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The RIDA® GENE Norovirus GI/GII assay, performed on the Applied Biosystems® 7500 Fast Dx System, is a real-time RT-PCR in vitro diagnostic test for the qualitative detection and differentiation of norovirus genogroup I (GI) and II (GII) RNA from raw or unpreserved stool specimens collected from individuals with signs and symptoms of acute gastroenteritis.

The RIDA®GENE Norovirus GUGII assay is intended for use as an aid in the differential diagnosis of norovirus genogroup I and II infections in patients symptomatic for gastroenteritis in conjunction with clinical evaluation. laboratory findings, and epidemiological information. The assay aids in the detection and identification of norovirus infections as the cause of acute gastroenteritis in sporadic cases as well as in the context of outbreaks.

Negative results do not preclude a norovirus infection and should not be used as the sole basis for diagnosis.

The RIDA®GENE Norovirus GI/GII assay, performed on the Applied Biosystems® 7500 Fast Dx System, is a real-time RT-PCR in vitro diagnostic test for the qualitative detection and differentiation of norovirus genogroup I (GI) and II (GII) RNA in human stool specimens. The assay also detects an internal control RNA (ICR, bacteriophage MS2) that is added to each sample prior to extraction. Sample preparation and amplification/real-time detection are completed on separate instruments. Each sample is pre-treated prior to extraction and sample processing is completed on the bioMérieux NucliSENS® easyMAG® instrument with bioMérieux NucliSENS® Nucleic Acid Extraction Reagents according to the manufacturer's instructions. The ICR serves to monitor inhibitors in the extracted specimen; it assures that adequate amplification has taken place and confirms that the nucleic acid extraction was sufficient.

Following processing, either extracted nucleic acids or extracted negative control (NC) or positive control (PC) material is added to the Master-Mix. The assay is performed on an Applied Biosystems® 7500 FAST Dx System. The detection is performed in a one-step real-time RT-PCR format where the reverse transcription is followed by the PCR in the same reaction tube under optimized conditions. The isolated RNA is transcribed into cDNA by a reverse transcriptase. Gene fragments specific for norovirus GI and GII are subsequently amplified by real-time PCR. The amplified targets (ORF1/ORF2 conserved junction region) are detected with hydrolysis (TaqMan®) probes, which are labeled at one end with a quencher and at the other end with a fluorescent reporter dye (fluorophore). In the presence of a target, the probe(s) hybridize to it and during the extension step the Taqpolymerase breaks the reporter-quencher proximity. Upon excitation by the Applied Biosystems® 7500 FAST Dx's halogen light source, the reporter emits a distinct fluorescent signal which is detected by the optical unit of the Applied Biosystems® 7500 FAST Dx System. Hence, depending on the target sequence present (genogroup GI, GII or both), one, two or none of the reporters on the norovirus specific probes emits light to be detected by the instrument. Fluorophores are chosen in a way that their excitation and emission wavelengths do not overlap and signals are readily discriminated by the software. The fluorescence signal increases with the amount of formed amplicons.

Here's an analysis of the RIDA®GENE Norovirus GI/GII assay's acceptance criteria and the study proving it meets them, based on the provided text:

Acceptance Criteria and Device Performance for RIDA®GENE Norovirus GI/GII Assay

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state pre-defined acceptance criteria (e.g., "PPA must be > X%, NPA must be > Y%") for clinical performance. Instead, it presents the results of the clinical study which are intended to demonstrate substantial equivalence to the predicate device. The values reported therefore are the "reported device performance."

However, based on the nature of diagnostic molecular assays, common acceptance metrics are Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) with a reference method. The clinical study results are presented against these metrics.

Note: The document only provides actual performance values rather than specified acceptance thresholds. The "acceptance criteria" here are implied to be that the performance is sufficiently good to demonstrate substantial equivalence.

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

• Sample Size for Clinical (Test) Set: A total of 1019 raw or unpreserved stool specimens were used for the clinical performance study.
  • This included 769 samples collected prospectively from February 2014 to April 2015. Out of these, 50 could not be used, leaving 719 samples.
  • Additionally, 332 retrospectively collected samples from various previous outbreaks were tested from September 2016 to April 2017. Out of these, 300 provided valid results.
  • The sum (719 + 300) does not exactly equal 1019, suggesting either some overlap or different accounting for invalid samples. However, the document clearly states "A total of 1019 study specimens consisted of raw or unpreserved stool specimens".
• Data Provenance:
  • Country of Origin: United States (multi-center study conducted at four institutions in the U.S.).
  • Retrospective or Prospective: A combination of prospective (769 samples) and retrospective (332 samples) data was used.

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

The ground truth for the clinical test set was established by a "composite reference method performed at the CDC (Atlanta, GA)."

• Number of Experts: Not specified. The reference method involved specialized laboratory testing at the CDC, but it doesn't state if expert adjudication (e.g., individual opinion or consensus) was part of establishing the final composite reference result beyond the laboratory procedure itself.
• Qualifications of Experts: Not specified beyond the fact that the testing was performed at the CDC, implying highly qualified laboratory personnel. The reference method itself ("conventional RT-PCR followed by bi-directional sequencing") is a highly technical and objective method, rather than a subjective expert interpretation.

4. Adjudication Method for the Test Set

The ground truth was a "composite comparator method that consisted of a combination of Center for Disease Control and Prevention (CDC) RT-PCR assays and bi-directional sequencing for norovirus." This describes a laboratory-based, objective reference standard, not a human consensus or adjudication process in the traditional sense that might be seen in imaging studies (e.g., "2+1" rule for radiologists). The final determination would have been based on the results of these specialized molecular tests.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not performed. This study focuses on the standalone performance of the RIDA®GENE Norovirus GI/GII assay against a laboratory reference standard. It does not involve human readers interpreting results, nor does it compare human performance with and without AI assistance.

6. Standalone (Algorithm Only Without Human-in-the-Loop Performance) Study

Yes, a standalone performance study was conducted. The document describes the "Clinical Performance" of the RIDA®GENE Norovirus GI/GII assay as a diagnostic test operating on its own (after sample preparation and instrument processing) to detect and differentiate norovirus GI and GII RNA. The results (PPA and NPA) are for the device's performance against the reference method without human interpretation of the assay's output influencing the direct comparison.

7. Type of Ground Truth Used for Clinical Test Set

The ground truth used for the clinical test set was a composite reference method consisting of:

• Conventional RT-PCR assays
• Bi-directional sequencing for both Region C and Region D of norovirus.

This is a laboratory-based, highly sensitive, and specific molecular gold standard.

8. Sample Size for the Training Set

The document does not specify the sample size used for the training set. This is a common characteristic of medical device submissions for molecular assays like RT-PCR kits, where the 'training' of the assay is typically based on optimizing primers, probes, and reaction conditions during development, using a variety of known positive and negative controls and clinical samples to establish analytical performance characteristics (like LoD, inclusivity, exclusivity). It's not a machine learning model that undergoes explicit "training" with a labeled dataset in the same way.

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

As noted above, a distinct "training set" with a formally established ground truth in the context of machine learning is not described. For the development and optimization of the assay:

• Analytical Sensitivity (LoD): Established using dilution series of native fecal samples (genogroup I and II) where norovirus RNA copy numbers were determined by standard curves using quantified transcripts. The genogroup of native samples was determined by conventional RT-PCR followed by bi-directional sequencing.
• Analytical Specificity (Cross-Reactivity): Evaluated against a panel of 69 known organisms (bacteria, fungus, viruses, parasites) whose identity was known.
• Analytical Reactivity (Inclusivity): Evaluated against 24 known norovirus genotypes (GI and GII strains) at low and high concentrations.

Thus, the ground truth for establishing analytical performance characteristics (which inform the assay's design and "training") relies on known, characterized isolates/strains and quantified reference materials, often confirmed by gold-standard molecular methods like sequencing.

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