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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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The RIDASCREEN® Norovirus 3td Generation test is a qualitative enzyme immunoassay (EIA) intended for the detection of selected genogroup I (GI.1, GL.2, GI.3, GI.4, GL.7) and genogroup II (GII.1, GII.2, GII.3, GII.4, GII.5, GII.6, GII.7, GII.8, GII.10, GII.12, GII.13, GII.14, GII.17) norovirus strains in human feces as an aid in investigating the cause of acute gastroenteritis outbreaks. The likelihood of detecting a norovirus outbreak by use of the RIDASCREEN® Norovirus 3td Generation test improves as the number of patients tested during an outbreak increases, as well as when quality of the specimens increases. Preliminary identification of norovirus as the cause of an acute gastroenteritis outbreak by RIDASCREEN® Norovirus 3td Generation testing should be confirmed by reference methods as appropriate, particularly if only a limited number of positive samples are associated with a suspected outbreak. Additional testing of negative samples by other methods should be performed if norovirus is strongly suspected as the cause of an acute gastroenteritis outbreak.

RIDASCREEN® Norovirus 3rd Generation test is a solid phase sandwich-type EIA for the detection of genogroups GI and GII noroviruses in stool samples. Microwell strips are coated with a mixture of GI and GII norovirus specific monoclonal antibodies. An aliquot of fecal suspension is added to the microwell together with biotinylated monoclonal norovirus antibodies. After washing, streptavidin peroxidase conjugate is added. Norovirus antigens that are present in the stool sample are captured in a sandwich complex of the immobilized antibodies, the norovirus antigens and the monoclonal antibodies conjugated with the biotin-streptavidin-peroxidase complex. Unbound streptavidin peroxidase conjugate is removed by washing and a chromogenic colorless substrate solution (hydrogen peroxide/TMB) is added. The substrate is hydrolyzed by any bound peroxidase, changing the chromogen to a blue color. Stopping the reaction with acid converts the blue to a yellow color indicating the presence of norovirus antigens.

Test results are read photometrically; intensity significantly above background levels is indicative of the presence of Norovirus antigen in the specimen or control. The RIDASCREEN® Norovirus 3rd Generation test does not identify specific norovirus strains.

Here's an analysis of the provided text, extracting the requested information about acceptance criteria and the device study:

1. A table of acceptance criteria and the reported device performance

The document does not explicitly state formal "acceptance criteria" against which the device's performance was measured. Instead, it reports the device's performance characteristics. If we were to infer acceptance criteria from the reported performance in the Overall Clinical Trial, a reasonable interpretation would be the achieved positive agreement and negative agreement.

Inferred Acceptance Criteria (based on reported performance):

2. Sample size used for the test set and the data provenance (e.g. country of origin of the data, retrospective or prospective)

• Sample Size (Test Set): 609 samples (after excluding 69 invalid samples and 2 equivocal samples from an initial collection of 680).
• Data Provenance:
  • Country of Origin: Three sites in the United States (Ohio, California, Cincinnati) and one site in Canada (Hamilton, Ontario).
  • Retrospective or Prospective: Prospective (samples were collected from patients presenting with acute gastroenteritis symptoms).

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts (e.g. radiologist with 10 years of experience)

The ground truth for the test set was established using a "Clinical Diagnostic Truth" algorithm based on a composite reference endpoint, not directly by experts. The reference methods included:

• Quantitative real-time RT-PCR (qRT-PCR)
• Conventional RT-PCR with bi-directional sequencing (Region D and Region B)
• Electron Microscopy (EM)

While these methods would be performed by trained laboratory personnel, the document does not specify the number or qualifications of experts (e.g., virologists, molecular biologists) who established the final "Clinical Diagnostic Truth." However, the reference testing was conducted at established public health and disease control laboratories: the California Dept. of Public Health (CPDH) and the National Calicivirus Laboratory, Centers for Disease Control and Prevention (CDC).

4. Adjudication method (e.g. 2+1, 3+1, none) for the test set

The ground truth was established by a rule-based algorithm, not an adjudication process involving multiple human readers in the traditional sense. The algorithm for "Clinical Diagnostic Truth" considered a sample positive if it was positive by:

• Region D RT-PCR OR
• Region B RT-PCR OR
• Electron Microscopy (EM)

If all three methods were negative, the sample was considered negative. There was an initial "Original Composite 'Clinical Diagnostic Truth' Algorithm" that included qRT-PCR, but this was revised due to concerns, and the final algorithm focused on Region D RT-PCR, Region B RT-PCR, and EM.

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, a multi-reader multi-case (MRMC) comparative effectiveness study was not done. This device is a diagnostic immunoassay for Norovirus detection, not an AI-assisted diagnostic tool designed to be used by human readers (like a radiologist reading an AI-analyzed image). Therefore, the concept of human readers improving with/without AI assistance does not apply.

6. If a standalone (i.e. algorithm only without human-in-the-loop performance) was done

Yes, the study primarily focused on standalone performance of the RIDASCREEN® Norovirus 3rd Generation EIA. The device is a qualitative enzyme immunoassay that provides a positive or negative result. The "Overall Clinical Trial Performance" table directly compares the results of the RIDASCREEN® assay against the "Clinical Diagnostic Truth" reference standard. There is no indication of human interpretation influencing the ELISA's output or any human-in-the-loop step being assessed as part of its performance.

7. The type of ground truth used (expert consensus, pathology, outcomes data, etc)

The ground truth used was a composite reference standard (referred to as "Clinical Diagnostic Truth"). This was established through a combination of molecular assays (conventional RT-PCR with bi-directional sequencing for regions B and D, and quantitative real-time RT-PCR initially) and electron microscopy (EM). This is a laboratory-based, objective ground truth, which is often considered highly reliable for viral detection.

8. The sample size for the training set

The document does not explicitly mention a "training set" in the context of device development or a formal machine learning model. For this device, an immunoassay, the concept of a "training set" as understood in AI/ML is not directly applicable. Performance characteristics (like precision, detection limit, cross-reactivity, and strain reactivity) were evaluated using panels of samples, but these are typically part of analytical validation rather than training a model.

The "Strain-reactivity" section mentions the sponsor studied 100 archived samples (80 positive across different genotypes, 10 negative, 10 other viruses) but this was for analytical characterization, not model training.

9. How the ground truth for the training set was established

As there's no explicitly defined "training set" in the AI/ML sense, a ground truth establishment method for it isn't described. For the analytical studies (e.g., LoD, strain reactivity) that used well-characterized samples, the ground truth was based on known viral concentrations or confirmed genotypes through methods like conventional RT-PCR followed by sequencing and electron microscopy.

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