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K Number
DEN140004
Device Name
QUIDEL MOLECULAR DIRECT HSV 1 +2/VZV ASSAY
Manufacturer
DIAGNOSTIC HYBRIDS, INC.
Date Cleared
2014-05-13

(81 days)

Product Code
PGI
Regulation Number
866.3309
Type
Post-NSE
Panel
Microbiology
Reference & Predicate Devices
N/A
Predicate For
N/A
AI/MLSaMDIVD (In Vitro Diagnostic)TherapeuticDiagnosticis PCCP AuthorizedThirdpartyExpeditedreview
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 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.

AI/ML Overview

Here's an analysis of the acceptance criteria and study details for the Lyra™ Direct HSV 1 + 2/VZV Assay, formatted as requested:

Acceptance Criteria and Device Performance for Lyra™ Direct HSV 1 + 2/VZV Assay

The Lyra™ Direct HSV 1 + 2/VZV Assay is an in vitro multiplex Real-Time PCR test for qualitative detection and differentiation of HSV-1, HSV-2, and VZV DNA from cutaneous or mucocutaneous lesion samples. The regulatory classification is Class II with special controls (21 CFR 866.3309).

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state pre-defined acceptance criteria with numerical thresholds in a single table. Instead, performance is presented through various analytical and clinical studies. We will infer the de facto acceptance criteria based on the demonstrated performance that led to the device's classification. For clinical performance, the comparable effectiveness is evaluated against established methods (ELVIS® HSV ID and D3 Typing Test for HSV and DSFA and Culture with DFA for VZV). Successful performance in these comparisons implies the acceptance criteria were met.

Inferred Acceptance Criteria Table and Reported Device Performance:

Performance MetricTarget AnalyteAcceptance Criteria (Inferred from successful study)Reported Device Performance (Life Technologies QuantStudio™ Dx)Reported Device Performance (Applied Biosystems® 7500 Fast Dx)Reported Device Performance (Cepheid SmartCycler® II)
CLINICAL PERFORMANCE (Cutaneous Lesions)
SensitivityHSV-1High, approaching 100%100% (24/24)100% (24/24)100% (24/24)
SpecificityHSV-1High, >95%98.4% (250/254)98.8% (252/254)98.8% (251/254)
SensitivityHSV-2High, approaching 100%100% (35/35)97.1% (34/35)100% (35/35)
SpecificityHSV-2High, >95%96.3% (234/243)96.7% (236/244)96.7% (235/243)
SensitivityVZVHigh, approaching 100%100% (27/27)100% (26/27)100% (27/27)
SpecificityVZVHigh, >90%95.9% (187/195)95.9% (189/196)94.9% (185/195)
CLINICAL PERFORMANCE (Mucocutaneous Lesions)
SensitivityHSV-1High, >95%97.1% (100/103)95.1% (98/103)98.1% (101/103)
SpecificityHSV-1High, >95%97.1% (527/543)98.2% (531/541)97.2% (525/540)
SensitivityHSV-2High, approaching 100%100% (95/95)97.9% (93/95)98.9% (94/95)
SpecificityHSV-2High, >95%96.2% (530/551)97.1% (533/549)97.1% (532/548)
SensitivityVZVHigh, approaching 100%100% (4/4)100% (4/4)100% (4/4)
SpecificityVZVHigh, >97%98.8% (423/428)99.3% (423/426)98.8% (420/425)
ANALYTICAL PERFORMANCE
Reproducibility (Detection Rate)Low Positive100% for all analytes90/90 (HSV-1), 90/90 (HSV-2), 89/90 (VZV)90/90 (HSV-1), 90/90 (HSV-2), 88/90 (VZV)90/90 (HSV-1), 89/90 (HSV-2), 88/90 (VZV)
Reproducibility (Negative Samples)Negative0% Detection0/90 (all analytes)0/90 (all analytes)0/90 (all analytes)
Limit of Detection (LoD)All AnalytesLoD within 3 doubling dilutions across platformsAchieved for various strains (see section 1.e for details)Achieved for various strains (see section 1.e for details)Achieved for various strains (see section 1.e for details)
Analytical ReactivityMultiple strainsDetection at near LoD concentrations (100% positivity for all tested strains at specified concentrations)All 16 tested strains were detectedAll 16 tested strains were detectedAll 16 tested strains were detected
Analytical Specificity (Cross-Reactivity/Inhibition)Diverse Microorganisms, Endogenous SubstancesNo cross-reactivity with negative samples, no inhibition of positive samplesNo cross-reactivity observed, no inhibition observedNo cross-reactivity observed, no inhibition observedNo cross-reactivity observed, no inhibition observed
Competitive InterferenceMultiple analytes in same sampleNo interference when multiple analytes present at varying concentrationsNo competitive interference observedNo competitive interference observedNo competitive interference observed
Carry-over ContaminationPCR productsNo cross-contamination from high positives to negative samplesNo cross-contamination (0/48 tested negatives positive)No cross-contamination (0/48 tested negatives positive)No cross-contamination (0/48 tested negatives positive)

Study Proving Acceptance Criteria Met:

The device's performance characteristics, including both analytical and clinical studies, were conducted to demonstrate it meets the requirements for a Class II designation with special controls.

2. Sample Sizes Used for the Test Set and Data Provenance

Test Set Sample Sizes (Clinical Study):

The clinical study was a multi-center study conducted between April 2013 and October 2013.

  • Life Technologies QuantStudio™ Dx:
    • Cutaneous Lesions: 279 specimens initially collected (278 for HSV-1/HSV-2, 222 for VZV after exclusions).
    • Mucocutaneous Lesions: 650 specimens initially collected (646 for HSV-1/HSV-2, 432 for VZV after exclusions).
  • Applied Biosystems® 7500 Fast Dx:
    • Cutaneous Lesions: 279 specimens (279 for HSV-1/HSV-2, 223 for VZV after exclusions).
    • Mucocutaneous Lesions: 650 specimens initially collected (644 for HSV-1/HSV-2, 430 for VZV after exclusions).
  • Cepheid SmartCycler® II:
    • Cutaneous Lesions: 279 specimens initially collected (278 for HSV-1/HSV-2, 222 for VZV after exclusions).
    • Mucocutaneous Lesions: 650 specimens initially collected (643 for HSV-1/HSV-2, 429 for VZV after exclusions).

Data Provenance:

  • Country of Origin: Not explicitly stated, but the submission is to the FDA, suggesting United States given the typical regulatory context for De Novo submissions. The multi-center nature implies data was collected from different clinical sites within the same regulatory jurisdiction.
  • Retrospective or Prospective: The clinical study description states: "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." This clearly indicates a prospective collection of specimens for the purpose of the study.

3. Number of Experts Used to Establish Ground Truth for the Test Set and Their Qualifications

The ground truth for the clinical test set was established using FDA-cleared comparator methods, not directly by human experts in the sense of independent adjudication.

  • For HSV-1 and HSV-2, the comparator method was the ELVIS® HSV ID and D3 Typing Test, an FDA-cleared cell culture system.
  • For VZV, the comparator method involved staining cells present in the samples with an FDA-cleared VZV detection reagent (DSFA) and culturing the specimen using a mixed cell culture (H&V mixed cells) followed by staining with the same FDA-cleared reagent used for DSFA.

Therefore, there were no direct human experts establishing ground truth in the typical sense of radiologists or pathologists. The ground truth was based on the results of established, FDA-cleared laboratory methods, operated by trained laboratory personnel.

4. Adjudication Method for the Test Set

The primary adjudication method for discrepancies between the Lyra™ Direct Assay and the comparator methods was the use of an additional RT-PCR assay.

  • For discrepancies in HSV-1 (e.g., Lyra™ positive, comparator negative), these cases were "positive by an additional RT-PCR assay."
  • Similarly for HSV-2 and VZV discrepancies, "positives were positive by an additional RT-PCR assay."
  • In some cases where Lyra™ was negative but the comparator was positive, these were also re-evaluated by an additional RT-PCR assay, with the statement "negatives were positive by an additional RT-PCR assay."

This acts as a tie-breaker or confirmatory test for discordant results, enhancing the robustness of the ground truth derived from the comparator methods.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done, If So, What Was the Effect Size of How Much Human Readers Improve with AI vs without AI Assistance

No, an MRMC comparative effectiveness study was not done. This device is an in vitro diagnostic (IVD) PCR assay, not an AI/CADe medical imaging device that assists human readers. Its performance is evaluated compared to established laboratory methods, not human interpretation of images. Therefore, the concept of "human readers improve with AI vs without AI assistance" is not applicable here.

6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) Was Done

Yes, the primary clinical study represents standalone performance. The Lyra™ Direct HSV 1 + 2/VZV Assay is an automated real-time PCR test. Its results (positive/negative for each virus) are generated directly by the instrument and its software, without human interpretation of raw signals to determine the final viral status. The clinical sensitivity and specificity reported are the performance of the algorithm/device alone compared to the ground truth.

7. The Type of Ground Truth Used (Expert Consensus, Pathology, Outcomes Data, etc.)

The ground truth used for the clinical study was based on FDA-cleared conventional laboratory methods:

  • Cell Culture with Immunofluorescence Staining (or similar detection):
    • HSV-1 and HSV-2: ELVIS® HSV ID and D3 Typing Test.
    • VZV: Direct Smear Fluorescent Antibody (DSFA) and mixed cell culture with DFA staining.
  • Confirmatory RT-PCR/molecular assay: Used as an adjudication method for discordant results between the Lyra™ Direct Assay and the primary comparator methods.

8. The Sample Size for the Training Set

The document does not explicitly describe a separate "training set" for the clinical evaluation in the way machine learning algorithms typically use them. For IVD devices like this, the "development" or "training" process involves optimizing the assay components (primers, probes, reaction conditions) and setting analytical cut-offs based on analytical studies (e.g., Limit of Detection, reactivity, specificity studies) using characterized samples.

The reproducibility and precision studies used simulated samples (medium positive, low positive, high negative, negative, 5x LoD, 2x LoD, <0.25x LoD) but these are for analytical validation rather than "training" a machine learning model.

The document implicitly refers to extensive internal development and optimization by the manufacturer (Quidel Corporation) which would involve internal data, but this data is not detailed as a "training set" in the context of the regulatory submission.

9. How the Ground Truth for the Training Set Was Established

Given the absence of a distinct "training set" described as such for a machine learning model, the concept of "ground truth for the training set" does not directly apply.

However, the analytical characteristics of the assay, which would be refined during development, were established using:

  • Quantified viral stocks (TCID50/mL): Used to determine the Limit of Detection (LoD).
  • Stocks of various HSV-1, HSV-2, and VZV strains: Used for analytical reactivity.
  • Various microorganisms and interfering substances: Used for analytical specificity and interference studies.

These samples, with their known viral content and concentrations, served as the "ground truth" for the analytical development and validation of the assay's performance parameters.

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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:
    1. Classification: Class II (special controls)
    1. Product code: PGI
    1. 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.

    1. Indication(s) for use:
      Same as intended use
    1. Special conditions for use statement(s):
      For prescription use only in accordance with 21 CFR 801.109.
    1. 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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TargetDye
HSV-1FAM
HSV-2CAL Fluor® Orange 560
VZVCAL Fluor® Red 610
PRCQuasar® 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:

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).
    1. CLSI MM13-A: Guidance for the Collection, Transport, Preparation and Storage of Specimens for Molecular Methods (Vol. 25. No. 31) (Dec 2005).
    1. CLSI EP7-A2: Guidance for Interference Testing in Clinical Chemistry (Vol. 25, No.27 Second Ed) (Nov 2005).
    1. CLSI EP12-A: Guidance for User Protocol for Evaluation of Qualitative Test Performance (Vol. 22, No. 14) (Sept 2002).
    1. CLSI MM6-A: Guidance for the Quantitative Molecular Methods for Infectious Diseases (Vol. 23, No.28) (Oct 2003).
    1. 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
PanelSite 1Site 2Site 3Combined Site Data
MemberIDRate ofDetectionAVECt%CVRate ofDetectionAVECt%CVRate ofDetectionAVECt%CVRate ofDetectionAVECt%CV
HSV-1HighNegative10/3037.04.916/3034.58.917/3034.07.643/9034.88.2
HSV-1LowPositive30/3029.81.630/3029.02.630/3029.53.690/9029.42.9
HSV-1ModeratePositive30/3028.31.930/3027.32.430/3027.93.790/9027.83.1
HSV-1Negative0/30N/AN/A0/29*N/AN/A0/30N/AN/A0/89*N/AN/A
HSV-2HighNegative30/3034.83.030/3031.94.728/3034.16.288/9033.66.0
HSV-2LowPositive30/3032.81.430/3032.02.330/3031.83.690/9032.22.9
HSV-2ModeratePositive30/3030.83.830/3029.82.830/3030.84.290/9030.53.9
HSV-2Negative0/30N/AN/A0/29*N/AN/A0/30N/AN/A0/89*N/AN/A
VZV HighNegative9/3037.44.210/3035.26.45/3034.46.624/9035.96.5
VZV LowPositive30/3029.21.730/3028.71.229/3029.95.589/9029.33.7
VZVModeratePositive30/3027.61.330/3027.21.030/3027.72.690/9027.52.9
VZVNegative0/30N/AN/A0/29*N/AN/A0/30N/AN/A0/89*N/AN/A
HSV-1PositiveControl30/3027.42.630/3025.10.930/3026.82.790/9026.47.0
HSV-2PositiveControl30/3026.76.630/3029.81.530/3025.43.790/9022.87.9
VZVPositiveControl30/3022.113.430/3020.00.830/3021.14.590/9021.19.2
NegativeControl0/30N/AN/A0/30N/AN/A0/30N/AN/A0/90N/AN/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
PanelMemberIDRate ofDetectionAVECt%CVRate ofDetectionAVECt%CVRate ofDetectionAVECt%CVRate ofDetectionAVECt%CV
HSV-1HighNegative9/3036.23.69/3035.010.112/2936.56.030/8936.17.6
HSV-1LowPositive30/3030.31.830/3030.33.430/3030.22.690/9030.32.7
HSV-1ModeratePositive30/3028.82.430/3028.52.630/3029.58.190/9028.95.4
HSV-1Negative0/30N/AN/A0/30N/AN/A0/30N/AN/A0/90N/AN/A
HSV-2HighNegative30/3035.43.730/3034.25.824/3036.06.084/9035.25.5
HSV-2LowPositive30/3032.84.230/3033.63.830/3032.75.990/9033.04.8
HSV-2ModeratePositive30/3031.22.130/3031.32.930/3031.94.390/9031.53.4
HSV-2Negative0/30N/AN/A0/30N/AN/A0/30N/AN/A0/90N/AN/A
VZV HighNegative0/30N/AN/A3/3033.86.20/30N/AN/A3/9033.86.2
VZV LowPositive29/3031.54.429/3031.54.630/3032.16.288/9031.75.2
VZVModeratePositive30/3029.42.530/3029.22.130/3030.99.190/9029.86.2
VZVNegative0/30N/AN/A0/30N/AN/A0/30N/AN/A0/90N/AN/A
HSV-1PositiveControl30/3027.111.630/3025.91.330/3026.22.290/9026.47.2
HSV-2PositiveControl30/3026.18.130/3024.91.230/3025.72.090/9025.65.2
VZVPositiveControl30/3023.613.230/3021.81.130/3022.11.790/9022.58.6
NegativeControl0/30N/AN/A0/30N/AN/A0/30N/AN/A0/90N/AN/A

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Reproducibility Results - Cepheid SmartCycler® II
PanelSite 1Site 2Site 3Combined Site Data
MemberIDRate ofDetectionAVECt%CVRate ofDetectionAVECt%CVRate ofDetectionAVECt%CVRate ofDetectionAVECt%CV
HSV-1HighNegative5/3037.81.95/3033.47.513/3035.32.423/3035.46.0
HSV-1LowPositive30/3031.31.330/3031.04.530/3031.52.490/9031.33.1
HSV-1ModeratePositive29/29*29.42.930/3028.94.230/3030.02.489/89*29.53.6
HSV-1Negative0/30N/AN/A0/30N/AN/A0/30N/AN/A0/90N/AN/A
HSV-2HighNegative24/3038.55.127/3036.66.517/3037.34.468/9037.45.8
HSV-2LowPositive30/3036.23.329/3035.82.530/3035.84.989/9036.03.7
HSV-2ModeratePositive29/29*33.61.630/3033.34.630/3033.83.489/89*33.62.5
HSV-2Negative0/30N/AN/A0/30N/AN/A0/30N/AN/A0/90N/AN/A
VZV HighNegative0/30N/AN/A1/30N/AN/A0/30N/AN/A1/90N/AN/A
VZV LowPositive29/3033.47.029/3032.72.830/3036.210.788/9034.18.9
VZVModeratePositive29/29*30.92.330/3030.61.130/3033.48.589/89*31.66.7
VZVNegative0/30N/AN/A0/30N/AN/A0/30N/AN/A0/90N/AN/A
HSV-1PositiveControl30/3027.68.630/3026.41.830/3027.42.290/9027.25.6
HSV-2PositiveControl30/3028.07.030/3026.51.830/3028.19.390/9027.57.3
VZVPositiveControl30/3024.712.130/3022.91.130/3023.71.990/9023.87.9
NegativeControl0/30N/AN/A0/30N/AN/A0/30N/AN/A0/90N/AN/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
TargetPositiveControl5X LoD2X LoD<0.25X LoDNegativeMatrix
Op 1ª Avg Ct25.027.729.436.1Neg
HSV-1Op 2° Avg Ct25.427.628.936.2Neg
Positivity (%)100100100રી0
Op 1 Avg Ct26.328.329.037.2Neg
HSV-2Op 2 Avg Ct26.328.229.037.6Neg
Positivity (%)100100100630
VZVOp 1 Avg Ct18.827.829.334.3Neg
Op 2 Avg Ct18.727.929.234.7Neg
Positivity (%)100100100રી0

ª Operator 1; b Operator 2

Applied Biosystems® 7500 Fast Dx Results Summary
TargetPositive Control5X LoD2X LoD≤0.25X LoDNegative Matrix
HSV-1Op 1 Avg Ct26.228.329.938.6Neg
Op 2 Avg Ct25.927.529.037.4Neg
Positivity (%)100100100320
HSV-2Op 1 Avg Ct25.429.130.137.2Neg
Op 2 Avg Ct25.828.429.636.8Neg
Positivity (%)100100100750
VZVOp 1 Avg Ct19.629.033.136.9Neg
Op 2 Avg Ct19.529.130.736.7Neg
Positivity (%)10010099250
Cepheid SmartCycler® II Results Summary
TargetPositiveControl5X LoD2X LoD≤0.25X LoDNegativeMatrix
HSV-1Op 1 Avg Ct25.630.832.837.7Neg
Op 2 Avg Ct25.530.932.636.4Neg
Positivity (%)100100100380
HSV-2Op 1 Avg Ct27.232.433.537.9Neg
Op 2 Avg Ct27.632.233.437.7Neg
Positivity (%)100100100650
VZVOp 1 Avg Ct21.030.531.939.5Neg
Op 2 Avg Ct21.030.732.540.2Neg
Positivity (%)100100100540

{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
TargetStorage Temperature
RT2-8°C-20°C
HSV-14hrs24hrs76 hrs
HSV-24hrs24hrs76 hrs
VZV1.5hrs24hrs76 hrs
Stability for RehydratedMaster Mix
Storage Temperature and Stability Claim
RT2-8°C-20°C
1 hr9 hrs72 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 andRoom Temp2° to 8°C-20°C-70°C
HSV-13211212
HSV-23211212
VZV3211212
Negative1744

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℃.

ControlCtValue(Ave.)Ct Average20° to 25°C(50 hours)Ct ShiftCt Average2° to 8°C(50 hours)CtShiftCt Average-20°C(32 days)Ct ShiftCt Average-20°C(32 days)Ct Shift
HSV-130.531.7+ 1.230.5028.9- 1.633.7+3.2
HSV-233.930.3- 3.635.7+1.834.6+0.734.8+0.9
VZV30.530.7+0.230.6+0.130.3-0.230.8+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
InstrumentVirus TCID50/mL
HSV-1MacintyreHSV-1QC 316HSV-2Strain GHSV-2QC CompVZVEllenVZV 130
Life TechnologyQuantStudio Dx2.18E+035.69E+032.44E+032.53E+021.68E+002.33E+01
AppliedBiosystems® 7500Fast Dx4.35E+031.14E+042.44E+035.05E+023.35E+002.33E+01
CepheidSmartCycler II5.44E+021.14E+042.44E+031.01E+038.38E-012.33E+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 316SFG-029Isolate 7
8.70E+038.70E+034.84E+038.70E+03
HSV-1PRCHSV-1PRCHSV-1PRCHSV-1PRC
130.030.332.429.038.429.432.329.6
230.029.833.830.038.730.332.029.8
330.030.233.830.038.630.231.729.7
Avg. Ct30.030.133.329.738.629.932.029.7
SD0.00.30.80.60.20.50.30.1
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 6Isolate 9Isolate 10MS
4.88E+03HSV-2PRCHSV-2PRCHSV-2PRCHSV-2PRC
129.230.631.826.231.026.130.826.626.426.1
229.430.332.226.331.326.331.026.628.226.2

{12}------------------------------------------------

HSV-2 Results
HSV-2 Strain and Test Concentration (TCID50/mL)
Replicate #Strain G (control)Isolate 6Isolate 9Isolate 10MS
4.88E+034.88E+034.88E+034.88E+034.88E+03
HSV-2PRCHSV-2PRCHSV-2PRCHSV-2PRCHSV-2PRC
329.330.232.326.430.826.230.325.828.226.1
Avg. Ct29.330.432.126.331.126.230.726.327.626.1
SD0.10.20.30.10.20.10.30.41.00.1
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#130(Control)4.67E+01AV-92-34.67E+01Ellen4.67E+01Isolate B4.67E+01Isolate D4.67E+01Strain 824.67E+01Strain 2754.67E+01
VZVPRCVZVPRCVZVPRCVZVPRCVZVPRCVZVPRCVZVPRC
130.232.129.026.527.026.129.826.526.526.428.426.927.626.3
230.830.729.826.226.926.327.426.727.726.527.926.428.026.5
330.830.529.526.126.926.329.226.327.426.828.526.727.826.4
Avg. Ct30.631.129.426.326.926.228.826.527.226.628.326.727.826.4
SD0.30.90.40.20.10.11.20.20.60.20.30.30.20.1
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
OrganismTest ConcentrationLyraTM Direct HSV 1 + 2/VZVAssay Result
NegativeMatrixHSV-1HSV-2VZV
Adenovirus 79.38E+05 TCID50/mLNegPosPosPos
Coronavirus OC431.79E+05 TCID50/mLNegPosPosPos

{13}------------------------------------------------

Analytical Specificity – Interfering or Cross-Reactive Microorganism
Lyra™ Direct HSV 1 + 2/VZV
Assay Result
OrganismTest ConcentrationNegativeMatrixHSV-1HSV-2VZV
Coxsackievirus B42.60E+06 TCID50/mLNegPosPosPos
Cytomegalovirus Towne VR-9771.55E+05 TCID50/mLNegPosPosPos
Echovirus 111.61E+05 TCID50/mLNegPosPosPos
HHV-61.46E+06 TCID50/mLNegPosPosPos
HHV-78.63E+05 TCID50/mLNegPosPosPos
HIV Virus1.05E+05 TCID50/mLNegPosPosPos
hMPV A11.24E+05 TCID50/mLNegPosPosPos
HSV-1 SFG0293.27E+05 TCID50/mLNegPosPosPos
HSV-1 Macintyre2.14E+05 TCID50/mLNegPosPosPos
HSV-2 MS6.44E+06 TCID50/mLNegPosPosPos
HSV-2 Strain G1.38E+05 TCID50/mLNegPosPosPos
InfluenzaA/Mexico/4108/20092.17E+06 TCID50/mLNegPosPosPos
Influenza B Hong Kong VR-7917.15E+05 TCID50/mLNegPosPosPos
Measles virus1.46E+05 TCID50/mLNegPosPosPos
Mumps virus2.07E+07 TCID50/mLNegPosPosPos
Parainfluenza Type 14.02E+05 TCID50/mLNegPosPosPos
Parainfluenza Type 33.25E+05 TCID50/mLNegPosPosPos
Parainfluenza Type 41.28E+05 TCID50/mLNegPosPosPos
RSV A Long7.13E+05 TCID50/mLNegPosPosPos
RSV B Washington1.01E+06 TCID50/mLNegPosPosPos
Rubella Virus9.45E+05 TCID50/mLNegPosPosPos
Acholeplasma laidlawi5.33E+06 cfu/mLNegPosPosPos
Bordetella bronchiseptica8.78E+07 cfu/mLNegPosPosPos
Bordetella pertussis1.41E+07 cfu/mLNegPosPosPos
Clostridium perfringens1.18E+06 cfu/mLNegPosPosPos
Mycoplasma hominis9.75E+06 cfu/mLNegPosPosPos
Mycoplasma hyorhinis4.95E+06 cfu/mLNegPosPosPos
Mycoplasma orale1.16E+07 cfu/mLNegPosPosPos
Mycoplasma pneumoniae7.50E+06 ccu/mLNegPosPosPos
Mycoplasma salivarium1.25E+07 cfu/mLNegPosPosPos
Enterovirus 703.83E+04 TCID50/mLNegPosPosPos
Epstein Barr Virus1.67E+04 TCID50/mLNegPosPosPos
HBV1.67E+03 TCID50/mLNegPosPosPos
HCV1.67E+04 TCID50/mLNegPosPosPos
HHV-86.95E+04 TCID50/mLNegPosPosPos
HPVNot AvailableNegPosPosPos
Parainfluenza Type 25.23E+04 TCID50/mLNegPosPosPos

{14}------------------------------------------------

Analytical Specificity – Interfering or Cross-Reactive Microorganism
Lyra™ Direct HSV 1 + 2/VZVAssay Result
OrganismTest ConcentrationNegativeMatrixHSV-1HSV-2VZV
VZV Ellen8.75E+02 TCID50/mLNegPosPosPos
VZV Webster1.85E+03 TCID50/mLNegPosPosPos
Chlamydia trachomatis5.00E+05 cfu/mLNegPosPosPos
Chlamydophila pneumoniae2.67E+03 cp/mLNegPosPosPos
Toxoplasma gondii1.10E+06 tachyzoites/mLNegPosPosPos
Trichomonas vaginalis2.75E+05trophozoites/mLNegPosPosPos
Acinetobacter calcoaceticus7.35E+08 cfu/mLNegPosPosPos
Bacteroides fragilis5.67E+05 cfu/mLNegPosPosPos
Candida albicans1.50E+06 cfu/mLNegPosPosPos
Candida glabrata1.50E+06 cfu/mLNegPosPosPos
Corynebacterium diphtheriae2.40E+08 cfu/mLNegPosPosPos
Enterococcus faecalis1.65E+08 cfu/mLNegPosPosPos
Escherichia coli4.35E+07 cfu/mLNegPosPosPos
Gardnerella vaginalis9.00E+06 cfu/mLNegPosPosPos
Haemophilis influenzae type A6.30E+08 cfu/mLNegPosPosPos
Klebsiella pneumoniae3.90E+07 cfu/mLNegPosPosPos
Lactobacillus acidophilus1.35E+06 cfu/mLNegPosPosPos
Legionella pneumophila2.85E+07 cfu/mLNegPosPosPos
Mobiluncus mulieris1.91E+08 cfu/mLNegPosPosPos
Moraxella cartarrhalis2.25E+07 cfu/mLNegPosPosPos
Neisseria gonorrhoeae2.40E+07 cfu/mLNegPosPosPos
Proteus mirabilis9.60E+07 cfu/mLNegPosPosPos
Pseudomonas aeruginosa5.25E+07 cfu/mLNegPosPosPos
Salmonella enteriditis4.05E+07 cfu/mLNegPosPosPos
Salmonella typhimurium3.45E+07 cfu/mLNegPosPosPos
Staphylococcus aureus1.38E+09 cfu/mLNegPosPosPos
Staphylococcus saprophyticus2.25E+08 cfu/mLNegPosPosPos
Streptococcus agalactiae1.65E+08 cfu/mLNegPosPosPos
Streptococcus pneumoniae2.10E+06 cfu/mLNegPosPosPos
Streptococcus pyogenes2.85E+08 cfu/mLNegPosPosPos
Ureaplasma uralyticumUnable to titerNegPosPosPos
Analytical Specificity - Interfering or Cross-Reactive Substances
Lyrat™ Direct HSV 1 + 2/VZV Assay Result
Substance NameSubstance FinalConcentrationNegativeMatrixHSV-1HSV-2VZV
Seminal Fluid7%NegativePositivePositivePositive
Cornstarch2.5 mg/mLNegativePositivePositivePositive
Acetamidophenol10 mg/mLNegativePositivePositivePositive
Feces0.219%NegativePositivePositivePositive
Chlorpheniramine5 mg/mLNegativePositivePositivePositive
Dextromethorphan5 mg/mLNegativePositivePositivePositive
Blood/EDTA0.625%NegativePositivePositivePositive
Female Urine7%NegativePositivePositivePositive
Male Urine7%NegativePositivePositivePositive
Acyclovir7 mg/mLNegativePositivePositivePositive
Albumin3.3 mg/mLNegativePositivePositivePositive
Casein7 mg/mLNegativePositivePositivePositive
KY Jelly7%NegativePositivePositivePositive
Douche7%NegativePositivePositivePositive
Miconazole 17%NegativePositivePositivePositive
Miconazole 37%NegativePositivePositivePositive
Tioconazole 1Approx. 7%NegativePositivePositivePositive
Preparation H7%NegativePositivePositivePositive
Lanacane3.5%NegativePositivePositivePositive
Listerine7%NegativePositivePositivePositive
Abreva7%NegativePositivePositivePositive
Carmex7%NegativePositivePositivePositive
Releev7%NegativePositivePositivePositive
Colgate7%NegativePositivePositivePositive
Mucin60 µg/mLNegativePositivePositivePositive
Leukocytes2.5e5 cells/mLNegativePositivePositivePositive

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 NumberCombined AnalytesHSV1HSV2VZVPRCInterference
Control 2x HSV-1N/A29.8NEGNEG25.8N/A
12x HSV-12x HSV-229.230.8NEG23.5NO
22x HSV-110x HSV-229.728.8NEG22.6NO
32x HSV-1100x HSV-229.326.2NEG22.8NO
42x HSV-12x VZV29.2NEG29.023.1NO
52x HSV-110x VZV29.9NEG26.724.2NO
62x HSV-1100x VZV29.6NEG23.423.6NO
162x HSV-11000x VZV29.8NEG20.322.7NO
172x HSV-110,000x VZV30.6NEG18.321.6NO
HSV-2: 2x Results Summary: Competitive Interference
Sample NumberCombined AnalytesHSV1HSV2VZVPRCInterference
Control 2x HSV-2N/ANEG32.1NEG25.6N/A
12x HSV-2 2x HSV-129.230.8NEG23.5NO
72x HSV-2 10x HSV-127.632.1NEG23.8NO
82x HSV-2 100x HSV-123.432.4NEG23.4NO
92x HSV-2 2x VZVNEG31.629.524.3NO
102x HSV-2 10x VZVNEG33.026.923.7NO
112x HSV-2 100x VZVNEG33.623.725.1NO
182x HSV-2 1000x VZVNEG29.820.522.3NO
192x HSV-2 10,000x VZVNEG29.118.422.7NO
VZV: 2x Results Summary: Competitive Interference
Sample NumberCombined AnalytesHSV1HSV2VZVPRCInterference

{17}------------------------------------------------

Control 2x VZVN/ANEGNEG29.026.0N/A
42x VZV2x HSV-129.2NEG29.023.1NO
92x VZV2x HSV-2NEG31.629.524.3NO
122x VZV10x HSV-127.5NEG29.224.2NO
132x VZV100x HSV-124.1NEG29.422.3NO
142x VZV10x HSV-2NEG29.429.124.6NO
152x VZV100x HSV-2NEG25.929.022.9NO
HSV-1: 2x Results Summary: Competitive Interference (Avg C₁)
Sample NumberCombined AnalytesHSV1HSV2VZVPRCInterference
Control 2x HSV-1N/A31.0NEGNEG26.2N/A
12x HSV-1 2x HSV-229.931.4NEG28.8NO
22x HSV-1 10x HSV-230.831.1NEG27.0NO
32x HSV-1 100x HSV-230.728.1NEG30.0NO
42x HSV-1 2x VZV30.3NEG30.229.0NO
52x HSV-1 10x VZV29.6NEG28.126.9NO
62x HSV-1 100x VZV31.2NEG25.228.2NO
162x HSV-1 1000x VZV29.8NEG21.730.1NO
172x HSV-1 10,000x VZV31.0NEG19.430.4NO
HSV-2: 2x Results Summary: Competitive Interference (Avg C₁)
Sample NumberCombined AnalytesHSV1HSV2VZVPRCInterference
Control 2x HSV-2N/ANEG32.4NEG25.6N/A
12x HSV-22x HSV-129.931.4NEG28.8NO
72x HSV-210x HSV-127.430.4NEG28.9NO
82x HSV-2100x HSV-124.033.5NEG27.4NO
92x HSV-22x VZVNEG31.130.428.9NO
102x HSV-210x VZVNEG31.528.029.3NO
112x HSV-2100x VZVNEG33.025.027.2NO
182x HSV-21000x VZVNEG29.321.630.2NO
192x HSV-210,000x VZVNEG28.819.330.3NO
VZV: 2x Results Summary: Competitive Interference (Avg Cₜ)
Sample NumberCombined AnalytesHSV1HSV2VZVPRCInterference
Control 2x VZVN/ANEGNEG31.126.3N/A
42x VZV 2x HSV-130.3NEG30.229.0NO
92x VZV 2x HSV-2NEG31.130.428.9NO
122x VZV 10x HSV-126.3NEG31.628.7NO
132x VZV 100x HSV-124.4NEG30.428.1NO

{18}------------------------------------------------

142x VZV10x HSV-2NEG28.730.227.1NO
152x VZV100x HSV-2NEG26.530.527.1NO
HSV-1: 2x Results Summary: Competitive Interference
Sample NumberCombined AnalytesHSV1HSV2VZVPRCInterference
Control 2x HSV-1N/A33.1NEGNEG28.8N/A
12x HSV-12x HSV-232.436.6NEG28.4NO
22x HSV-110x HSV-233.032.8NEG28.8NO
32x HSV-1100x HSV-232.929.2NEG28.5NO
42x HSV-12x VZV33.1NEG31.728.8NO
52x HSV-110x VZV33.6NEG28.728.7NO
62x HSV-1100x VZV32.6NEG25.428.4NO
162x HSV-11000x VZV32.6NEG23.031.5NO
172x HSV-110,000x VZV34.4NEG20.431.9NO
HSV-2: 2x Results Summary: Competitive Interference
Sample NumberCombined AnalytesHSV1HSV2VZVPRCInterference
Control 2x HSV-2N/ANEG36.2NEG28.7N/A
12x HSV-1 2x HSV-232.436.6NEG28.4NO
72x HSV-2 10x HSV-128.835.3NEG28.5NO
82x HSV-2 100x HSV-127.335.8NEG28.4NO
92x HSV-2 2x VZVNEG34.931.028.9NO
102x HSV-2 10x VZVNEG35.428.728.2NO
112x HSV-2 100x VZVNEG35.625.928.4NO
182x HSV-2 1000x VZVNEG32.823.031.2NO
192x HSV-2 10,000x VZVNEG32.820.431.8NO
202x HSV-2 1000x HSV-123.333.1NEG30.3NO
VZV: 2x Results Summary: Competitive Interference
Sample NumberCombined AnalytesHSV1HSV2VZVPRCInterference
Control 2x VZVN/ANEGNEG31.928.7N/A
42x VZV2x HSV-133.1NEG31.728.8NO
92x VZV2x HSV-2NEG34.931.028.9NO
122x VZV10x HSV-130.0NEG31.828.4NO
132x VZV100x HSV-127.2NEG31.528.0NO
142x VZV10x HSV-2NEG32.731.528.5NO
152x VZV100x HSV-2NEG28.732.428.4NO
212x VZV1000x HSV-123.3NEG31.329.7NO

{19}------------------------------------------------

i. Sample stability studies

This study was performed to assess whether fresh and frozen samples provided comparable results when tested in the Lyra™ Direct HSV 1 + 2/VZV Assay. The study was performed on the Applied Biosystems® 7500 Fast Dx using a panel of contrived specimens at near LoD levels (2x and 5x) for each virus. Five (5) panels were created that contained randomized 2x LoD, 5x LoD, and negative samples. Each panel was prepared with 30 aliquots of 5x LoD and 30 aliquots of 2x LoD for each virus and 10 aliquots of negative matrix. Panel 1 was tested on the day of preparation. Panels 2 and 3 were stored at -20°C and then tested on day 7 and day 8, respectively. Panels 4 and 5 were stored at 2° to 8°C and then tested on day 7 and day 8, respectively. Each panel member was tested in singlet. Positive and Negative Controls were analyzed on each instrument run. The Positive Controls were 5x LoD HSV-1, HSV-2 and VZV. The Negative Control was negative matrix. For each panel, one set of Positive Controls and one Negative Control was prepared.

Summary of Results from the Applied Biosystems® 7500 Fast Dx
Panel2xHSV-15xHSV-12xHSV-25xHSV-22xVZV5xVZVNegative
-20°CDAY 8Mean Ct ofpositives29.627.930.929.530.828.926.7
Ct Std. Dev0.90.81.21.20.70.40.5
CV %3.12.84.04.22.31.51.7
2-8°CDAY 7Number detected#/3030/3030/3030/3030/3030/3030/3010/10
% Detection100%100%100%100%100%100%100%
Mean Ct ofpositives30.028.330.428.930.929.027.4
2-8°CDAY 8Ct Std. Dev0.80.61.61.01.00.20.7
CV %2.72.15.33.53.30.82.5
Number detected#/3030/3030/3030/3030/3030/3030/3010/10
% Detection100%100%100%100%100%100%100%
Mean Ct ofpositives29.628.330.428.631.329.125.9
Ct Std. Dev0.80.61.51.52.00.40.9
CV %2.62.04.95.46.51.33.3

There was >95% agreement between the results obtained with the contrived specimens on the day of preparation and after storage at 29 to 8°C for 7 and 8 days. There was >95% agreement between the results obtained with the contrived specimens on the day of preparation and after storage at -20℃ for 7 and 8 days. Based on this study, specimens may be stored at either 2° to 8°C or -20°C for up to 7days with no effect on the performance of the Lyra™ Direct HSV 1 + 2/VZV Assay.

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Comparison of Transport Media j.

The following study was performed to determine whether various transport media impact the performance in the Lyra™ Direct HSV 1 + 2/VZV Assay on the Applied Biosystems 7500® Fast Dx instrument. HSV-1, HSV-2, and VZV virus stocks were diluted to 2x LoD concentrations using negative matrix collected in five different media: UTM. M4. M4-RT. M5. and M6. The acceptance criteria for this study were met for all media. The Lyra™ Direct HSV 1 + 2/VZV Assay can be used with samples collected in UTM, M4, M4RT, M5, or M6 transport media.

  • k. Carry-over Contamination
    An evaluation of potential carry-over contamination during the sample processing and amplification steps was performed on each instrument. The study was conducted using a 96-sample panel consisting of 48 high positives and 48 negative specimens. Each high positive specimen contained 1.00E+05 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 5days, cross-contamination and amplicon carry-over did not occur with the Lyra™ Direct HSV 1+2/VZV Assay on any of the three instruments.

    1. Comparison studies:
    • a. Method comparison with predicate device:

Not applicable. Refer to the Clinical Studies section of this document.

  • b. Matrix comparison:
    Not applicable.

    1. Clinical studies:
      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 locations. Each specimen was also processed and inoculated into two (2) different cell culture systems within 72 hours of collection: one system for the isolation and identification of HSV-1 and HSV-2 and the other system for the isolation and identification of VZV. Cells isolated from the specimens were also stained for the presence of VZV.

The specimens were categorized as cutaneous (skin lesion, penis), or mucocutaneous

{21}------------------------------------------------

Combined Study – Age and Gender Distribution (Cutaneous)
GenderFemaleMale
Age
< 5 years616
6 to 21 years1812
22 to 59 years7298
≥ 60 years3720
Total133146

(anorectal, vaginal/cervical, and oral lesion). The gender and age demographics for each category are listed below.

Performance on the 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 the 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
Comparator: ELVIS® HSV ID and D3 Typing Test
LyraTM Direct HSV 1 + 2/VZV AssayPositiveNegativeTotal
Positive244*28
Negative0250250
Total24254278
Sensitivity24/24100%86.2% to 100%95% CI
Specificity250/25498.4%96.0% to 99.4%
  • One (1) of the four (4) 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 the Lyra™ Direct

{22}------------------------------------------------

HSV-2
Comparator: ELVIS® HSV ID and D3 Typing Test
Lyra™ Direct HSV 1 +2/VZV AssayPositiveNegativeTotal
Positive359*44
Negative0234250
Total35243278
95% CI
Sensitivity35/35100%90.1% to 100%

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.

234/243 * Nine (9) of the nine (9) positives were positive by an additional RT-PCR assay.

96.3%

93.1% to 98.0%

Two-hundred and seventy-nine (279) active cutaneous lesion specimens were cultured for VZV and were also tested with the subject device for VZV viral DNA. The detection and isolation of VZV was performed by staining cells present in the samples with a FDAcleared VZV detection reagent (DSFA) and by culturing the specimen for 96-hours using a mixed cell culture (H&V mixed cells) consisting of MRC-5 cells (human diploid fibroblast) and CV-1 cells (african green monkey kidney), and staining the cultures with the same FDA-cleared reagent used for DSFA. 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 the Lyra™ Direct HSV 1 + 2/VZV Assay. Thus, a total of 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
Comparator: DSFA and Culture with DFA
Lyra™ Direct HSV 1 +2/VZV AssayPositiveNegativeTotal
Positive278*35
Negative0187187
Total27195222
95% CI
Sensitivity27/27100%87.5% to 100%
Specificity187/19595.9%92.1% to 97.9%
  • Seven (7) of the eight (8) positives were positive by an additional RT-PCR assay.

Mucocutaneous Lesions

Specificity

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 the Lyra™ Direct HSV 1 + 2/VZV Assay. These four (4) specimens have been excluded from further analysis. The

{23}------------------------------------------------

HSV-1
Comparator: ELVIS® HSV ID and D3 Typing Test
Lyra™ Direct HSV 1 +2/VZV AssayPositiveNegativeTotal
Positive10016*116
Negative3**527530
Total103543646
95% CI
Sensitivity100/10397.1%91.8% to 99.0%
Specificity527/54397.1%95.3% to 98.2%

table below summarizes the HSV-1 results for the remaining six-hundred forty-six (646) specimens.

  • Thirteen (13) of the sixteen (16) positives were positive by an additional RT-PCR assav.

** Three (3) of the three (3) positives 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 one (1) specimen was invalid in the 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 six-hundred forty-six (646) specimens.

HSV-2
Comparator: ELVIS® HSV ID and D3 Typing Test
Lyra™ Direct HSV 1 +2/VZV AssayPositiveNegativeTotal
Positive9521*116
Negative0530530
Total95551646
95% CI
Sensitivity95/95100%96.1% to 100%
Specificity530/55196.2%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 and were also tested with the subject device for VZV viral DNA. The detection and isolation of VZV was performed by staining cells present in the samples with a FDAcleared VZV detection reagent (DSFA) and by culturing the specimen for 96-hours using a mixed cell culture (H&V mixed cells) consisting of MRC-5 cells (human diploid fibroblast) and CV-1 cells (african green monkey kidney), and staining the cultures with the same FDA-cleared reagent used for DSFA. Due the presence of either HSV-1 or HSV-2, two hundred seventeen (217) specimens have been excluded from analysis. One

{24}------------------------------------------------

(1) specimen was invalid in the 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.

VZV
Comparator: DSFA and Culture with DFA
Lyra™ Direct HSV 1 +2/VZV AssayPositiveNegativeTotal
Positive45*9
Negative0423423
Total4428432
95% CI
Sensitivity4/4100%51.0% to 100%
Specificity423/42898.8%97.3% to 99.5%
  • Five (5) of the five (5) positives were positive by an additional RT-PCR assay.

Performance on the 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.

HSV-1
Comparator: ELVIS® HSV ID and D3 Typing Test
Lyra™ Direct HSV 1 +2/VZV AssayPositiveNegativeTotal
Positive243*27
Negative0252252
Total24255279
95% CI
Sensitivity24/24100%86.2% to 100%
Specificity252/25498.8%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.

{25}------------------------------------------------

HSV-2
Comparator: ELVIS® HSV ID and D3 Typing Test
LyraTM Direct HSV 1 +2/VZV AssayPositiveNegativeTotal
Positive348*42
Negative1**236251
Total35244279
95% CI
Sensitivity34/3597.1%85.5% to 99.5%
Specificity236/24496.7%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 and were also tested with the subject device for VZV viral DNA. The detection and isolation of VZV was performed by staining cells present in the samples with a FDAcleared VZV detection reagent (DSFA) and by culturing the specimen for 96-hours using a mixed cell culture (H&V mixed cells) consisting of MRC-5 cells (human diploid fibroblast) and CV-1 cells (african green monkey kidney), and staining the cultures with the same FDA-cleared reagent used for DSFA. 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
Comparator: DSFA and Culture with DFA
Lyra™ Direct HSV 1 +2/VZV AssayPositiveNegativeTotal
Positive267*33
Negative1**189190
Total27196223
95% CI
Sensitivity26/27100%87.5% to 100%
Specificity189/19695.9%92.1% to 97.9%
  • 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 the 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 six-hundred forty-four (644) specimens.

{26}------------------------------------------------

HSV-1
Comparator: ELVIS® HSV ID and D3 Typing Test
Lyra™ Direct HSV 1 +2/VZV AssayPositiveNegativeTotal
Positive9810*108
Negative5**531536
Total103541644
95% CI
Sensitivity98/10395.1%89.1% to 97.9%
Specificity531/54198.2%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 the 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 six-hundred forty-four (644) specimens.

HSV-2
Comparator: ELVIS® HSV ID and D3 Typing Test
LyraTM Direct HSV 1 +2/VZV AssayPositiveNegativeTotal
Positive9316*109
Negative2**533535
Total95549644
95% CI
Sensitivity93/9597.9%92.6% to 99.4%
Specificity533/54997.1%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 and were also tested with the subject device for VZV viral DNA. The detection and isolation of VZV was performed by staining cells present in the samples with a FDAcleared VZV detection reagent (DSFA) and by culturing the specimen for 96-hours using a mixed cell culture (H&V mixed cells) consisting of MRC-5 cells (human diploid fibroblast) and CV-1 cells (african green monkey kidney), and staining the cultures with the same FDA-cleared reagent used for DSFA. 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 the 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.

{27}------------------------------------------------

VZV
Comparator: DSFA and Culture with DFA
Lyra™ Direct HSV 1 +2/VZV AssayPositiveNegativeTotal
Positive43*7
Negative0423423
Total4426430
95% CI
Sensitivity4/4100%51.0% to 100%
Specificity423/42699.3%98.0% to 99.8%
  • Three (3) of the three (3) positives were positive by an additional RT-PCR assay.

Performance on the Cepheid SmartCycler® II

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 the 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
Comparator: ELVIS® HSV ID and D3 Typing Test
LyraTM Direct HSV 1 +2/VZV AssayPositiveNegativeTotal
Positive243*27
Negative0251251
Total24254278
95% CI
Sensitivity24/24100%86.2% to 100%
Specificity251/25498.8%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 the 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.

{28}------------------------------------------------

HSV-2
Comparator: ELVIS® HSV ID and D3 Typing Test
Lyra™ Direct HSV 1 +2/VZV AssayPositiveNegativeTotal
Positive358*43
Negative0235235
Total35243278
95% CI
Sensitivity35/35100%90.1% to 100%
Specificity235/24396.7%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 and were also tested with the subject device for VZV viral DNA. The detection and isolation of VZV was performed by staining cells present in the samples with a FDAcleared VZV detection reagent (DSFA) and by culturing the specimen for 96-hours using a mixed cell culture (H&V mixed cells) consisting of MRC-5 cells (human diploid fibroblast) and CV-1 cells (african green monkey kidney), and staining the cultures with the same FDA-cleared reagent used for DSFA. 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 the Lyra™ Direct HSV 1 + 2/VZV Assay. The table below details the VZV results for the two-hundred and twenty-two (222) specimens.

VZV
Comparator: DSFA and Culture with DFA
Lyra™ Direct HSV 1 +2/VZV AssayPositiveNegativeTotal
Positive2710*37
Negative0185185
Total27195222
95% CI
Sensitivity27/27100%87.5% to 100%
Specificity185/19594.9%90.8% to 97.2%
  • Six (6) of the ten (10) 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 four (4) specimens were invalid in the 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 six-hundred forty-three (643) specimens.

{29}------------------------------------------------

HSV-1
Comparator: ELVIS® HSV ID and D3 Typing Test
LyraTM Direct HSV 1 +2/VZV AssayPositiveNegativeTotal
Positive10115*116
Negative2**525527
Total103540643
95% CI
Sensitivity101/10398.1%93.2% to 99.5%
Specificity525/54097.2%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 the 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 six-hundred forty-three (643) specimens.

HSV-2
Comparator: ELVIS® HSV ID and D3 Typing Test
Lyra™ Direct HSV 1 +2/VZV AssayPositiveNegativeTotal
Positive9416*110
Negative1**532533
Total95548643
95% CI
Sensitivity94/9598.9%94.3% to 99.8%
Specificity532/54897.1%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 and were also tested with the subject device for VZV viral DNA. The detection and isolation of VZV was performed by staining cells present in the samples with a FDAcleared VZV detection reagent (DSFA) and by culturing the specimen for 96-hours using a mixed cell culture (H&V mixed cells) consisting of MRC-5 cells (human diploid fibroblast) and CV-1 cells (african green monkey kidney), and staining the cultures with the same FDA-cleared reagent used for DSFA. 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 the Lyra™ Direct HSV 1 + 2/VZV Assay. These two hundred twenty-one (221) specimens have been excluded from analysis. The table below

{30}------------------------------------------------

VZV
Comparator: DSFA and Culture with DFA
Lyra™ Direct HSV 1 +2/VZV AssayPositiveNegativeTotal
Positive45*9
Negative0420423
Total4425429
95% CI
Sensitivity4/4100%51.0% to 100%
Specificity420/42598.8%97.3% to 99.5%

details the VZV results for the remaining four-hundred and twenty-nine (429) specimens.

  • Five (5) of the five (5) positives were positive by an additional RT-PCR assay.

4. 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 virus detected on the three instruments based on 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 for each of 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, 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.

Expected Values (Cutaneous) (N=279)*
HSV- 1HSV-2VZV
AgeTotal #Total PositivePrevalenceTotal #Total PositivePrevalenceTotal #Total PositivePrevalence
< 5 years2214.5%2214.5%2229.1%
6 to 21 years30826.7%3013.3%3013.3%
22 to 59 years170148.2%1703319.4%1702112.4%
> 60 years*5658.9%56916.1%561221.4%

Life Technologies QuantStudio™ Dx

*1 Specimen was invalid when tested on the Life Technologies QuantStudio™ Dx. It has been removed from analysis.

Expected Values (Cutaneous) (N=279)*
HSV- 1HSV-2VZV
Total #Total PositivePrevalenceTotal #Total PositivePrevalenceTotal #Total PositivePrevalence
skin lesion213*2411.3%213*2712.7%213*3516.4%

{31}------------------------------------------------

genital - penis6.2%26.2%1.5%
1. 8 74Carlos Company of Children Company of Children Company Company Company Company Company Company Company Company Company Company Company Company Company Company Comments of Chi(Comments of the may be and the comments of the comments of

*1 Specimen was invalid when tested on the Life Technologies QuantStudio™ Dx. It has been removed from analysis.

Expected Values (Mucocutaneous) (N=650)*
HSV- 1HSV-2VZV
AgeTotal #Total PositivePrevalenceTotal #Total PositivePrevalenceTotal #Total PositivePrevalence
< 5 years18527.8%180N/A180N/A
6 to 21 years1302620.0%1302519.2%13010.8%
22 to 59 years*4488118.1%4488118.1%44861.3%
> 60 years5347.5%531018.9%5323.8%

*1 Specimen was invalid when tested on the Life Technologies QuantStudio™ Dx. It has been removed from analysis.

Expected Values (Mucocutaneous) (N=650)*
HSV- 1HSV-2VZV
Total #Total PositivePrevalenceTotal #Total PositivePrevalenceTotal #Total PositivePrevalence
anorectal26415.4%26623.1%2613.8%
genital –vaginal/cervical473*7515.9%473*10722.6%473*40.8%
Nares9222.2%90N/A90N/A
Ocular60N/A60N/A6116.6%
oral lesion1353525.9%13532.2%13532.2%

*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- 1HSV-2VZV
. AgeTotal#TotalPositivePrevalenceTotal #TotalPositivePrevalenceTotal #TotalPositivePrevalence
< 5 years224.5%224.5%2229.1%
6 to 21 years30826.7%3026.7%303.3%
22 to 59 years170148.2%1703118.2%1702011.8%
> 60 years57ব7.0%57814.0%571017.5%
Expected Values (Cutaneous) (N=279)
HSV- 1HSV-2VZV
Total #TotalPositivePrevalenceTotal #TotalPositivePrevalenceTotal #TotalPositivePrevalence
skin lesion2142411.2%2142612.1%2143315.4%
genital - penis6534.6%651624.6%650N/A
Expected Values (Mucocutaneous) (N=650)*
AgeHSV-1HSV-2VZV

{32}------------------------------------------------

Total1 #TotalPositivePrevalenceTotal #TotalPositivePrevalenceTotal #TotalPositivePrevalence
< 5 years18527.8%180N/A180N/A
6 to 21 years1302519.2%1302418.5%13010.8%
22 to 59 years*4467516.8%4467516.8%44651.1%
> 60 years5335.7%531018.9%5311.9%
  • Three (3) specimens were invalid when tested on the Applied Biosystems® 7500 Fast Dx. They have been removed from analysis.
Expected Values (Mucocutaneous) (N=656)*
HSV- 1HSV-2VZV
Total #Total PositivePrevalenceTotal #Total PositivePrevalenceTotal #Total PositivePrevalence
anorectal26415.4%26623.1%2613.8%
genital –vaginal/cervical471*7014.9%471*10221.7%471*30.6%
Nares9111.1%90N/A90N/A
Ocular60N/A60N/A6116.6%
oral lesion1353324.4%13510.7%13521.5%
  • 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- 1HSV-2VZV
AgeTotal #Total PositivePrevalenceTotal #Total PositivePrevalenceTotal #Total PositivePrevalence
< 5 years2214.5%2214.5%2229.1%
6 to 21 years30826.7%3013.3%3013.3%
22 to 59 years*169158.9%1693218.9%1692313.6%
> 60 years5735.3%57915.8%571119.3%

*One (1) specimen was invalid when tested on the Cepheid SmartCycler® II. It has been removed from analysis.

Expected Values (Cutaneous) (N=279)*
HSV- 1HSV-2VZV
Total #TotalPositivePrevalenceTotal #TotalPositivePrevalenceTotal #TotalPositivePrevalence
skin lesion213*2210.3%213*2612.2%213*3717.4%
genital - penis6557.7%651726.2%650N/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- 1HSV-2VZV
AgeTotal #Total PositivePrevalenceTotal #Total PositivePrevalenceTotal #Total PositivePrevalence
≤ 5 years18527.8%180N/A180N/A
6 to 21 years1302922.3%1302418.5%13010.8%
22 to 59 years*4457817.5%4457617.1%44561.3%
≥ 60 years5347.5%531018.9%5323.8%

*Four (4) specimens were invalid when tested on the Cepheid SmartCycler® II. They have been removed from analysis.

{33}------------------------------------------------

Expected Values (Mucocutaneous) (N=650)*
HSV-1HSV-2VZV
Total #Total PositivePrevalenceTotal #Total PositivePrevalenceTotal #Total PositivePrevalence
anorectal26415.4%26623.1%2613.8%
genital –vaginal/cervical470*7516.0%470*10321.9%470*40.9%
nares9222.2%90N/A90N/A
ocular60N/A60N/A6116.6%
oral lesion1353525.9%13510.7%13532.2%

*Four (4) specimens were invalid when tested on the Cepheid SmartCycler® II. They have been removed from
analysis.

{34}------------------------------------------------

M. Instrument Names:

The manufacturer intends to market this assay for use with three instrument platforms:

  • Life Technologies QuantStudio™ Dx -
  • Applied Biosystems® 7500 Fast Dx -
  • Cepheid SmartCycler® II System -

N. System Description:

    1. Modes of Operation:
    1. Modes of Operation

For this application, Quidel assessed the software for each of the three systems and provided documentation on these platforms per FDA Guidance for Industry, FDA Reviewers and Compliance- Off-The-Shelf Software Use in Medical Devices. The firm's assessment determined that the software packages, when used as intended, presents a Moderate Level of Concern. Ouidel states thev have full access to the instruments with the current software. Each instrument is under a quality service plan with the manufacturers that includes both maintenance and software upgrades and will be in effect for the life of the Lyra™ Direct HSV 1+2/VZV Assay.The Applied Biosystems 7500 Fast Dx Real-Time PCR Instrument with the SDS Software version 1.4 is a real-time nucleic acid amplification and detection system that measures fluorescence signal output from dual-labeled hydrolysis probes. The Sequence Detection Software (SDS) version 1.4 for the 7500 Fast Dx Instrument is used for instrument control, data collection and data analysis. The software can analyze cycle-by-cycle real-time signals from the sample. Life Technologies QuantStudio™ Dx uses fluorescent-based polymerase chain reaction (PCR) reagents to provide quantitative detection of target nucleic acid sequences (targets) using real-time analysis. After a test run, the version 1.0 software uses calibration data to determine the location and intensity of the fluorescent signals in each read, the dye associated with each fluorescent signal, and the significance of the signal. The Cepheid SmartCycler II with the Software version 3.0b is a real-time nucleic acid amplification and detection system that measures fluorescence signal output from dual-labeled hydrolysis probes. Software version 3.0b is used for instrument control, data collection and data analysis. The software can measure cycle-by-cycle real-time signals from the sample.

    1. Specimen Identification: Not Applicable
    1. Specimen Sampling 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° to 8°C or -20°C for up to 7 days prior to processing. The performance of the assay was evaluated using: M4, M4-RT, M5, M6, and UTM. No

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significant difference in assay performance was seen between the five different types of viral transport media. Prepared samples can be stored for 48 hours when stored at 2º to 8°C, 31 days when stored at either -20°C or -70°C.

    1. Calibration:
      Calibration is not required or recommended.
    1. 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.
    1. 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.
    1. 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.
    1. Software:

FDA has reviewed applicant's Hazard Analysis and Software Development processes for this line of product types:

Yes___X______ or No___________________________________________________________________________________________________________________________________________________________

O. Other Supportive Instrument Performance Characteristics Data Not Covered In the "Performance Characteristics" Section above:

Not Applicable

P. Proposed Labeling:

The labeling is sufficient and satisfies the requirements of 21 CFR Part 809.10, 21 CFR 801.109, and the special controls.

O. Identified Potential Risks and Required Mitigation Measures

Identified Potential RisksRequired Mitigation Measures

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Risk of false resultsSpecial controls (1), (2), and (3)
Failure to correctly interpret test resultsSpecial controls (4) and (5)
Failure to correctly operate theinstrumentSpecial controls (6) and (7)

R. Benefit/Risk Analysis

Summary
Summary ofthe Benefit(s)When used for the proposed intended use, the benefits to the clinician and the patientinclude: (1) establishment of the device performance in a manner that demonstratesconsistent accurate test results; and 2) ability to use a well validated device to diagnoseHSV 1, HSV 2 and VZV in cutaneous and mucocutaneous lesion swab specimens,which will allow prompt initiation of disease specific treatment when that is indicated.
Summary ofthe Risk(s)The risks associated with the device, when used as intended, are those related to therisk of false test results, failure to correctly interpret the test results and failure tocorrectly operate the instrument. Inaccurate test results may lead to error or delay inthe diagnosis of HSV 1, HSV 2 and VZV infections, error or delay in appropriatetreatment of these infections, failure or delay in implementing infection controlmeasures, unnecessary use of anti-viral therapy, and delay in establishing the patient'strue diagnosis.
Summary ofOtherFactorsHSV 1, HSV 2 and VZV infections may present as acute clinical episodes that arelikely to recur. In most patients these infections are mild, but in certain populationsthey can be severe and life threatening. Severe infections are treated with antiviraldrugs such as acyclovir, but initiation of therapy in the first few days of the infection iscritical for a successful outcome.The analytical studies conducted by the sponsor were robust while the clinical studywas limited by the number of VZV positive mucocutaneous lesion specimens availableto evaluate the device. The data provided is adequate to demonstrate the device'sperformance characteristics. No post-market information is available.

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ConclusionsThe probable benefits of this device outweigh the probable risks associated with itsuse. There are no substantial clinical concerns with the classification of this device inClass II given the combination of general and special controls.
Do theprobablebenefitsoutweigh theprobable risks?

S. Conclusion:

The information provided in this de novo submission is sufficient to classify this device into class II under regulation 21 CFR 866.3309 with special controls. FDA believes that special controls, along with the applicable general controls, provide reasonable assurance of the safety and effectiveness of the device type. The device is classified under the following:

Device Type:Herpes virus nucleic acid-based cutaneous and mucocutaneous lesionpanel
Class:II (special controls)
Regulation:21 CFR 866.3309

(a) Identification. A herpes virus nucleic acid- based cutaneous and mucocutaneous 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.

(b) 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.
    1. 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.
    1. 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.

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    1. 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.
    1. 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)."
    1. 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.
    1. The risk management activities performed as part of the manufacturer's 21 CFR 820.30 design controls 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.

§ 866.3309 Herpes virus nucleic acid-based cutaneous and mucocutaneous lesion panel.

(a)
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.(b)
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 820.30 design controls 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.