VERIGENE RESPIRATORY VIRUS NUCLEIC ACID TEST ON THE VERIGENE SP SYSTEM (RVNATSP)
Device Facts
| Record ID | K092566 |
|---|---|
| Device Name | VERIGENE RESPIRATORY VIRUS NUCLEIC ACID TEST ON THE VERIGENE SP SYSTEM (RVNATSP) |
| Applicant | Nanosphere, Inc. |
| Product Code | OCC · Microbiology |
| Decision Date | Oct 9, 2009 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.3980 |
| Device Class | Class 2 |
Intended Use
The Verigene® Respiratory Virus Nucleic Acid Test on the Verigene SP System (RVNATsp) is a qualitative multiplex in vitro diagnostic test for the detection and identification of Influenza A Virus, Influenza B Virus, and Respiratory Syncytial Virus (RSV) nucleic acids purified from nasopharyngeal swab specimens obtained from patients symptomatic for viral upper respiratory infection. The test is intended to be used on the Verigene® SP System as an aid in the differential diagnosis of Influenza A, Influenza B, and RSV infections. The test is not intended to detect Influenza C virus. Negative results do not preclude influenza virus or RSV infection and should not be used as the sole basis for treatment or other management decisions. It is recommended that negative test results be confirmed by culture. Performance characteristics for Influenza A Virus were established when Influenza A/H3 and A/H1 were the predominant Influenza A viruses in circulation. When other Influenza A viruses are emerging, performance characteristics may vary. If infection with a novel Influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent Influenza viruses and sent to state or local health department for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.
Device Story
Verigene SP System provides sample-to-result testing for Influenza A, Influenza B, and RSV. Input: nasopharyngeal swab specimens. Process: automated on-board sample extraction, multiplex RT-PCR amplification, and hybridization of amplicons to gold-labeled probes on a microarray-based chip. Detection: signal enhancement with elemental silver via Verigene Processor. Output: qualitative detection/identification of viral targets. Used in clinical settings; operated by laboratory personnel. System automates fluid transfer and mixing previously performed manually. Results aid clinicians in differential diagnosis of respiratory infections; negative results require confirmation by culture.
Clinical Evidence
Bench testing only. Analytical sensitivity (LoD) compared to predicate using serial dilutions of Influenza A, B, and RSV strains; results were identical. Precision/reproducibility studies conducted at three sites over multiple days showed high agreement (98.7-100%) with expected results. Method comparison study (n=558 tests) demonstrated 97.9% PPA and 100% NPA compared to the predicate system. Discordant results were limited to low-positive Influenza A samples, which resolved upon repeat testing.
Technological Characteristics
System includes Verigene Reader and Processor SP. Uses magnetic bead-based isolation, multiplex RT-PCR, and gold nanoparticle probe hybridization on a microarray chip. Automated fluid transfer via internal pipettor. Connectivity: up to 32 SP units per Reader. Software version 2.0 (Processor) and 1.5.6 (Reader).
Indications for Use
Indicated for symptomatic patients with suspected viral upper respiratory infection to detect and identify Influenza A, Influenza B, and RSV nucleic acids in nasopharyngeal swab specimens.
Regulatory Classification
Identification
A respiratory viral panel multiplex nucleic acid assay is a qualitative in vitro diagnostic device intended to simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or viral culture. The detection and identification of a specific viral nucleic acid from individuals exhibiting signs and symptoms of respiratory infection aids in the diagnosis of respiratory viral infection when used in conjunction with other clinical and laboratory findings. The device is intended for detection and identification of a combination of the following viruses:(1) Influenza A and Influenza B; (2) Influenza A subtype H1 and Influenza A subtype H3; (3) Respiratory Syncytial Virus subtype A and Respiratory Syncytial Virus subtype B; (4) Parainfluenza 1, Parainfluenza 2, and Parainfluenza 3 virus; (5) Human Metapneumovirus; (6) Rhinovirus; and (7) Adenovirus.
Special Controls
*Classification.* Class II (special controls). The special controls are:(1) FDA's guidance document entitled “Class II Special Controls Guidance Document: Respiratory Viral Panel Multiplex Nucleic Acid Assay;” (2) For a device that detects and identifies Human Metapneumovirus, FDA's guidance document entitled “Class II Special Controls Guidance Document: Testing for Human Metapneumovirus (hMPV) Using Nucleic Acid Assays;” and (3) For a device that detects and differentiates Influenza A subtype H1 and subtype H3, FDA's guidance document entitled “Class II Special Controls Guidance Document: Testing for Detection and Differentiation of Influenza A Virus Subtypes Using Multiplex Nucleic Acid Assays.” See § 866.1(e) for the availability of these guidance documents.
Predicate Devices
- Verigene® Respiratory Virus Nucleic Acid Test on the Verigene System (K083088)
Related Devices
- K103209 — VERIGENE RESPIRATORY VIRUS NUCLEIC ACID TEST +(RVNAT+) · Nanosphere, Inc. · Jan 10, 2011
- K083088 — VERIGENE RESPIRATORY VIRUS NUCLEIC ACID TEST · Nanosphere, Inc. · May 1, 2009
- K143653 — Verigene Respiratory Pathogens Flex Nucleic Acid Test (RP Flex) · Nanosphere, Inc. · Sep 4, 2015
Submission Summary (Full Text)
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K092566
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Image /page/0/Picture/1 description: The image shows the word "Nanosphere" in a sans-serif font. To the left of the word is a circular logo that is divided into two halves. The left half of the logo is filled with a pattern of small dots, while the right half is filled with a pattern of horizontal lines.
OCT - 9 2009
# Summary of 510(k)
| 510(k)<br>numbers: | K092566: Verigene® Respiratory Virus Nucleic Acid Test on the Verigene® SP System (RVNATSP) |
|-------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Summary<br>preparation<br>date: | October 7, 2009 |
| Submitted<br>by: | Nanosphere, Inc.<br>4088 Commercial Avenue<br>Northbrook, IL 60062<br>Phone: 847-400-9000 Fax: 847-400-9199 |
| Contact: | Gregory W. Shipp, M.D.<br>Chief Medical Officer<br>VP, Medical and Regulatory Affairs and Quality Assurance |
| Proprietary<br>names: | For instrument:<br>Verigene® SP System<br>For the assay:<br>Verigene® Respiratory Virus Nucleic Acid Test on the Verigene® SP System (RVNATSP) |
| Common<br>names: | For the instrument:<br>Bench-top molecular diagnostics workstation<br>For the assays:<br>Respiratory panel<br>Respiratory virus panel<br>Respiratory viruses<br>Influenza A assay<br>Influenza B assay<br>RSV assay<br>Influenza A/B and RSV assay |
| Comparison<br>VRNAT and<br>RVNATSP: | Additional claim to the 510(k) application, K083088 is described. The Verigene Respiratory Virus Nucleic<br>Acid Test (VRNAT) will add the Verigene SP System to the labeling as a cleared system. The safety and<br>effectiveness of the Verigene® Respiratory Virus Nucleic Acid Test on the Verigene SP System<br>(RVNATSP) is demonstrated with analytical and method comparison studies.<br>The FDA-cleared VRNAT (K083088) is a qualitative test based on identifying virus-specific nucleic acids<br>for Influenza A virus, Influenza B virus, and respiratory syncytial virus (RSV). Within this test, the steps of<br>sample preparation (or nucleic acid extraction), target amplification, and hybridization test and analysis<br>take place on three separate instruments (Table 1). The RVNATSP is the same assay with additional<br>system claims for the Verigene SP System, a modified Verigene® System that allows sample preparation,<br>target amplification, and hybridization test and analysis using a single system (Table 1). |
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| Table 1. | | | | |
|---------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------|--------------------|
| | Sample Preparation | Target Amplification | Hybridization Test and Analysis | |
| | VRNAT<br>(cleared, K083088) | NucliSENS EasyMAG<br>(bioMerieux) | Thermocycler | Verigene System |
| | RVNATSP | Verigene SP System | Verigene SP System | Verigene SP System |
| | The RVNATSP is designed to identify virus-specific nucleic acids for Influenza A virus, Influenza B virus, and respiratory syncytial virus (RSV). The RVNATSP involves: | | | |
| | 1) | Sample Preparation – magnetic bead-based viral RNA extraction from nasopharyngeal swab specimens obtained from symptomatic patients; | | |
| | 2) | Target Amplification – Multiplex RT-PCR-based amplification of the eluted viral RNA targets to generate virus-specific amplicons; | | |
| | 3) | Verigene Hybridization Test and Analysis - detection and identification of virus-specific amplicons by using gold nanoparticle probe-based technology. | | |
| | The entire RVNATSP is performed on the Verigene® SP System, which is a bench-top molecular diagnostics workstation that consists of two instruments, the Verigene SP Processor and the Verigene Reader. The Verigene SP Processor performs the assay steps on each sample by using a robotic pipettor to transfer and mix reagents within and between separate testing modules designed for nucleic acid extraction, target amplification, and the Verigene Hybridization test. The Verigene hybridization test module is the same as in the previous Verigene System with added modules for nucleic acid extraction and RT-PCR target amplification. Key functions of the Verigene SP Processor include: | | | |
| | 1) | Reading of the barcode identification label on inserted Test Consumables to maintain positive identification of patient samples throughout processing. | | |
| | 2) | Facilitation of nucleic acid extraction, multiplex RT-PCR target amplification, and the Verigene Hybridization Test. | | |
| Device description: | 3) | Real-time communication of test processing status to the Reader. | | |
| | The Verigene Reader is the same instrument as in the FDA-cleared VRNAT. It is a free-standing instrument with a touch screen control panel and a wand-based barcode scanner. It utilizes a graphical user interface to guide the user through the process of ordering tests and reporting results. There are no serviceable parts and no user calibration is required. Interaction with the touch screen is minimized through barcode use. This instrument also serves as the reader of the Test Cartridges using advanced optics. The key functions of the Verigene Reader include: | | | |
| | 1) | Entry and tracking of specimen identification numbers via manual keyboard input or via barcode-reader wand. | | |
| | 2) | Test selection for each specimen. | | |
| | 3) | Automated transfer of specimen processing instructions on Test Cartridge-specific basis to linked Processor unit(s). A single Reader unit can control up to 32 Processor units. | | |
| | 4) | Automated imaging and analysis of Test Cartridges. | | |
| | 5) | Results display. | | |
| | 6) | Results report generation. | | |
| | RVNATSP consumables within each single-use disposable test kit include: (i) Tip Holder Assembly; (ii) Extraction Tray; (iii) Amplification Tray; and (iv) RV Test Cartridge. The kit components are inserted into the corresponding module of the Verigene SP Processor prior to each test, and the sample is added to the Extraction Tray. Patient information is entered into the Reader to initiate the test procedure. | | | |
| | 1) | Tip Holder Assembly – The robotic pipettor picks up pipettes from the Tip Holder Assembly. The pipettes are used for mixing and transferring reagents within the test procedure. | | |
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2) Extraction Tray – Nucleic acids are extracted from the sample by using magnetic bead-based methods within the Extraction Tray. Each Tray contains reagents for a single extraction procedure. A robotic pipette transfers reagents to designated wells within the Extraction Tray to affect the steps of lysis, capture of nucleic acids onto the magnetic beads, washing, and eluting the isolated nucleic acids from the magnetic beads. 3) Amplification Tray – The isolated nucleic acids are amplified by using multiplex RT-PCR within the Amplification Tray. Each Tray contains reagents for a single multiplex RT-PCR procedure. A robotic pipette transfers the reagents to a specific well within the Amplification Tray. A set thermal profile is then initiated to perform all of the amplification related steps including UDG-based decontamination, reverse transcription, and multiplex PCR in a single tube. Upon completion, an aliquot of the amplified sample is mixed with hybridization buffer containing the virus specific mediator probes. The sample is then transferred to the Test Cartridge. 4) RV Test Cartridge for Verigene Hybridization Test - The virus-specific amplicons are detected and identified within a Test Cartridge by using specific nucleic acid probes in conjunction with gold nanoparticle probe-based detection technology. Each Test Cartridge is a self-contained, laboratory consumable that consists of two parts. The upper housing of each cartridge is called the "reagent pack" and contains reservoirs filled with the detection reagents. When in place with the 'substrate holder', the reagent pack creates an air-tight hybridization chamber surrounding the region of the substrate containing a target-specific capture array. As each step of the test is completed, old reagents are moved out of the hybridization chamber and new reagents are added from the reagent pack via microfluidic channels and pumps. Once the test is complete, the Test Cartridge is removed from the Verigene SP Processor unit and the reagent pack is snapped off and discarded. The remaining slide is now ready for imaging and analysis in the Verigene Reader. 5) End-point detection on the Verigene Reader: The test slide is inserted into the Verigene Reader wherein it is illuminated along its side. The gold-silver aggregates at the test sites scatter the light, which is in turn captured by a photosensor. The relative intensity arising from each arrayed test site is tabulated. Net signals, defined as the absolute signal intensities with background signals subfracted, are compared with thresholds determined by negative controls within the slide in order to arrive at a decision regarding the presence or absence of target. These results are linked to the test and patient information entered at the beginning of each test session to provide a comprehensive results file. The RVNAT so is a qualitative multiplex in vitro diagnostic test for the detection and identification of Influenza A Virus, Influenza B Virus, and Respiratory Syncytial Virus (RSV) nucleic acids purified from nasopharyngeal swab specimens obtained from patients symptomatic for viral upper respiratory infection. The test is intended to be used on the Verigene® SP System as an aid in the differential diagnosis of Influenza A, Influenza B, and RSV infections. The test is not intended to detect Influenza C virus. Neqative results do not preclude influenza virus or RSV infection and should not be used as the sole basis for treatment or other management decisions. It is recommended that negative test results be confirmed by culture. Intended uses: Performance characteristics for Influenza A Virus were established when Influenza A/H3 and A/H1 were the predominant Influenza A viruses in circulation. When other Influenza A viruses are emerging, performance characteristics may vary. If infections with a novel Influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to state or local health department for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens. The cleared Verigene® Respiratory Virus Nucleic Acid Test (VRNAT), K083088 is claimed as the predicate Predicate device (Table 2). It is a multiplex in vitro diagnostic test for the rapid and qualifative detection and device: discrimination of Influenza A Virus, Influenza B Virus, and Respiratory Syncytial Virus (RSV).
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| Feature | VRNAT (Predicate, K083088) | Verigene RVNATSP |
|-------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Intended use | The Verigene® Respiratory Virus Nucleic Acid Test<br>on Verigene SP System (RVNAT gp) is a qualitative<br>multiplex in vitro diagnostic test for the detection and<br>identification of Influenza A Virus, Influenza B Virus,<br>and Respiratory Syncytial Virus (RSV) nucleic acids<br>purified from nasopharyngeal swab specimens<br>obtained from patients symptomatic for viral upper<br>respiratory infection. The test is intended to be used<br>on the Verigene® SP System as an aid in the<br>differential diagnosis of Influenza A, Influenza B, and<br>RSV infections. The test is not intended to detect<br>Influenza C virus.<br>Negative results do not preclude influenza virus or<br>RSV infection and should not be used as the sole<br>basis for treatment or other management decisions. It<br>is recommended that negative results be confirmed by<br>culture.<br>Performance characteristics for Influenza A Virus were<br>established when Influenza A/H3 and A/H1 were the<br>predominant Influenza A viruses in circulation. When<br>other Influenza A viruses are emerging, performance<br>characteristics may vary.<br>If infection with a novel Influenza A virus is suspected<br>based on current clinical and epidemiological<br>screening criteria recommended by public health<br>authorities, specimens should be collected with<br>appropriate infection control precautions for novel<br>virulent influenza viruses and sent to state or local<br>health department for testing. Viral culture should not<br>be attempted in these cases unless a BSL 3+ facility is<br>available to receive and culture specimens. | Same test on Verigene SP System |
| l argets | Intluenza A, Influenza B, RSV | ldentical |
| Specimen | Nasopharyngeal swabs in sample matrix | Identical |
| Sample<br>preparation | Automated extraction of nucleic acids performed<br>externally on the NucliSENS EasyMAG (bioMerieux)<br>using silica coated magnetic beads and chaotropic<br>salts using chemistry based on U.S. Patent 5,234,809. | Automated extraction of nucleic acids<br>performed on the Verigene SP Processor<br>using silica coated magnetic beads and<br>chaotropic salts licensing the identical<br>patent chemistry. |
| Sample size | 200 µL | Identical |
| Quality<br>control | Internal procedural quality control, external quality<br>control solutions | Identical |
| Amplification<br>method | Multiplex RT-PCR: Target Amplification by RT-PCR is<br>performed on an external thermocycler | Multiplex RT-PCR: Identical reagents;<br>performed within the added Amplification<br>Module on the Verigene SP Processor |
| | M-MLV Reverse Transcriptase | Identical |
| Pipetting | | |
| Pipetting | Pipetting between the three steps is performed by the user. | Robotic pipettor added to automate fluid transfer steps. |
| Detection<br>Method | Verigene Hybridization Test is performed in the hybridization module housed in the Processor of the Verigene® System by using single-use Test Cartridges | Identical |
| Decision<br>algorithm | Target-specific signal intensities are compared to a signal threshold and ratioed against positive and negative controls for a decision. | Identical |
| Results | Positive or negative qualitative results | Identical |
| Reader | Provides the user interface, controls the Processor, performs image analysis, and provides results. | Identical |
| Software | A custom embedded software application running under the Micro-C/OS real-time operating system. | Architecture is the same. Additional software programming to control the Extraction and Amplification Modules. |
| Reagent<br>storage | Test Cartridge: 2 - 8 °C<br>Amplification Kit: -20 °C | Test Cartridge and Extraction Tray: 2 - 8 °C.<br>Amplification Tray: -20 °C |
| Assay<br>Performance | As in the cleared VRNAT (K083088). | Identical Limits of Detection as the cleared VRNAT.<br>Precision/Reproducibility - Clinically and statistically equivalent.<br>Method Comparison - Clinically and statistically equivalent |
# Table 2. Similarities and Differences between the Cleared and the New System.
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#### Performance Characteristics of the RVNATse
Analytical and method comparison studies to establish the performance of the test on the Verigene SP System are described.
- A. Comparison of Analytical Sensitivity
The analytical sensitivity of the RVNAT o was compared to the cleared VRNAT (K083088) by determining the Limit of Detection (LOD) of Influenza B, RSV A, and RSV B viruses. Strains with established titers were used for each virus stock was serially diluted into a sample matrix (Universal Transport Media, Copan), and each concentration was tested in quadruplicate using the RVNAT ge. The LOD was confirmed by performing an additional 20 replicates for each strain in order to demonstrate that the virus was detected ≥95% of the time. The LOD for each virus was identical to the LOD observed with the same strains on the cleared VRNAT (K083088) (Table 3).
| Limits of Detection | Concentration |
|---------------------------------------|---------------|
| Influenza A (A/Wisconsin/67/05) | 2 TCID50/mL |
| Influenza B (B/Florida/04/2006) | 50 TCID50/mL |
| RSV A (Strain Long) | 10 TCID50/mL |
| RSV B (B-1 Wild Type (B WV/14617/85)) | 2 TCID50/mL |
| | | Table 3. Limit of Detection |
|--|--|-----------------------------|
| | | |
- B. Carryover and Crossover Contamination Studies
High positive samples of Influenza A, Influenza B, RSV B were alternated with high neqative samples for all four viruses. Based on the collective data, there was no evidence of crosscontamination from any of the test steps including sample extraction, multiplex RT-PCR step, and the Verigene Hybridization Test.
- C. Precision/Reproducibility Studies Comparison between the RVNATso and the cleared VRNAT (K083088)
The Precision/Reproducibility Studies were performed at each of three sites. At Site 1, the Reproducibility Study was part of a larger precision/Reproducibility Studies for the RVNAT go were conducted exactly as for the previously cleared VRNAT (K083088) to allow equivalency comparisons. As before, eight unique samples were created by diluting known concentrations of viral particles with Viral Transport Medium (Table 4). Since the Analytical Sensitivity of the RVNATs was identical to the cleared VRNAT, the same strains and levels were used in the studies. Each strain was represented at 3 distinct concentrations: high negative, low positive, and moderate positive.
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| Unique Samples | Viral Strains and Levels |
|----------------|------------------------------------------------------------|
| 1 | Influenza A - High Negative; Influenza B - High Negative |
| 2 | RSV A - High Negative; RSV B - High Negative |
| 3 | Influenza A - Low Positive |
| 4 | Influenza B - Low Positive |
| 5 | RSV A - Low Positive |
| 6 | RSV B - Low Positive |
| 7 | Influenza A - Moderate Positive; RSV A - Moderate Positive |
| 8 | Influenza B - Moderate Positive; RSV B - Moderate Positive |
| | | | | Table 4. Sample panel for the Precision/Reproducibility Studies. | |
|--|--|--|--|------------------------------------------------------------------|--|
|--|--|--|--|------------------------------------------------------------------|--|
The Precision Study (Site 1) tested the sample set over 12 non-consecutive days. On each test day, two operators performed the RVNAT و in duplicate for each sample sets per day total). In the reproducibility study performed by sites 2 and 3, the sample set was tested in triplicate daily by 2 operators on each of five non-consecutive days.
Performance characteristics of the RVNAT go in the Precision/Reproducibility Studies were equivalent to those for the cleared VRNAT (Table 5).
| | | Agreement of 'Observed Results' to 'Expected Results' | | | | | | | | | | | |
|-------------|----------------------|-------------------------------------------------------|--------|--------|--------------|----------------|-----------------|--------|--------|--------|--------------|----------------|-----------------|
| | | New RVNATSP | | | | | Cleared VRNAT | | | | | | |
| | Panel Member | Site 1 | Site 2 | Site 3 | All<br>Sites | %<br>Agreement | 95% Score<br>CI | Site 1 | Site 2 | Site 3 | All<br>Sites | %<br>Agreement | 95% Score<br>CI |
| Influenza A | High<br>Negative | 48/48 | 15/15 | 15/15 | 78/78 | 100% | 95.3 - 100% | 46/48 | 14/15 | 15/15 | 75/78 | 96% | 89.3 - 98.7% |
| | Low<br>Positive | 48/48 | 15/15 | 15/15 | 78/78 | 100% | 95.3 - 100% | 48/48 | 14/15 | 15/15 | 77/78 | 98.7% | 93.1 - 99.8% |
| | Moderate<br>Positive | 48/48 | 15/15 | 15/15 | 78/78 | 100% | 95.3 - 100% | 48/48 | 15/15 | 15/15 | 78/78 | 100.0% | 95.3 - 100% |
| Influenza B | High<br>Negative | 48/48 | 15/15 | 15/15 | 78/78 | 100% | 95.3 - 100% | 48/48 | 15/15 | 15/15 | 78/78 | 100% | 95.3 - 100% |
| | Low<br>Positive | 47/48 | 15/15 | 15/15 | 77/78 | 98.7% | 93.1 - 99.8% | 47/48 | 15/15 | 15/15 | 77/78 | 98.7% | 93.1 - 99.8% |
| | Moderate<br>Positive | 48/48 | 15/15 | 15/15 | 78/78 | 100% | 95.3 - 100% | 48/48 | 15/15 | 15/15 | 78/78 | 100% | 95.3 - 100% |
| RSV A | High<br>Negative | 48/48 | 15/15 | 14/15 | 77/78 | 99% | 93.1 - 99.8% | 48/48 | 13/15 | 15/15 | 76/78 | 97% | 91.1 - 99.3% |
| | Low<br>Positive | 47/48 | 15/15 | 15/15 | 77/78 | 98.7% | 93.1 - 99.8% | 47/48 | 15/15 | 15/15 | 77/78 | 98.7% | 93.1 - 99.8% |
| | Moderate<br>Positive | 48/48 | 15/15 | 15/15 | 78/78 | 100.0% | 95.3 - 100% | 48/48 | 15/15 | 15/15 | 78/78 | 100.0% | 95.3 - 100% |
| RSV B | High<br>Negative | 48/48 | 15/15 | 15/15 | 78/78 | 100% | 95.3 - 100% | 46/48 | 14/15 | 14/15 | 74/78 | 95% | 87.5 - 98.0% |
| | Low<br>Positive | 48/48 | 15/15 | 15/15 | 78/78 | 100% | 95.3 - 100% | 48/48 | 15/15 | 15/15 | 78/78 | 100% | 95.3 - 100% |
| | Moderate<br>Positive | 48/48 | 15/15 | 15/15 | 78/78 | 100% | 95.3 - 100% | 48/48 | 15/15 | 15/15 | 78/78 | 100.0% | 95.3 - 100% |
Table 5. Comparison of Precision/Reproducibility data from the new RVNAT م to the cleared VRNAT
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#### D. Method Comparison Studies for RVNAT sp and the cleared VRNAT (K083088)
A sample set representing Influenza B, and RSV was prepared by diluting culture positive nasopharyngeal swab samples with negative samples (Table 6). Dilutions were aimed to yield viral load levels close to the low positive levels for each virus type. A total of 62 unique samples were diluted, aliquoted, and frozen. The sample set was tested at the internal site (Site 1) using the cleared VRNAT, and at all three sites using the RVNATs for a total of 62x4=248 unique tests.
Each sample set yielded a total of 186 decisions from 62 unique samples as each test provides a decision of 'Detected' or 'Not Detected' for each of the 3 viruses, Influenza B, and RSV. Of the 62 samples, 3 samples had dual infections where 2 viruses were present.
| Sample Set | Positives | Negatives | Totals |
|-------------|-----------|-----------|--------|
| Influenza A | 15 | 47 | 62 |
| Influenza B | 16 | 46 | 62 |
| RSV A/B | 34 | 28 | 62 |
| Total | 65* | 121 | 186 |
Table 6. Method Comparison Study Sample Set.
*3 samples had 2 viruses raising the total number of positives from 62 to 65
Decisions on the RVNAT of or each site were compared to the cleared VRNAT. The Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) are provided in Tables 7 - 10. The two systems are equivalent based on the collected data.
| All Viruses | | VRNAT (Old System) | | | |
|-------------------------|----------|--------------------|----------|-------|---------------------------------------|
| | | Positive | Negative | Total | |
| RVNATSP<br>(New System) | Positive | 65 | 0 | 65 | PPA 100.0 %<br>(95%CI=94.4% - 100.0%) |
| | Negative | 0 | 121 | 121 | NPA 100.0%<br>(95%CI=96.9% - 100.0%) |
| | Total | 65 | 121 | 186 | |
Table 8. RVNAT se Method Comparison Data collected at Site 2
| All Viruses | | VRNAT (Old System) | | | |
|-------------------------|----------|--------------------|----------|-------|--------------------------------------|
| | | Positive | Negative | Total | |
| RVNATSP<br>(New System) | Positive | 64 | 0 | 64 | PPA 98.5 %<br>(95%CI=91.8% - 99.7%) |
| | Negative | 1a | 121 | 122 | NPA 100.0%<br>(95%CI=96.9% - 100.0%) |
| | Total | 65 | 121 | 186 |…
