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The Verigene® Respiratory Virus Plus Nucleic Acid Test (RV+) on the Verigene® System is a qualitative nucleic acid multiplex test intended to simultaneously detect and identify multiple respiratory virus nucleic acids in nasopharyngeal (NP) swab specimens from individuals with signs and symptoms of respiratory tract infection. The following virus types and subtypes are identified using the RV+: Influenza A, Influenza A subtype H1, Influenza A subtype H3, 2009 H1N1, Influenza B, Respiratory Syncytial Virus (RSV) subtype A, and RSV subtype B. The test is not intended to detect Influenza C virus. Detecting and identifying specific viral nucleic acids from individuals exhibiting signs and symptoms of respiratory infection aids in the diagnosis of respiratory viral infection, if used in conjunction with other clinical and laboratory findings.

Negative results for Influenza A, Influenza B, or RSV do not preclude influenza virus or RSV infection and should not be used as the sole basis for diagnosis, treatment, or patient management decisions. Conversely, positive results do not rule-out bacterial infection or co-infection with other viruses. The agent detected may not be the definite cause of disease. The use of additional laboratory testing and clinical presentation must be considered in order to obtain the final diagnosis of respiratory viral infection.

Performance characteristics for Influenza A Virus were established when Influenza A/H3, A/H1, and 2009 H1N1 were the predominant Influenza A viruses circulating. These characteristics may vary when other Influenza A viruses are emerging.

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 used specifically 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 entire RV+ test is performed on the Verigene® System, which is a bench-top molecular diagnostics workstation that consists of two instruments, the Verigene Processor SP and the Verigene Reader. The Verigene Processor SP 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 original Verigene System with added modules for nucleic acid extraction and RT-PCR target amplification. Key functions of the Verigene Processor SP 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. 3) Real-time communication of test processing status to the Reader.

The Verigene Reader is the same instrument as in the FDA-cleared RVNATSP. 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 SP 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.

RV+ 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 Processor SP 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.
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 and subtype-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 Processor SP 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 subtracted, 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.

Here's an analysis of the acceptance criteria and the study proving the device meets those criteria, based on the provided text:

Acceptance Criteria and Device Performance for Verigene® Respiratory Virus Plus Nucleic Acid Test (RV+)

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state pre-defined acceptance criteria in terms of specific sensitivity and specificity thresholds. Instead, it presents the "Performance Characteristics" from a methods comparison study, and the acceptance is implied by the FDA's clearance of the device (K103209). Therefore, the "acceptance criteria" here are interpreted as the observed performance deemed acceptable for FDA clearance, and the "reported device performance" refers to the results from the clinical methods comparison study.

Reproducibility/Precision Study:
The document also presents reproducibility data, interpreted as another form of acceptance criteria for device performance stability.

• Total Agreement for all panel members across all 3 sites: 97.2% - 100% (95% CI range from 90.3% - 99.7% to 95.0% - 100.0%).
• "No Call" rate: 1.6% (14/864)
• "Pre-analytical error" failure rate: 0.2% (2/864)

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

• Sample Size for Test Set: 1022 prospectively-collected specimens were used for the methods comparison study.
• Data Provenance:
  • Country of Origin: Not explicitly stated, but the study was conducted at "three collection hospital sites" and shipped to "Nanosphere" (located in Northbrook, IL, USA) for processing and then to "testing sites." Given the FDA submission from a US-based company, it is highly probable the data is from the USA.
  • Retrospective or Prospective: The samples were collected prospectively during the 2008-2009 and 2009-2010 respiratory seasons. Residual specimens were then de-identified, frozen, and shipped to Nanosphere, then shipped to testing sites.

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

• Number of Experts: Not explicitly stated as "experts." The ground truth was established using a combination of methods:
  • Culture-based methods confirmed with an FDA-cleared Direct Fluorescent Antibody (DFA) test for Influenza A, Influenza B, and RSV.
  • Bidirectional sequencing for Influenza A subtyping (H3, H1, 2009 H1N1) and RSV subtyping (RSV A, RSV B).
  • For discordant results between RV+ and culture/DFA, bidirectional sequencing and/or NAAT (Nucleic Acid Amplification Test) was used for resolution.
• Qualifications of Experts: Not specified. The reference methods mentioned are laboratory-based standard tests, implying qualified laboratory personnel perform these tests, but no specific "expert" role or qualifications (e.g., years of experience) are detailed.

4. Adjudication Method for the Test Set

The adjudication method for the test set involved a multi-step process for resolving discrepancies:

• Initial comparison of RV+ results against culture-based methods confirmed with FDA-cleared DFA.
• For Influenza A subtyping and RSV subtyping, bidirectional sequencing was used as the primary comparative method.
• For discordant results between RV+ and culture/DFA, further clarification was sought using bi-directional sequencing and/or NAAT.
• Specific notes for individual discrepancies (e.g., footnote comments below the tables) illustrate this process, where sequencing or repeat testing with NAAT/culture informed the final ground truth. This resembles a tie-breaker adjudication approach for discordant results.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done

• No, a multi-reader multi-case (MRMC) comparative effectiveness study was NOT mentioned. This study focuses on the standalone performance of the RV+ device against established laboratory methods, not on comparing human reader performance with and without AI assistance. The device itself is a molecular diagnostic test, not an AI-powered image analysis tool for human readers.

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

• Yes, a standalone performance study was conducted. The methods comparison study evaluated the RV+ system (which includes sample preparation, target amplification, and a Verigene Hybridization Test with automated imaging and analysis, generating results automatically) as a direct comparison against standard laboratory ground truth methods. The Verigene System is described as a "bench-top molecular diagnostics workstation" that automates various steps and provides "results display" and "results report generation." This indicates a standalone performance evaluation of the device as a diagnostic tool without continuous human-in-the-loop adjustments to the test outcome.

7. The Type of Ground Truth Used

The ground truth used was a composite gold standard primarily consisting of:

• Culture-based methods confirmed with an FDA-cleared DFA test: For general detection of Influenza A, Influenza B, and RSV.
• Bidirectional sequencing: For subtyping of Influenza A (H3, H1, 2009 H1N1) and RSV (RSV A, RSV B), and for resolution of discordant results.
• NAAT (Nucleic Acid Amplification Test): Used for further clarification in cases of culture/DFA discordance.

This approach uses a combination of established laboratory diagnostic techniques.

8. The Sample Size for the Training Set

The document does not report a separate training set or its sample size for the Verigene® Respiratory Virus Plus Nucleic Acid Test (RV+). The provided information focuses on the validation studies (analytical and methods comparison) for the final trained/developed device. Molecular diagnostic tests like this one are typically developed through iterative optimization and then validated, rather than having a distinct "training set" in the machine learning sense for the final product evaluation.

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

Since a distinct "training set" is not reported in the context of device development as per the provided text, the method for establishing its ground truth is also not applicable or reported. The focus is on the performance evaluation of the already developed device using the methods comparison and reproducibility studies.

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