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The Quidel Molecular RSV + hMPV assay is a multiplex Real Time RT-PCR assay for the in vitro qualitative detection and identification of respiratory syncytial virus and human metapneumovirus viral RNA extracted from nasal and nasopharyngeal swabs specimens from patients with signs and symptoms of respiratory infection. This in vitro diagnostic test is intended to aid in the differential diagnosis of respiratory syncytial virus and human metapneumovirus infections. This test is not intended to differentiate the four genetic sub-lineages of hMPV.

Negative results do not preclude RSV or hMPV infection and should not be used as the sole basis for diagnosis, treatment or other patient management decisions.

The Quidel Molecular RSV + hMPV Assay detects viral nucleic acids that have been extracted from a patient sample using the NucliSENS® easyMAG® automated extraction platform. A multiplex RT-PCR reaction is carried out under optimized conditions in a single tube generating amplicons for each of the target viruses present in the sample. This reaction is performed utilizing either the Cepheid SmartCycler® II or the Applied Biosystems 7500 Fast DX. Identification of RSV and hMPV and the PRC occurs by the use of target-specific primers and fluorescent-labeled probes that hybridize to conserved regions in the genomes of RSV and hMPV and the PRC.

Here's a summary of the acceptance criteria and study details for the Quidel Molecular RSV + hMPV Assay, based on the provided text:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria provided in the document are primarily for analytical performance (LoD, Reproducibility, Inclusivity, Specificity) and clinical performance (Sensitivity and Specificity/Positive and Negative Percent Agreement). The clinical performance is reported compared to predicate devices or established methods.

Device: Quidel Molecular RSV + hMPV Assay

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

• Clinical Performance Test Set Samples:
  • Total samples collected: 1014 specimens (414 fresh, 600 frozen) for RSV comparison.
  • RSV testing (SmartCycler II): 1009 specimens after excluding contaminated cell cultures.
  • hMPV testing (SmartCycler II): 951 specimens after excluding invalid comparative device results.
  • RSV testing (7500 Fast Dx): 1007 specimens after excluding contaminated cell cultures and invalid subject method results.
  • hMPV testing (7500 Fast Dx): 946 specimens after excluding invalid comparative and subject method results.
• Data Provenance: The samples were collected prospectively during the 2012 respiratory virus season (January to March 2012) from symptomatic patients at four sites across the United States. The study specifically used "fresh (414) and frozen (600) swab specimens."

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

The document does not explicitly state the number or qualifications of experts used. However, the ground truth for RSV was established using "DSFA & Cell Culture w/DFA" (Direct Specimen Fluorescent Antibody and Cell Culture with DFA), which implies interpretation by trained laboratory personnel or specialists, although their specific qualifications or number are not detailed. For hMPV, the ground truth was established by comparing it to the "FDA Cleared hMPV molecular test" (Gen-Probe Prodesse Pro hMPV+, K082688), which is a molecular diagnostic method rather than expert interpretation of raw data.

4. Adjudication Method for the Test Set

• RSV Discrepant Results:
  • For the SmartCycler II, all 21 originally discordant specimens (QM RSV + hMPV positive, reference method negative) were positive for RSV by an FDA-cleared RT-PCR assay and by bi-directional sequence analysis.
  • For the 7500 Fast Dx, 25 of 28 originally discordant specimens were positive by an FDA-cleared RT-PCR assay, and 27 of 28 were positive by bi-directional sequence analysis.
• hMPV Discrepant Results:
  • For both the SmartCycler II and the 7500 Fast Dx, all originally discordant specimens (QM RSV + hMPV positive, reference method negative) were positive for hMPV by bi-directional sequence analysis.

This indicates a form of post-hoc adjudication or discrepancy resolution using additional, more definitive molecular methods (FDA-cleared RT-PCR and bi-directional sequencing) for cases where the subject device and the initial reference method disagreed.

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, this was not an MRMC study. The device is an in vitro diagnostic (molecular assay) for direct detection of viral RNA, not an imaging device requiring human reader interpretation or AI assistance in interpretation. Therefore, a multi-reader multi-case comparative effectiveness study on human reader improvement with or 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 clinical performance study evaluates the standalone performance of the Quidel Molecular RSV + hMPV Assay. The results are presented as the device's agreement (sensitivity, specificity, positive/negative percent agreement) compared directly to the reference methods, without human interpretation of the assay results impacting the reported performance metrics. The assay itself provides a qualitative (positive/negative) result based on its internal thresholding (e.g., fluorescence achieved by 50 cycles on SmartCycler II or 35 cycles on ABI 7500 Fast Dx).

7. The Type of Ground Truth Used

• For RSV: The ground truth for clinical performance was established using Direct Specimen Fluorescent Antibody (DSFA) and Cell Culture with DFA.
• For hMPV: The ground truth for clinical performance was established using an FDA Cleared hMPV molecular test (Gen-Probe Prodesse Pro hMPV+).
• For discrepant results: Bi-directional sequence analysis and/or another FDA-cleared RT-PCR assay were used as definitive ground truth.

8. The Sample Size for the Training Set

The document does not explicitly state a sample size for a "training set" in the context of machine learning or algorithm development. For this type of molecular diagnostic assay, analytical studies (LoD, inclusivity, specificity) and clinical validation are performed. The LoD study involved replicates of serially diluted viral cultures, and inclusivity/specificity studies used panels of various strains/organisms. These analytical studies are analogous to "training" or "development" data in that they inform and validate the assay's operational parameters, but they are not framed as a classic machine learning training set.

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

Given that this is a molecular diagnostic assay, the "ground truth" for establishing analytical parameters (like LoD, inclusivity, and specificity) is based on:

• Quantified viral cultures (TCID50/mL): Used for LoD studies, where the exact concentration of virus is known.
• Known viral strains or bacterial/yeast cultures: Used for inclusivity (known to contain the target virus) and specificity (known to contain other organisms to test for cross-reactivity) studies. The identity and concentration of these cultures are established through standard microbiological and virological techniques.

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The Diagnostic Hybrids, Inc. device, D3 DFA Metapneumovirus Identification Kit, is intended for the qualitative detection and identification of human metapneumovirus (hMPV) in nasal and nasopharyngeal swabs and aspirates/washes or cell culture. The assay detects hMPV antigens by immunofluorescence using a blend of three monoclonal antibodies (MAbs), from patients with signs and symptoms of acute respiratory infection. This assay detects but is not intended to differentiate the four recognized genetic sub-lineages of hMPV.

Negative results do not preclude hMPV infection and should not be used as the sole basis for diagnosis, treatment or other management decisions. It is recommended that specimens found to be negative after examination of the direct specimen results be confirmed by an FDA cleared hMPV molecular assay.

The D DFA Metapneumovirus Identification Kit uses a blend of three hMPV antigen-specific murine MAbs that are directly labeled with fluorescein for detection of hMPV. The reagent detects but does not differentiate between the four recognized subtypes of hMPV (subtypes A1, A2, B1, and B2).

Kit Components:

• 1. Metapneumovirus DFA Reagent, 5-mL. One dropper bottle containing a blend (see below for MAb discussion) of fluorescein-labeled murine monoclonal antibodies directed against MPV. The buffered, stabilized, aqueous solution contains Evans Blue as a counter-stain and 0.1% sodium azide as a preservative.
• 2. hMPV Antigen Control Slides, 5 slides. Five individually packaged control slides, each with a well containing cell culture-derived MPV positive cells and a well containing cell culture-derived negative cells. Each slide is intended to be stained only one time. Control material has been treated to be non-infectious; however normal laboratory precautions are required when the material is handled.
• 3. 40X PBS Concentrate, 25-mL. One bottle containing a 40X concentrate consisting of 4% sodium azide (0.1% sodium azide after dilution to 1X using de-mineralized water) in PBS.
• 4. Mounting Fluid, 7-mL. One dropper bottle containing an aqueous, buffer-stabilized solution of glycerol and 0.1% sodium azide.

The cells to be tested, derived from a clinical specimen or cell culture, are placed onto a glass slide, allowed to air dry and are fixed in acetone. The Metapneumovirus DFA Reagent is added to the cells which are then incubated for 15 to 30 minutes at 35℃ to 37℃ in a humidified chamber or humidified incubator. The stained cclls are then washed with the diluted phosphate buffered saline (PBS), a drop of the supplied Mounting Fluid is added and a coverslip is placed on the prepared cells. The cells are examined using a fluorescence microscope. The hMPV infected cells will fluoresce apple-green. Uninfected cells will contain no fluorescence but will be stained red by the Evans Blue counter-stain.

It is recommended that specimens found to contain no fluorescent cells after examination of the direct specimen be confirmed by an FDA cleared hMPV molecular assay.

Here's an analysis of the acceptance criteria and the study that proves the device meets them, based on the provided text:

Device Name: D3 DFA Metapneumovirus Identification Kit

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are implied by the performance results required for clearance. As this is not a modern AI/ML device, the performance metrics are clinical sensitivity and specificity, or Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA). Acceptance usually implies that these metrics meet certain thresholds, particularly with lower bounds of the 95% Confidence Interval (CI) demonstrating adequate performance.

For Direct Specimen Testing (Clinical Studies Sites 1-3):

*Note: For Study Site 3, Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) were reported instead of sensitivity and specificity due to the comparator method used.

For Cultured Cells Testing (Clinical Study Site 4):

For Reproducibility (Analytical Performance):

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

The clinical performance studies used the following sample sizes for the test set:

• Study Site 1: 1482 fresh nasal wash/nasopharyngeal aspirate specimens.
• Study Site 2: 368 fresh nasal/nasopharyngeal swab specimens.
• Study Site 3: 32 fresh nasal wash/nasopharyngeal aspirate specimens and 66 fresh nasal/nasopharyngeal swab specimens.
• Study Site 4 (Cultured Cells): 74 freeze-thawed nasopharyngeal swab specimens that were cultured.

Data Provenance: The data was collected during prospective studies at 3 geographically diverse U.S. clinical laboratories (Study Sites 1-3) during the 2005-2006 and 2006-2007 respiratory virus seasons (December 2005 - April 2006 and December 2006 - March 2007). Study Site 4, for cultured cells, was performed at DHI during the 2007-2008 respiratory virus season (January - April 2008). Specimens were "excess, remnants of respiratory specimens that were prospectively collected from symptomatic individuals suspected of respiratory infection, and were submitted for routine care or analysis by each site, and that otherwise would have been discarded." Individual specimens were de-linked from all patient identifiers. All clinical sites were granted waivers of informed consent by their IRBs.

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

The document does not explicitly state the number of experts or their qualifications for establishing the ground truth. It describes the ground truth as composite comparator methods or a single comparator assay in different sites:

• Sites 1 and 2: Ground truth was a composite comparator method consisting of viral culture and a validated real-time RT-PCR comparator assay with bi-directional sequencing analysis. "True" hMPV positive was defined as positive by viral culture OR positive by RT-PCR with sequencing matching hMPV. "True" hMPV negative was defined as negative by both viral culture and RT-PCR.
• Site 3 and 4: Ground truth was based solely on the validated hMPV real-time RT-PCR followed by bi-directional sequencing analysis comparator assay. Positive was defined by sequencing data matching hMPV, and negative by negative RT-PCR.

While these methods are considered reference standards in microbiology, the document does not specify human expert involvement in interpreting these results or adjudicating discrepancies, beyond the inherent expertise in running and interpreting these laboratory assays.

4. Adjudication Method for the Test Set

The document does not explicitly detail an "adjudication method" in the sense of multiple human readers resolving disagreements, as would be typical for image-based AI studies. Instead, the ground truth itself is a carefully defined reference standard.

• For Sites 1 and 2, the ground truth was a composite definition:
  • Positive: Viral culture positive OR Real-time RT-PCR positive with bi-directional sequencing matching hMPV.
  • Negative: Viral culture negative AND Real-time RT-PCR negative.
• For Sites 3 and 4, the ground truth was based on the hMPV real-time RT-PCR followed by bi-directional sequencing analysis comparator assay. Positive was defined by acceptable sequencing data matching hMPV, and negative by negative RT-PCR.

This structure inherently handles potential discrepancies between methods by prioritizing certain outcomes (e.g., a positive by either method for the composite ground truth).

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 involving human readers and AI assistance was not done. This device is a diagnostic kit (an immunofluorescence assay), not an AI-powered system designed to assist human readers in interpreting complex data. The clinical studies evaluated the standalone performance of the kit itself against a reference standard.

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

Yes, the studies described are essentially standalone performance studies for the D3 DFA Metapneumovirus Identification Kit. The kit's results (fluorescence detection by microscopy) were compared directly against the established ground truth without any involvement of a human-in-the-loop for interpreting the kit's results in the context of an AI system. The "algorithm" here is the biochemical and optical detection mechanism of the DFA test combined with human interpretation of fluorescence under a microscope, as per standard laboratory practice.

7. The Type of Ground Truth Used

The type of ground truth used varied slightly across study sites but primarily involved molecular and classical microbiological methods:

• Clinical Study Sites 1 and 2: Composite Ground Truth combining:
  • Viral Culture: A classical microbiological method for isolating and identifying viruses.
  • Validated Real-time RT-PCR followed by bi-directional sequencing analysis: A molecular method to detect hMPV nucleic acid, with sequencing confirming the identity.
• Clinical Study Sites 3 and 4: Molecular Ground Truth based solely on a validated hMPV real-time RT-PCR followed by bi-directional sequencing analysis. This method is considered a highly specific and sensitive reference standard.

8. The Sample Size for the Training Set

No information about a "training set" for an algorithm is provided. This device is a diagnostic kit (DFA assay), not an AI/ML model that undergoes a training phase.

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

As there is no "training set" for an AI/ML algorithm in this context, this question is not applicable. The device relies on direct antigen detection via immunofluorescence, not on learned patterns from a training dataset.

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