 2/VZV Assay kit consists of the following:
- Rehydration Solution -
- Process Buffer Part M5050 (contains the PRC) -
- Lyra™ Direct HSV 1 + 2/VZV Master Mix Part M5012 -
- Lyophilized Contents:
- o DNA polymerase enzyme
- Primers and probes o
- dNTPs O
- o Stabilizers
Materials Required But Not Provided:
- Micropipettors (range between 2 to 20 uL and 20 to 200 uL) -
- Non-aerosol pipette tips -
- Life Technologies OuantStudio™ Dx or the Applied Biosystems® 7500 Fast Dx -
- Applied Biosystems Fast Dx 96 well PCR plate -
- Optical plate films -
- -Plate centrifuge for Applied Biosystems 96 well plate
- Dry heating block, capable of heating 1.5 mL tubes at 60℃ for 5 minutes -
- Empty microcentrifuge tubes -
### Or
- Micropipettors (range between 2 to 20 uL and 20 to 200 uL) -
- -Non-aerosol pipette tips
- Cepheid SmartCycler® II -
- 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
Optional Materials:
- Positive controls for HSV-1, HSV-2 and VZV (i.e., Lyra™ Direct HSV 1 + 2/VZV -Control Set which serves as an external assay control)
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# J. Standard/Guidance Documents Referenced:
- 1. Guidance for Industry and FDA Staff Establishing the Performance Characteristics of In Vitro Diagnostic Devices for the Detection or Detection and Differentiation of Influenza Viruses (July 15, 2011) http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocument s/ucm079171.htm
- 2. Guidance for Industry and FDA Staff Class II Special Controls Guidance Document: Respiratory Viral Panel Multiplex Nucleic Acid Assay (October 9, 2009) http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocument s/ucm180307.htm
- 3. Guidance on Informed Consent for In Vitro Diagnostic Device Studies Leftover Human Specimens that are Not Individually Identifiable (April 2006) http://www.fda.gov/cdrh/oivd/guidance/1588.pdf.
- 4. Guidance for Industry and Food and Drug Administration Staff eCopy Program for Medical Device (December 2012) http://www.fda.gov/downloads/MedicalDevices/DeviceRegulationandGuidance/Guidanc eDocuments/UCM313794.pdf
- 5. Guidance for Industry, FDA Reviewers and Compliance on Off-The-Shelf Software Use in Medical Devices (September 9, 1999) http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocument s/ucm073778.htm
The following documents were referenced in one or more of the Guidance Documents above:
- 1. CLSI EP17-A: Guidance for Protocols for Determination of Limits of Detection and Limits of Quantitation (Vol. 2, No. 34) (Oct 2004).
- 2. CLSI MM13-A: Guidance for the Collection, Transport, Preparation and Storage of Specimens for Molecular Methods (Vol. 25. No. 31) (Dec 2005).
- 3. CLSI EP7-A2: Guidance for Interference Testing in Clinical Chemistry (Vol. 25, No.27 Second Ed) (Nov 2005).
- 4. CLSI EP12-A: Guidance for User Protocol for Evaluation of Qualitative Test Performance (Vol. 22, No. 14) (Sept 2002).
- 5. CLSI MM6-A: Guidance for the Quantitative Molecular Methods for Infectious Diseases (Vol. 23, No.28) (Oct 2003).
- 6. CLSI EP5-A2: Guidance for Evaluation of Precision Performance of Quantitative Measurement Methods (Vol. 24, No. 25 Second Ed.) (Aug 2004).
# K. Test Principle:
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
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fluorescence is achieved on the Life Technologies QuantStudio™ Dx, the Applied Biosystems® 7500 Fast Dx, or the Cepheid SmartCycler® II System, the sample is reported as positive for the detected nucleic acid.
The following is a summary of the procedure:
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 uL of each clinical specimen from the original collection tube and place into a clean microcentrifuge tube. Heat at 60°C for 5 minutes, then remove from heat and add 25 uL of Process Buffer within 60 minutes according to the detailed instructions for use. The Process Buffer contains a buffer and diluents as well as the PRC. The PRC serves to monitor in the prepared specimen, assures that adequate amplification has taken place, and confirms that the overall process was performed correctly.
Rehydration of Master Mix: Rehydrate the lyophilized Master Mix using 135 uL of Rehydration Solution. The Master Mix contains oligonucleotide primers, fluorophore and quencher-labeled probes targeting conserved regions of HSV-1, and VZV, as well as the PRC sequence.
Nucleic Acid Amplification and Detection: Add 15 uL of the rehydrated Master Mix to each reaction tube or plate well. Then add 5 uL of nucleic acids (specimen with PRC) to the plate well or appropriately labeled reaction tube. Place the plate or tube into either the QuantStudio™ Dx, Applied Biosystems® 7500 Fast Dx or 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 targets.
# L. Performance Characteristics (if/when applicable):
# 1. Analytical performance:
# a. Reproducibility/ Precision
Reproducibility: The reproducibility of the Lyra™ Direct HSV 1 + 2/VZV Assay was evaluated at 3 laboratory sites (two external, one in-house). Reproducibility was assessed using a panel of 6 simulated samples that include medium positive, low positive, high negative and negative HSV-1, HSV-2, and VZV samples.
The panels and controls were processed and tested on the Life Technologies QuantStudio™ Dx, the Applied Biosystems® 7500 Fast Dx, and 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).
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| Reproducibility Results - Life Technologies QuantStudio™ Dx | | | | | | | | | | | | |
|-------------------------------------------------------------|----------------------|-----------|---------|----------------------|-----------|---------|------------------------------|-----------|---------|----------------------|-----------|-----|
| Panel | | Site 1 | | | Site 2 | | Site 3<br>Combined Site Data | | | | | |
| Member<br>ID | Rate of<br>Detection | AVE<br>Ct | %<br>CV | Rate of<br>Detection | AVE<br>Ct | %<br>CV | Rate of<br>Detection | AVE<br>Ct | %<br>CV | Rate of<br>Detection | AVE<br>Ct | %CV |
| HSV-1<br>High<br>Negative | 10/30 | 37.0 | 4.9 | 16/30 | 34.5 | 8.9 | 17/30 | 34.0 | 7.6 | 43/90 | 34.8 | 8.2 |
| HSV-1<br>Low<br>Positive | 30/30 | 29.8 | 1.6 | 30/30 | 29.0 | 2.6 | 30/30 | 29.5 | 3.6 | 90/90 | 29.4 | 2.9 |
| HSV-1<br>Moderate<br>Positive | 30/30 | 28.3 | 1.9 | 30/30 | 27.3 | 2.4 | 30/30 | 27.9 | 3.7 | 90/90 | 27.8 | 3.1 |
| HSV-1<br>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<br>High<br>Negative | 30/30 | 34.8 | 3.0 | 30/30 | 31.9 | 4.7 | 28/30 | 34.1 | 6.2 | 88/90 | 33.6 | 6.0 |
| HSV-2<br>Low<br>Positive | 30/30 | 32.8 | 1.4 | 30/30 | 32.0 | 2.3 | 30/30 | 31.8 | 3.6 | 90/90 | 32.2 | 2.9 |
| HSV-2<br>Moderate<br>Positive | 30/30 | 30.8 | 3.8 | 30/30 | 29.8 | 2.8 | 30/30 | 30.8 | 4.2 | 90/90 | 30.5 | 3.9 |
| HSV-2<br>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<br>Negative | 9/30 | 37.4 | 4.2 | 10/30 | 35.2 | 6.4 | 5/30 | 34.4 | 6.6 | 24/90 | 35.9 | 6.5 |
| VZV Low<br>Positive | 30/30 | 29.2 | 1.7 | 30/30 | 28.7 | 1.2 | 29/30 | 29.9 | 5.5 | 89/90 | 29.3 | 3.7 |
| VZV<br>Moderate<br>Positive | 30/30 | 27.6 | 1.3 | 30/30 | 27.2 | 1.0 | 30/30 | 27.7 | 2.6 | 90/90 | 27.5 | 2.9 |
| VZV<br>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<br>Positive<br>Control | 30/30 | 27.4 | 2.6 | 30/30 | 25.1 | 0.9 | 30/30 | 26.8 | 2.7 | 90/90 | 26.4 | 7.0 |
| HSV-2<br>Positive<br>Control | 30/30 | 26.7 | 6.6 | 30/30 | 29.8 | 1.5 | 30/30 | 25.4 | 3.7 | 90/90 | 22.8 | 7.9 |
| VZV<br>Positive<br>Control | 30/30 | 22.1 | 13.4 | 30/30 | 20.0 | 0.8 | 30/30 | 21.1 | 4.5 | 90/90 | 21.1 | 9.2 |
| Negative<br>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.
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| Reproducibility Results - Applied Biosystems® 7500 Fast Dx | | | | | | | | | | | | |
|------------------------------------------------------------|----------------------|-----------|---------|----------------------|-----------|---------|----------------------|-----------|---------|----------------------|-----------|-----|
| Panel<br>Member<br>ID | Rate of<br>Detection | AVE<br>Ct | %<br>CV | Rate of<br>Detection | AVE<br>Ct | %<br>CV | Rate of<br>Detection | AVE<br>Ct | %<br>CV | Rate of<br>Detection | AVE<br>Ct | %CV |
| HSV-1<br>High<br>Negative | 9/30 | 36.2 | 3.6 | 9/30 | 35.0 | 10.1 | 12/29 | 36.5 | 6.0 | 30/89 | 36.1 | 7.6 |
| HSV-1<br>Low<br>Positive | 30/30 | 30.3 | 1.8 | 30/30 | 30.3 | 3.4 | 30/30 | 30.2 | 2.6 | 90/90 | 30.3 | 2.7 |
| HSV-1<br>Moderate<br>Positive | 30/30 | 28.8 | 2.4 | 30/30 | 28.5 | 2.6 | 30/30 | 29.5 | 8.1 | 90/90 | 28.9 | 5.4 |
| HSV-1<br>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<br>High<br>Negative | 30/30 | 35.4 | 3.7 | 30/30 | 34.2 | 5.8 | 24/30 | 36.0 | 6.0 | 84/90 | 35.2 | 5.5 |
| HSV-2<br>Low<br>Positive | 30/30 | 32.8 | 4.2 | 30/30 | 33.6 | 3.8 | 30/30 | 32.7 | 5.9 | 90/90 | 33.0 | 4.8 |
| HSV-2<br>Moderate<br>Positive | 30/30 | 31.2 | 2.1 | 30/30 | 31.3 | 2.9 | 30/30 | 31.9 | 4.3 | 90/90 | 31.5 | 3.4 |
| HSV-2<br>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<br>Negative | 0/30 | N/A | N/A | 3/30 | 33.8 | 6.2 | 0/30 | N/A | N/A | 3/90 | 33.8 | 6.2 |
| VZV Low<br>Positive | 29/30 | 31.5 | 4.4 | 29/30 | 31.5 | 4.6 | 30/30 | 32.1 | 6.2 | 88/90 | 31.7 | 5.2 |
| VZV<br>Moderate<br>Positive | 30/30 | 29.4 | 2.5 | 30/30 | 29.2 | 2.1 | 30/30 | 30.9 | 9.1 | 90/90 | 29.8 | 6.2 |
| VZV<br>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<br>Positive<br>Control | 30/30 | 27.1 | 11.6 | 30/30 | 25.9 | 1.3 | 30/30 | 26.2 | 2.2 | 90/90 | 26.4 | 7.2 |
| HSV-2<br>Positive<br>Control | 30/30 | 26.1 | 8.1 | 30/30 | 24.9 | 1.2 | 30/30 | 25.7 | 2.0 | 90/90 | 25.6 | 5.2 |
| VZV<br>Positive<br>Control | 30/30 | 23.6 | 13.2 | 30/30 | 21.8 | 1.1 | 30/30 | 22.1 | 1.7 | 90/90 | 22.5 | 8.6 |
| Negative<br>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 |
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| Reproducibility Results - Cepheid SmartCycler® II | | | | | | | | | | | | |
|---------------------------------------------------|----------------------|-----------|---------|----------------------|-----------|---------|----------------------|-----------|---------|----------------------|--------------------|-----|
| Panel | | Site 1 | | | Site 2 | | | Site 3 | | | Combined Site Data | |
| Member<br>ID | Rate of<br>Detection | AVE<br>Ct | %<br>CV | Rate of<br>Detection | AVE<br>Ct | %<br>CV | Rate of<br>Detection | AVE<br>Ct | %<br>CV | Rate of<br>Detection | AVE<br>Ct | %CV |
| HSV-1<br>High<br>Negative | 5/30 | 37.8 | 1.9 | 5/30 | 33.4 | 7.5 | 13/30 | 35.3 | 2.4 | 23/30 | 35.4 | 6.0 |
| HSV-1<br>Low<br>Positive | 30/30 | 31.3 | 1.3 | 30/30 | 31.0 | 4.5 | 30/30 | 31.5 | 2.4 | 90/90 | 31.3 | 3.1 |
| HSV-1<br>Moderate<br>Positive | 29/29* | 29.4 | 2.9 | 30/30 | 28.9 | 4.2 | 30/30 | 30.0 | 2.4 | 89/89* | 29.5 | 3.6 |
| HSV-1<br>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<br>High<br>Negative | 24/30 | 38.5 | 5.1 | 27/30 | 36.6 | 6.5 | 17/30 | 37.3 | 4.4 | 68/90 | 37.4 | 5.8 |
| HSV-2<br>Low<br>Positive | 30/30 | 36.2 | 3.3 | 29/30 | 35.8 | 2.5 | 30/30 | 35.8 | 4.9 | 89/90 | 36.0 | 3.7 |
| HSV-2<br>Moderate<br>Positive | 29/29* | 33.6 | 1.6 | 30/30 | 33.3 | 4.6 | 30/30 | 33.8 | 3.4 | 89/89* | 33.6 | 2.5 |
| HSV-2<br>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<br>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<br>Positive | 29/30 | 33.4 | 7.0 | 29/30 | 32.7 | 2.8 | 30/30 | 36.2 | 10.7 | 88/90 | 34.1 | 8.9 |
| VZV<br>Moderate<br>Positive | 29/29* | 30.9 | 2.3 | 30/30 | 30.6 | 1.1 | 30/30 | 33.4 | 8.5 | 89/89* | 31.6 | 6.7 |
| VZV<br>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<br>Positive<br>Control | 30/30 | 27.6 | 8.6 | 30/30 | 26.4 | 1.8 | 30/30 | 27.4 | 2.2 | 90/90 | 27.2 | 5.6 |
| HSV-2<br>Positive<br>Control | 30/30 | 28.0 | 7.0 | 30/30 | 26.5 | 1.8 | 30/30 | 28.1 | 9.3 | 90/90 | 27.5 | 7.3 |
| VZV<br>Positive<br>Control | 30/30 | 24.7 | 12.1 | 30/30 | 22.9 | 1.1 | 30/30 | 23.7 | 1.9 | 90/90 | 23.8 | 7.9 |
| Negative<br>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.
Precision: For the Precision/Within Laboratory Repeatability study, a blinded four-member panel consisting of HSV-1, HSV-2, and VZV positive and negative samples was tested by two (2) operators (Op 1 and Op 2), twice a day (2X) for twelve (12) days on all three instruments.
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| Life Technologies QuantStudio™ Dx Results Summary | | | | | | | | | |
|---------------------------------------------------|----------------|---------------------|--------|--------|------------|--------------------|--|--|--|
| Target | | Positive<br>Control | 5X LoD | 2X LoD | <0.25X LoD | Negative<br>Matrix | | | |
| | Op 1ª Avg Ct | 25.0 | 27.7 | 29.4 | 36.1 | Neg | | | |
| HSV-1 | Op 2° Avg Ct | 25.4 | 27.6 | 28.9 | 36.2 | Neg | | | |
| | Positivity (%) | 100 | 100 | 100 | રી | 0 | | | |
| | Op 1 Avg Ct | 26.3 | 28.3 | 29.0 | 37.2 | Neg | | | |
| HSV-2 | Op 2 Avg Ct | 26.3 | 28.2 | 29.0 | 37.6 | Neg | | | |
| | Positivity (%) | 100 | 100 | 100 | 63 | 0 | | | |
| VZV | Op 1 Avg Ct | 18.8 | 27.8 | 29.3 | 34.3 | Neg | | | |
| | Op 2 Avg Ct | 18.7 | 27.9 | 29.2 | 34.7 | Neg | | | |
| | Positivity (%) | 100 | 100 | 100 | રી | 0 | | | |
ª Operator 1; b Operator 2
| Applied Biosystems® 7500 Fast Dx Results Summary | | | | | | |
|--------------------------------------------------|----------------|------------------|--------|--------|------------|-----------------|
| Target | | Positive Control | 5X LoD | 2X LoD | ≤0.25X LoD | Negative Matrix |
| HSV-1 | Op 1 Avg Ct | 26.2 | 28.3 | 29.9 | 38.6 | Neg |
| | Op 2 Avg Ct | 25.9 | 27.5 | 29.0 | 37.4 | Neg |
| | Positivity (%) | 100 | 100 | 100 | 32 | 0 |
| HSV-2 | Op 1 Avg Ct | 25.4 | 29.1 | 30.1 | 37.2 | Neg |
| | Op 2 Avg Ct | 25.8 | 28.4 | 29.6 | 36.8 | Neg |
| | Positivity (%) | 100 | 100 | 100 | 75 | 0 |
| VZV | Op 1 Avg Ct | 19.6 | 29.0 | 33.1 | 36.9 | Neg |
| | Op 2 Avg Ct | 19.5 | 29.1 | 30.7 | 36.7 | Neg |
| | Positivity (%) | 100 | 100 | 99 | 25 | 0 |
| Cepheid SmartCycler® II Results Summary | | | | | | |
|-----------------------------------------|----------------|---------------------|--------|--------|------------|--------------------|
| Target | | Positive<br>Control | 5X LoD | 2X LoD | ≤0.25X LoD | Negative<br>Matrix |
| HSV-1 | Op 1 Avg Ct | 25.6 | 30.8 | 32.8 | 37.7 | Neg |
| | Op 2 Avg Ct | 25.5 | 30.9 | 32.6 | 36.4 | Neg |
| | Positivity (%) | 100 | 100 | 100 | 38 | 0 |
| HSV-2 | Op 1 Avg Ct | 27.2 | 32.4 | 33.5 | 37.9 | Neg |
| | Op 2 Avg Ct | 27.6 | 32.2 | 33.4 | 37.7 | Neg |
| | Positivity (%) | 100 | 100 | 100 | 65 | 0 |
| VZV | Op 1 Avg Ct | 21.0 | 30.5 | 31.9 | 39.5 | Neg |
| | Op 2 Avg Ct | 21.0 | 30.7 | 32.5 | 40.2 | Neg |
| | Positivity (%) | 100 | 100 | 100 | 54 | 0 |
{9}------------------------------------------------
- b. Linearity/assay reportable range:
Not applicable.
- c. Traceability, Stability, Expected values (controls, calibrators, or methods):
### Kit Stability:
The stability of the Lyra Direct HSV 1 + 2/VZV Assay is currently being assessed in a real-time study using 3 production lots of the kits. All kits used in the study are being stored at 2° to 8°C. The test protocol utilizes 2x LoD virus dilutions of HSV-1, HSV-2, and VZV. These dilutions are being tested in triplicate according to the product insert on the Applied Biosystems® 7500 Fast Dx. The Ct values for each analyte (HSV-1, HSV-2, and VZV) generated at each time point is being compared to the Ct values generated at the time of production of the kits. The Ct values at a stability time point must be within 3 Cts of the result at the initial time point for a kit to pass. The real-time testing data collected thus far demonstrates stability up to 243 days at 2° to 8°C. All time points have passed for each analyte.
### Rehydrated Master Mix Stability:
A study was performed on the Applied Biosystems® 7500 Fast Dx to determine the effect of time and temperature on rehydrated Master Mix stability. The Lyra™ DirectHSV 1 + 2/VZV Master Mix was rehydrated, stored at -20°C, 2° to 8°C and room temperature (RT) (20° to 25°C) for various times and then tested with samples at 2x LoD HSV-1, HSV-2 and VZV. Based on the data generated in this study, the final stability claims for rehydrated Lyra™ Direct HSV 1 + 2/VZV Master Mix are summarized in the tables below:
| HSV/VZV Rehydrated Master Mix Stability Summary | | | |
|-------------------------------------------------|---------------------|-------|--------|
| Target | Storage Temperature | | |
| | RT | 2-8°C | -20°C |
| HSV-1 | 4hrs | 24hrs | 76 hrs |
| HSV-2 | 4hrs | 24hrs | 76 hrs |
| VZV | 1.5hrs | 24hrs | 76 hrs |
| Stability for RehydratedMaster Mix | | |
|-----------------------------------------|-------|--------|
| Storage Temperature and Stability Claim | | |
| RT | 2-8°C | -20°C |
| 1 hr | 9 hrs | 72 hrs |
# Processed Sample Stability:
The stability of processed samples was evaluated when stored at room temperature, 2 to 8°C. -20°C. and -70°C for various periods of time. This study was conducted on the Applied Biosystems 7500 Fast Dx. HSV-1, HSV-2, and VZV stocks were diluted to 2x LoD, processed, and then stored at the various temperatures listed below.
{10}------------------------------------------------
| Number of Processed Samples Required for Each Storage Condition | | | | |
|-----------------------------------------------------------------|--------------------------|-----------|-------|-------|
| | Control and<br>Room Temp | 2° to 8°C | -20°C | -70°C |
| HSV-1 | 3 | 21 | 12 | 12 |
| HSV-2 | 3 | 21 | 12 | 12 |
| VZV | 3 | 21 | 12 | 12 |
| Negative | 1 | 7 | 4 | 4 |
HSV-1, HSV-2, and VZV processed samples tested positive with little or no Ct shift (see table below) at all tested time points and for all storage conditions. Based on results at the tested time points, processed samples are stable for 48 hours when stored at room temperature (20-25℃). 48 hours when stored at 2° to 8℃. 31 days when stored at -20℃, and 31 days when stored at -70℃.
| | Control<br>Ct<br>Value<br>(Ave.) | Ct Average<br>20° to 25°C<br>(50 hours) | Ct Shift | Ct Average<br>2° to 8°C<br>(50 hours) | Ct<br>Shift | Ct Average<br>-20°C<br>(32 days) | Ct Shift | Ct Average<br>-20°C<br>(32 days) | Ct Shift |
|-------|----------------------------------|-----------------------------------------|----------|---------------------------------------|-------------|----------------------------------|----------|----------------------------------|----------|
| HSV-1 | 30.5 | 31.7 | + 1.2 | 30.5 | 0 | 28.9 | - 1.6 | 33.7 | +3.2 |
| HSV-2 | 33.9 | 30.3 | - 3.6 | 35.7 | +1.8 | 34.6 | +0.7 | 34.8 | +0.9 |
| VZV | 30.5 | 30.7 | +0.2 | 30.6 | +0.1 | 30.3 | -0.2 | 30.8 | +0.3 |
- d. Assay cut-off:
See limit of detection.
- e. Detection limit (LoD)
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 two strains of HSV-1, two strains of HSV-2 and two strains of VZV serially diluted in a negative matrix. Testing was performed concurrently using the same serial dilutions on each instrument. Analytical sensitivity (LoD) is defined as the lowest concentration at which 95% of all replicates tested positive.
{11}------------------------------------------------
| Limit of Detection | | | | | | |
|----------------------------------------|--------------------|-----------------|-------------------|------------------|--------------|----------|
| Instrument | Virus TCID50/mL | | | | | |
| | HSV-1<br>Macintyre | HSV-1<br>QC 316 | HSV-2<br>Strain G | HSV-2<br>QC Comp | VZV<br>Ellen | VZV 130 |
| Life Technology<br>QuantStudio Dx | 2.18E+03 | 5.69E+03 | 2.44E+03 | 2.53E+02 | 1.68E+00 | 2.33E+01 |
| Applied<br>Biosystems® 7500<br>Fast Dx | 4.35E+03 | 1.14E+04 | 2.44E+03 | 5.05E+02 | 3.35E+00 | 2.33E+01 |
| Cepheid<br>SmartCycler II | 5.44E+02 | 1.14E+04 | 2.44E+03 | 1.01E+03 | 8.38E-01 | 2.33E+01 |
The data from this LoD study support the claim that the performance of the assay is substantially equivalent on the three platforms (the LoD for each virus tested is within three doubling dilutions on the platforms).
#### f. Analytical Reactivity
A study was performed on the Applied Biosystems® 7500 Fast Dx to verify that the Lyra™ Direct HSV 1 + 2/VZV Assay detects multiple strains of HSV-1, HSV-2 and VZV at concentrations near the limit of detection.
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.
| HSV-1 Results | | | | | | | | |
|---------------|---------------------|------|----------|------|----------|------|-----------|------|
| Replicate # | MacIntyre (Control) | | QC 316 | | SFG-029 | | Isolate 7 | |
| | 8.70E+03 | | 8.70E+03 | | 4.84E+03 | | 8.70E+03 | |
| | HSV-1 | PRC | HSV-1 | PRC | HSV-1 | PRC | HSV-1 | PRC |
| 1 | 30.0 | 30.3 | 32.4 | 29.0 | 38.4 | 29.4 | 32.3 | 29.6 |
| 2 | 30.0 | 29.8 | 33.8 | 30.0 | 38.7 | 30.3 | 32.0 | 29.8 |
| 3 | 30.0 | 30.2 | 33.8 | 30.0 | 38.6 | 30.2 | 31.7 | 29.7 |
| Avg. Ct | 30.0 | 30.1 | 33.3 | 29.7 | 38.6 | 29.9 | 32.0 | 29.7 |
| SD | 0.0 | 0.3 | 0.8 | 0.6 | 0.2 | 0.5 | 0.3 | 0.1 |
| CV % | 0.0% | 0.9% | 2.4% | 1.9% | 0.4% | 1.7% | 1.0% | 0.4% |
| HSV-2 Results | | | | | | | | | | |
|---------------|-------------------------------------------------|----------|-----------|------|-----------|------|------------|------|-------|------|
| Replicate # | HSV-2 Strain and Test Concentration (TCID50/mL) | | | | | | | | | |
| | Strain G (control) | | Isolate 6 | | Isolate 9 | | Isolate 10 | | MS | |
| | | 4.88E+03 | HSV-2 | PRC | HSV-2 | PRC | HSV-2 | PRC | HSV-2 | PRC |
| 1 | 29.2 | 30.6 | 31.8 | 26.2 | 31.0 | 26.1 | 30.8 | 26.6 | 26.4 | 26.1 |
| 2 | 29.4 | 30.3 | 32.2 | 26.3 | 31.3 | 26.3 | 31.0 | 26.6 | 28.2 | 26.2 |
{12}------------------------------------------------
| HSV-2 Results | | | | | | | | | | |
|---------------|-------------------------------------------------|----------|-----------|----------|-----------|------|------------|------|-------|------|
| | HSV-2 Strain and Test Concentration (TCID50/mL) | | | | | | | | | |
| Replicate # | Strain G (control) | | Isolate 6 | | Isolate 9 | | Isolate 10 | | MS | |
| | 4.88E+03 | 4.88E+03 | 4.88E+03 | 4.88E+03 | 4.88E+03 | | | | | |
| | HSV-2 | PRC | HSV-2 | PRC | HSV-2 | PRC | HSV-2 | PRC | HSV-2 | PRC |
| 3 | 29.3 | 30.2 | 32.3 | 26.4 | 30.8 | 26.2 | 30.3 | 25.8 | 28.2 | 26.1 |
| Avg. Ct | 29.3 | 30.4 | 32.1 | 26.3 | 31.1 | 26.2 | 30.7 | 26.3 | 27.6 | 26.1 |
| SD | 0.1 | 0.2 | 0.3 | 0.1 | 0.2 | 0.1 | 0.3 | 0.4 | 1.0 | 0.1 |
| CV % | 0.4% | 0.7% | 0.8% | 0.4% | 0.7% | 0.3% | 1.1% | 1.6% | 3.6% | 0.2% |
| | VZV Results | | | | | | | | | | | | | |
|----------------|-----------------------------------------------|------|---------------------|------|-------------------|------|-----------------------|------|-----------------------|------|-----------------------|------|------------------------|------|
| | VZV Strain and Test Concentration (TCID50/mL) | | | | | | | | | | | | | |
| Replicate<br># | 130<br>(Control)<br>4.67E+01 | | AV-92-3<br>4.67E+01 | | Ellen<br>4.67E+01 | | Isolate B<br>4.67E+01 | | Isolate D<br>4.67E+01 | | Strain 82<br>4.67E+01 | | Strain 275<br>4.67E+01 | |
| | VZV | PRC | VZV | PRC | VZV | PRC | VZV | PRC | VZV | PRC | VZV | PRC | VZV | PRC |
| 1 | 30.2 | 32.1 | 29.0 | 26.5 | 27.0 | 26.1 | 29.8 | 26.5 | 26.5 | 26.4 | 28.4 | 26.9 | 27.6 | 26.3 |
| 2 | 30.8 | 30.7 | 29.8 | 26.2 | 26.9 | 26.3 | 27.4 | 26.7 | 27.7 | 26.5 | 27.9 | 26.4 | 28.0 | 26.5 |
| 3 | 30.8 | 30.5 | 29.5 | 26.1 | 26.9 | 26.3 | 29.2 | 26.3 | 27.4 | 26.8 | 28.5 | 26.7 | 27.8 | 26.4 |
| Avg. Ct | 30.6 | 31.1 | 29.4 | 26.3 | 26.9 | 26.2 | 28.8 | 26.5 | 27.2 | 26.6 | 28.3 | 26.7 | 27.8 | 26.4 |
| SD | 0.3 | 0.9 | 0.4 | 0.2 | 0.1 | 0.1 | 1.2 | 0.2 | 0.6 | 0.2 | 0.3 | 0.3 | 0.2 | 0.1 |
| 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% |
# g. Analytical specificity:
Cross-Reactivity and Inhibition by Other Microorganisms:
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. The study evaluated the potential cross reactivity with the assay (using negative samples) and the potential inhibition by other microorganisms as follows. Each potentially interfering or cross-reactive microorganism was tested in negative matrix (for crossreactivity) or in the presence of 2x LoD HSV-1, HSV-2 and VZV viruses (for inhibition). Clinically relevant levels of viruses and bacteria are typically 10°cfu/ml or higher for bacteria and 10 pfu/ml or higher for viruses.
| Analytical Specificity – Interfering or Cross-Reactive Microorganism | | | | | |
|----------------------------------------------------------------------|--------------------|---------------------------------------------|-------|-------|-----|
| Organism | Test Concentration | LyraTM Direct HSV 1 + 2/VZV<br>Assay Result | | | |
| | | Negative<br>Matrix | HSV-1 | HSV-2 | VZV |
| Adenovirus 7 | 9.38E+05 TCID50/mL | Neg | Pos | Pos | Pos |
| Coronavirus OC43 | 1.79E+05 TCID50/mL | Neg | Pos | Pos | Pos |
{13}------------------------------------------------
| Analytical Specificity – Interfering or Cross-Reactive Microorganism | | | | | | | | | |
|----------------------------------------------------------------------|--------------------|----------------------------|-------|-------|-----|--|--|--|--|
| | | Lyra™ Direct HSV 1 + 2/VZV | | | | | | | |
| | | Assay Result | | | | | | | |
| Organism | Test Concentration | Negative<br>Matrix | HSV-1 | HSV-2 | VZV | | | | |
| | | | | | | | | | |
| Coxsackievirus B4 | 2.60E+06 TCID50/mL | Neg | Pos | Pos | Pos | | | | |
| Cytomegalovirus Towne VR-<br>977 | 1.55E+05 TCID50/mL | Neg | Pos | Pos | Pos | | | | |
| Echovirus 11 | 1.61E+05 TCID50/mL | Neg | Pos | Pos | Pos | | | | |
| HHV-6 | 1.46E+06 TCID50/mL | Neg | Pos | Pos | Pos | | | | |
| HHV-7 | 8.63E+05 TCID50/mL | Neg | Pos | Pos | Pos | | | | |
| HIV Virus | 1.05E+05 TCID50/mL | Neg | Pos | Pos | Pos | | | | |
| hMPV A1 | 1.24E+05 TCID50/mL | Neg | Pos | Pos | Pos | | | | |
| HSV-1 SFG029 | 3.27E+05 TCID50/mL | Neg | Pos | Pos | Pos | | | | |
| HSV-1 Macintyre | 2.14E+05 TCID50/mL | Neg | Pos | Pos | Pos | | | | |
| HSV-2 MS | 6.44E+06 TCID50/mL | Neg | Pos | Pos | Pos | | | | |
| HSV-2 Strain G | 1.38E+05 TCID50/mL | Neg | Pos | Pos | Pos | | | | |
| Influenza<br>A/Mexico/4108/2009 | 2.17E+06 TCID50/mL | Neg | Pos | Pos | Pos | | | | |
| Influenza B Hong Kong VR-<br>791 | 7.15E+05 TCID50/mL | Neg | Pos | Pos | Pos | | | | |
| Measles virus | 1.46E+05 TCID50/mL | Neg | Pos | Pos | Pos | | | | |
| Mumps virus | 2.07E+07 TCID50/mL | Neg | Pos | Pos | Pos | | | | |
| Parainfluenza Type 1 | 4.02E+05 TCID50/mL | Neg | Pos | Pos | Pos | | | | |
| Parainfluenza Type 3 | 3.25E+05 TCID50/mL | Neg | Pos | Pos | Pos | | | | |
| Parainfluenza Type 4 | 1.28E+05 TCID50/mL | Neg | Pos | Pos | Pos | | | | |
| RSV A Long | 7.13E+05 TCID50/mL | Neg | Pos | Pos | Pos | | | | |
| RSV B Washington | 1.01E+06 TCID50/mL | Neg | Pos | Pos | Pos | | | | |
| Rubella Virus | 9.45E+05 TCID50/mL | Neg | Pos | Pos | Pos | | | | |
| Acholeplasma laidlawi | 5.33E+06 cfu/mL | Neg | Pos | Pos | Pos | | | | |
| Bordetella bronchiseptica | 8.78E+07 cfu/mL | Neg | Pos | Pos | Pos | | | | |
| Bordetella pertussis | 1.41E+07 cfu/mL | Neg | Pos | Pos | Pos | | | | |
| Clostridium perfringens | 1.18E+06 cfu/mL | Neg | Pos | Pos | Pos | | | | |
| Mycoplasma hominis | 9.75E+06 cfu/mL | Neg | Pos | Pos | Pos | | | | |
| Mycoplasma hyorhinis | 4.95E+06 cfu/mL | Neg | Pos | Pos | Pos | | | | |
| Mycoplasma orale | 1.16E+07 cfu/mL | Neg | Pos | Pos | Pos | | | | |
| Mycoplasma pneumoniae | 7.50E+06 ccu/mL | Neg | Pos | Pos | Pos | | | | |
| Mycoplasma salivarium | 1.25E+07 cfu/mL | Neg | Pos | Pos | Pos | | | | |
| Enterovirus 70 | 3.83E+04 TCID50/mL | Neg | Pos | Pos | Pos | | | | |
| Epstein Barr Virus | 1.67E+04 TCID50/mL | Neg | Pos | Pos | Pos | | | | |
| HBV | 1.67E+03 TCID50/mL | Neg | Pos | Pos | Pos | | | | |
| HCV | 1.67E+04 TCID50/mL | Neg | Pos | Pos | Pos | | | | |
| HHV-8 | 6.95E+04 TCID50/mL | Neg | Pos | Pos | Pos | | | | |
| HPV | Not Available | Neg | Pos | Pos | Pos | | | | |
| Parainfluenza Type 2 | 5.23E+04 TCID50/mL | Neg | Pos | Pos | Pos | | | | |
{14}------------------------------------------------
| Analytical Specificity – Interfering or Cross-Reactive Microorganism | | | | | |
|----------------------------------------------------------------------|----------------------------------|-------------------------------------------------------------------|----------|----------|----------|
| Lyra™ Direct HSV 1 + 2/VZV<br>Assay Result | | | | | |
| Organism | Test Concentration | Negative<br>Matrix | HSV-1 | HSV-2 | VZV |
| VZV Ellen | 8.75E+02 TCID50/mL | Neg | Pos | Pos | Pos |
| VZV Webster | 1.85E+03 TCID50/mL | Neg | Pos | Pos | Pos |
| Chlamydia trachomatis | 5.00E+05 cfu/mL | Neg | Pos | Pos | Pos |
| Chlamydophila pneumoniae | 2.67E+03 cp/mL | Neg | Pos | Pos | Pos |
| Toxoplasma gondii | 1.10E+06 tachyzoites/mL | Neg | Pos | Pos | Pos |
| Trichomonas vaginalis | 2.75E+05<br>trophozoites/mL | Neg | Pos | Pos | Pos |
| Acinetobacter calcoaceticus | 7.35E+08 cfu/mL | Neg | Pos | Pos | Pos |
| Bacteroides fragilis | 5.67E+05 cfu/mL | Neg | Pos | Pos | Pos |
| Candida albicans | 1.50E+06 cfu/mL | Neg | Pos | Pos | Pos |
| Candida glabrata | 1.50E+06 cfu/mL | Neg | Pos | Pos | Pos |
| Corynebacterium diphtheriae | 2.40E+08 cfu/mL | Neg | Pos | Pos | Pos |
| Enterococcus faecalis | 1.65E+08 cfu/mL | Neg | Pos | Pos | Pos |
| Escherichia coli | 4.35E+07 cfu/mL | Neg | Pos | Pos | Pos |
| Gardnerella vaginalis | 9.00E+06 cfu/mL | Neg | Pos | Pos | Pos |
| Haemophilis influenzae type A | 6.30E+08 cfu/mL | Neg | Pos | Pos | Pos |
| Klebsiella pneumoniae | 3.90E+07 cfu/mL | Neg | Pos | Pos | Pos |
| Lactobacillus acidophilus | 1.35E+06 cfu/mL | Neg | Pos | Pos | Pos |
| Legionella pneumophila | 2.85E+07 cfu/mL | Neg | Pos | Pos | Pos |
| Mobiluncus mulieris | 1.91E+08 cfu/mL | Neg | Pos | Pos | Pos |
| Moraxella cartarrhalis | 2.25E+07 cfu/mL | Neg | Pos | Pos | Pos |
| Neisseria gonorrhoeae | 2.40E+07 cfu/mL | Neg | Pos | Pos | Pos |
| Proteus mirabilis | 9.60E+07 cfu/mL | Neg | Pos | Pos | Pos |
| Pseudomonas aeruginosa | 5.25E+07 cfu/mL | Neg | Pos | Pos | Pos |
| Salmonella enteriditis | 4.05E+07 cfu/mL | Neg | Pos | Pos | Pos |
| Salmonella typhimurium | 3.45E+07 cfu/mL | Neg | Pos | Pos | Pos |
| Staphylococcus aureus | 1.38E+09 cfu/mL | Neg | Pos | Pos | Pos |
| Staphylococcus saprophyticus | 2.25E+08 cfu/mL | Neg | Pos | Pos | Pos |
| Streptococcus agalactiae | 1.65E+08 cfu/mL | Neg | Pos | Pos | Pos |
| Streptococcus pneumoniae | 2.10E+06 cfu/mL | Neg | Pos | Pos | Pos |
| Streptococcus pyogenes | 2.85E+08 cfu/mL | Neg | Pos | Pos | Pos |
| Ureaplasma uralyticum | Unable to titer | Neg | Pos | Pos | Pos |
| | | Analytical Specificity - Interfering or Cross-Reactive Substances | | | |
| | | Lyrat™ Direct HSV 1 + 2/VZV Assay Result | | | |
| Substance Name | Substance Final<br>Concentration | Negative<br>Matrix | HSV-1 | HSV-2 | VZV |
| Seminal Fluid | 7% | Negative | Positive | Positive | Positive |
| Cornstarch | 2.5 mg/mL | Negative | Positive | Positive | Positive |
| Acetamidophenol | 10 mg/mL | Negative | Positive | Positive | Positive |
| Feces | 0.219% | Negative | Positive | Positive | Positive |
| Chlorpheniramine | 5 mg/mL | Negative | Positive | Positive | Positive |
| Dextromethorphan | 5 mg/mL | Negative | Positive | Positive | Positive |
| Blood/EDTA | 0.625% | Negative | Positive | Positive | Positive |
| Female Urine | 7% | Negative | Positive | Positive | Positive |
| Male Urine | 7% | Negative | Positive | Positive | Positive |
| Acyclovir | 7 mg/mL | Negative | Positive | Positive | Positive |
| Albumin | 3.3 mg/mL | Negative | Positive | Positive | Positive |
| Casein | 7 mg/mL | Negative | Positive | Positive | Positive |
| KY Jelly | 7% | Negative | Positive | Positive | Positive |
| Douche | 7% | Negative | Positive | Positive | Positive |
| Miconazole 1 | 7% | Negative | Positive | Positive | Positive |
| Miconazole 3 | 7% | Negative | Positive | Positive | Positive |
| Tioconazole 1 | Approx. 7% | Negative | Positive | Positive | Positive |
| Preparation H | 7% | Negative | Positive | Positive | Positive |
| Lanacane | 3.5% | Negative | Positive | Positive | Positive |
| Listerine | 7% | Negative | Positive | Positive | Positive |
| Abreva | 7% | Negative | Positive | Positive | Positive |
| Carmex | 7% | Negative | Positive | Positive | Positive |
| Releev | 7% | Negative | Positive | Positive | Positive |
| Colgate | 7% | Negative | Positive | Positive | Positive |
| Mucin | 60 µg/mL | Negative | Positive | Positive | Positive |
| Leukocytes | 2.5e5 cells/mL | Negative | Positive | Positive | Positive |
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 crossreactivity of this organism with 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™ Directassay conditions. Therefore, the presence of the
{15}------------------------------------------------
organism should not interfere with the assay.
None of the seventy (70) microorganisms listed above cross-reacted with the the assay or interfered with the ability of the assay to detect 2x LoD HSV1, HSV2 or VZV.
Interfering 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 twentysix (26) clinically relevant levels of potentially interfering substances that might be present in lesion specimens.
None of the twenty-six (26) potentially interfering substances interfered with the
{16}------------------------------------------------
detection of 2x LoD HSV-1, HSV-2, or VZV, or were cross-reactive with the Lyra™ Direct HSV 1 + 2/VZV Assay.
### Competitive Interference:
To evaluate whether competitive interference exists in the Lyra™ Direct HSV 1 + 2/VZV Assay when HSV-1, HSV-2 and VZV are present in the same reaction a study was performed on all three instruments. HSV-1, HSV-2 and VZV stocks at 2x LoD concentrations were tested in the presence of varying amounts of another analyte in order to determine if competitive interference exists.
No competitive interference was observed on any instrument with 2x LoD concentrations of HSV-1, HSV-2 and VZV when multiple two-analyte combinations of HSV-1, HSV-2 and VZV were tested in the same reaction with 2x, 10x, 100x, 1000x, and 10,000x LoD concentrations.
| Sample Number | Combined Analytes | HSV1 | HSV2 | VZV | PRC | Interference |
|------------------|-------------------------|------|------|------|------|--------------|
| Control 2x HSV-1 | N/A | 29.8 | NEG | NEG | 25.8 | N/A |
| 1 | 2x HSV-1<br>2x HSV-2 | 29.2 | 30.8 | NEG | 23.5 | NO |
| 2 | 2x HSV-1<br>10x HSV-2 | 29.7 | 28.8 | NEG | 22.6 | NO |
| 3 | 2x HSV-1<br>100x HSV-2 | 29.3 | 26.2 | NEG | 22.8 | NO |
| 4 | 2x HSV-1<br>2x VZV | 29.2 | NEG | 29.0 | 23.1 | NO |
| 5 | 2x HSV-1<br>10x VZV | 29.9 | NEG | 26.7 | 24.2 | NO |
| 6 | 2x HSV-1<br>100x VZV | 29.6 | NEG | 23.4 | 23.6 | NO |
| 16 | 2x HSV-1<br>1000x VZV | 29.8 | NEG | 20.3 | 22.7 | NO |
| 17 | 2x HSV-1<br>10,000x VZV | 30.6 | NEG | 18.3 | 21.6 | NO |
| HSV-2: 2x Results Summary: Competitive Interference | | | | | | | |
|-----------------------------------------------------|----------------------|------|------|------|------|--------------|--|
| Sample Number | Combined Analytes | HSV1 | HSV2 | VZV | PRC | Interference | |
| Control 2x HSV-2 | N/A | NEG | 32.1 | NEG | 25.6 | N/A | |
| 1 | 2x HSV-2 2x HSV-1 | 29.2 | 30.8 | NEG | 23.5 | NO | |
| 7 | 2x HSV-2 10x HSV-1 | 27.6 | 32.1 | NEG | 23.8 | NO | |
| 8 | 2x HSV-2 100x HSV-1 | 23.4 | 32.4 | NEG | 23.4 | NO | |
| 9 | 2x HSV-2 2x VZV | NEG | 31.6 | 29.5 | 24.3 | NO | |
| 10 | 2x HSV-2 10x VZV | NEG | 33.0 | 26.9 | 23.7 | NO | |
| 11 | 2x HSV-2 100x VZV | NEG | 33.6 | 23.7 | 25.1 | NO | |
| 18 | 2x HSV-2 1000x VZV | NEG | 29.8 | 20.5 | 22.3 | NO | |
| 19 | 2x HSV-2 10,000x VZV | NEG | 29.1 | 18.4 | 22.7 | NO | |
| VZV: 2x Results Summary: Competitive Interference | | | | | | |
|---------------------------------------------------|-------------------|------|------|-----|-----|--------------|
| Sample Number | Combined Analytes | HSV1 | HSV2 | VZV | PRC | Interference |
{17}------------------------------------------------
| Control 2x VZV | | N/A | NEG | NEG | 29.0 | 26.0 | N/A |
|----------------|--------|------------|------|------|------|------|-----|
| 4 | 2x VZV | 2x HSV-1 | 29.2 | NEG | 29.0 | 23.1 | NO |
| 9 | 2x VZV | 2x HSV-2 | NEG | 31.6 | 29.5 | 24.3 | NO |
| 12 | 2x VZV | 10x HSV-1 | 27.5 | NEG | 29.2 | 24.2 | NO |
| 13 | 2x VZV | 100x HSV-1 | 24.1 | NEG | 29.4 | 22.3 | NO |
| 14 | 2x VZV | 10x HSV-2 | NEG | 29.4 | 29.1 | 24.6 | NO |
| 15 | 2x VZV | 100x HSV-2 | NEG | 25.9 | 29.0 | 22.9 | NO |
| HSV-1: 2x Results Summary: Competitive Interference (Avg C₁) | | | | | | |
|--------------------------------------------------------------|----------------------|------|------|------|------|--------------|
| Sample Number | Combined Analytes | HSV1 | HSV2 | VZV | PRC | Interference |
| Control 2x HSV-1 | N/A | 31.0 | NEG | NEG | 26.2 | N/A |
| 1 | 2x HSV-1 2x HSV-2 | 29.9 | 31.4 | NEG | 28.8 | NO |
| 2 | 2x HSV-1 10x HSV-2 | 30.8 | 31.1 | NEG | 27.0 | NO |
| 3 | 2x HSV-1 100x HSV-2 | 30.7 | 28.1 | NEG | 30.0 | NO |
| 4 | 2x HSV-1 2x VZV | 30.3 | NEG | 30.2 | 29.0 | NO |
| 5 | 2x HSV-1 10x VZV | 29.6 | NEG | 28.1 | 26.9 | NO |
| 6 | 2x HSV-1 100x VZV | 31.2 | NEG | 25.2 | 28.2 | NO |
| 16 | 2x HSV-1 1000x VZV | 29.8 | NEG | 21.7 | 30.1 | NO |
| 17 | 2x HSV-1 10,000x VZV | 31.0 | NEG | 19.4 | 30.4 | NO |
| HSV-2: 2x Results Summary: Competitive Interference (Avg C₁) | | | | | | | |
|--------------------------------------------------------------|-------------------------|------|------|------|------|--------------|--|
| Sample Number | Combined Analytes | HSV1 | HSV2 | VZV | PRC | Interference | |
| Control 2x HSV-2 | N/A | NEG | 32.4 | NEG | 25.6 | N/A | |
| 1 | 2x HSV-2<br>2x HSV-1 | 29.9 | 31.4 | NEG | 28.8 | NO | |
| 7 | 2x HSV-2<br>10x HSV-1 | 27.4 | 30.4 | NEG | 28.9 | NO | |
| 8 | 2x HSV-2<br>100x HSV-1 | 24.0 | 33.5 | NEG | 27.4 | NO | |
| 9 | 2x HSV-2<br>2x VZV | NEG | 31.1 | 30.4 | 28.9 | NO | |
| 10 | 2x HSV-2<br>10x VZV | NEG | 31.5 | 28.0 | 29.3 | NO | |
| 11 | 2x HSV-2<br>100x VZV | NEG | 33.0 | 25.0 | 27.2 | NO | |
| 18 | 2x HSV-2<br>1000x VZV | NEG | 29.3 | 21.6 | 30.2 | NO | |
| 19 | 2x HSV-2<br>10,000x VZV | NEG | 28.8 | 19.3 | 30.3 | NO | |
| VZV: 2x Results Summary: Competitive Interference (Avg Cₜ) | | | | | | |
|------------------------------------------------------------|-------------------|------|------|------|------|--------------|
| Sample Number | Combined Analytes | HSV1 | HSV2 | VZV | PRC | Interference |
| Control 2x VZV | N/A | NEG | NEG | 31.1 | 26.3 | N/A |
| 4 | 2x VZV 2x HSV-1 | 30.3 | NEG | 30.2 | 29.0 | NO |
| 9 | 2x VZV 2x HSV-2 | NEG | 31.1 | 30.4 | 28.9 | NO |
| 12 | 2x VZV 10x HSV-1 | 26.3 | NEG | 31.6 | 28.7 | NO |
| 13 | 2x VZV 100x HSV-1 | 24.4 | NEG | 30.4 | 28.1 | NO |
{18}------------------------------------------------
| 14 | 2x VZV | 10x HSV-2 | NEG | 28.7 | 30.2 | 27.1 | NO |
|----|--------|------------|-----|------|------|------|----|
| 15 | 2x VZV | 100x HSV-2 | NEG | 26.5 | 30.5 | 27.1 | NO |
| HSV-1: 2x Results Summary: Competitive Interference | | | | | | |
|-----------------------------------------------------|-------------------------|------|------|------|------|--------------|
| Sample Number | Combined Analytes | HSV1 | HSV2 | VZV | PRC | Interference |
| Control 2x HSV-1 | N/A | 33.1 | NEG | NEG | 28.8 | N/A |
| 1 | 2x HSV-1<br>2x HSV-2 | 32.4 | 36.6 | NEG | 28.4 | NO |
| 2 | 2x HSV-1<br>10x HSV-2 | 33.0 | 32.8 | NEG | 28.8 | NO |
| 3 | 2x HSV-1<br>100x HSV-2 | 32.9 | 29.2 | NEG | 28.5 | NO |
| 4 | 2x HSV-1<br>2x VZV | 33.1 | NEG | 31.7 | 28.8 | NO |
| 5 | 2x HSV-1<br>10x VZV | 33.6 | NEG | 28.7 | 28.7 | NO |
| 6 | 2x HSV-1<br>100x VZV | 32.6 | NEG | 25.4 | 28.4 | NO |
| 16 | 2x HSV-1<br>1000x VZV | 32.6 | NEG | 23.0 | 31.5 | NO |
| 17 | 2x HSV-1<br>10,000x VZV | 34.4 | NEG | 20.4 | 31.9 | NO |
| HSV-2: 2x Results Summary: Competitive Interference | | | | | | | |
|-----------------------------------------------------|----------------------|------|------|------|------|--------------|--|
| Sample Number | Combined Analytes | HSV1 | HSV2 | VZV | PRC | Interference | |
| Control 2x HSV-2 | N/A | NEG | 36.2 | NEG | 28.7 | N/A | |
| 1 | 2x HSV-1 2x HSV-2 | 32.4 | 36.6 | NEG | 28.4 | NO | |
| 7 | 2x HSV-2 10x HSV-1 | 28.8 | 35.3 | NEG | 28.5 | NO | |
| 8 | 2x HSV-2 100x HSV-1 | 27.3 | 35.8 | NEG | 28.4 | NO | |
| 9 | 2x HSV-2 2x VZV | NEG | 34.9 | 31.0 | 28.9 | NO | |
| 10 | 2x HSV-2 10x VZV | NEG | 35.4 | 28.7 | 28.2 | NO | |
| 11 | 2x HSV-2 100x VZV | NEG | 35.6 | 25.9 | 28.4 | NO | |
| 18 | 2x HSV-2 1000x VZV | NEG | 32.8 | 23.0 | 31.2 | NO | |
| 19 | 2x HSV-2 10,000x VZV | NEG | 32.8 | 20.4 | 31.8 | NO | |
| 20 | 2x HSV-2 1000x HSV-1 | 23.3 | 33.1 | NEG | 30.3 | NO | |
| VZV: 2x Results Summary: Competitive Interference | | | | | | | |
|---------------------------------------------------|-------------------|-------------|------|------|------|------|--------------|
| Sample Number | Combined Analytes |…
