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The Lyra RSV + hMPV Assay is a multiplex Real-Time PCR (RT-PCR) assay for the qualitative detection and identification of respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) ribonucleic acid (RNA) extracted from nasal and nasopharyngeal swab specimens from patients with signs and symptoms of respiratory infection. This in vitro diagnostic test is intended to aid in the differential diagnosis of RSV and hMPV infections in humans in conjunction with clinical and epidemiological risk factors. This test is not intended to differentiate the two subtypes of RSV or the four genetic sub-lineages of hMPV.

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

Conversely, positive results do not rule-out bacterial 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.

The Lyra RSV + hMPV Assay can be performed using ether the Life Technologies QuantStudio Dx RT-PCR Instrument, the Applied Biosystems 7500 Fast Dx RT-PCR Instrument, or the Cepheid SmartCycler II System.

The Lyra RSV + hMPV Assay detects viral nucleic acids that have been extracted from a patient sample using the NucliSENS® easyMAG® or NucliSENS® EMAG® automated extraction platform. A multiplex RT-PCR reaction is then performed 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, the Applied Biosystems 7500 Fast DX, or the Life Technologies QuantStudio" 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.

The acceptance criteria and the study proving the device meets these criteria for the Lyra RSV + hMPV Assay are detailed below. It's important to note that the provided documents primarily describe a change to an existing device (the addition of a new extraction platform, BioMerieux NucliSENS EMAG) and compare its performance to the previously cleared predicate device. Therefore, the "acceptance criteria" and "reported device performance" are framed around this equivalency.

1. Table of Acceptance Criteria and Reported Device Performance

Based on the information provided, the overall acceptance criterion is equivalent performance of the modified Lyra RSV + hMPV Assay (with the new extraction platform) to the predicate device (Quidel RSV+hMPV Assay using the original extraction platform). The performance is assessed through "non-clinical and clinical verification and validation activities."

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

The exact sample sizes for the "Limit of Detection Equivalency Study" and "Clinical Equivalence Study" are not explicitly stated in the provided 510(k) summary.

• Test Set Sample Size: Not specified.
• Data Provenance: Not specified (e.g., country of origin, retrospective or prospective). The nature of "clinical equivalence study" typically implies prospective or retrospectively collected clinical samples, but details are lacking.

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

This information is not provided in the given text. For a PCR-based assay like this, ground truth is typically established using a highly sensitive and specific reference method (e.g., another validated PCR assay or sequencing), rather than expert clinical consensus in the traditional sense of image interpretation.

4. Adjudication Method for the Test Set

This information is not provided in the given text. Given that this is a molecular diagnostic assay, adjudication methods like N+1 for expert review (common in imaging studies) are generally not applicable. Instead, the "ground truth" would be determined by the reference method itself.

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

No, an MRMC comparative effectiveness study was not done. This type of study is primarily relevant for medical imaging interpretation where different human readers interpret cases, often with and without AI assistance. This device is a molecular diagnostic assay (RT-PCR) and does not involve human interpretation of complex images in the same way.

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

Yes, implicitly. The device itself is an in vitro diagnostic test, specifically a Real-Time PCR assay. Its performance (qualitative detection of RNA) is inherently "standalone" as it produces a result without direct human interpretive input during the assay itself. The studies ("Limit of Detection Equivalency Study" and "Clinical Equivalence Study") would evaluate the accuracy of the assay's output against a reference method, which is a standalone performance assessment.

7. The Type of Ground Truth Used

The type of ground truth used is not explicitly stated but can be inferred to be a highly reliable reference method, likely another validated molecular diagnostic test (e.g., gold standard PCR assay or culture/sequencing) for detecting RSV and hMPV. For Limit of Detection studies, ground truth would be established by precisely quantifying viral RNA in samples.

8. The Sample Size for the Training Set

This information is not applicable in the context of this device. The Lyra RSV + hMPV Assay is a Real-Time PCR assay, which is a biochemical reaction-based test, not an AI/Machine Learning algorithm that requires a "training set" in the traditional sense. The "training" for such an assay involves optimization of reagents, primers, probes, and reaction conditions.

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

This information is not applicable as there is no "training set" in the context of an AI/ML algorithm for this device. The development of the assay involves establishing analytical ground truth through various laboratory experiments to ensure the primers and probes are specific and sensitive to the target viruses, and that the internal control functions correctly.

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The Prodesse® Pro hMPV®+ Assay is a Real-Time PCR (RT-PCR) in vitro diagnostic test for the qualitative detection of human Metapneumovirus (hMPV) nucleic acid isolated and purified from nasopharyngeal swab (NP) specimens obtained from individuals exhibiting signs and symptoms of acute respiratory infection. This Assay targets a highly conserved region of the Nucleocapsid gene of hMPV. The detection of hMPV nucleic acid from symptomatic patients aids in the diagnosis of human respiratory hMPV infection if used in conjunction with other clinical and laboratory findings. This test is not intended to differentiate the four 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.

The Pro hMPV+ Assay enables detection of human Metapneumovirus and internal control nucleic acid. Nasopharyngeal swab specimens are collected from patients with signs and symptoms of a respiratory infection using a polyester, rayon or nylon tipped swab and placed into viral transport medium.

A Universal Internal Control (UIC) is added to each sample prior to nucleic acid isolation to monitor for inhibitors present in the specimens. The isolation and purification of the nucleic acids is performed using either a MagNA Pure LC Instrument (Roche) and the MagNA Pure Total Nucleic Acid Isolation Kit (Roche) or a NucliSENS® easyMAG™ System (bioMérieux) and the Automated Magnetic Extraction Reagents (bioMérieux).

The purified nucleic acids are added to Pro hMPV+ Supermix along with enzymes included in the Pro hMPV+ Assay Kit. The Pro hMPV+ Supermix contains oligonucleotide primers complementary to a highly conserved region of the Nucleocapsid gene of hMPV and a targetspecific oligonucleotide probe dual-labeled with a reporter dye attached to the 5'-end and a quencher dve attached to nucleotide #7 from the 5 end.

Reverse transcription of the RNA in the sample into complementary DNA (cDNA) and subsequent amplification of DNA is performed in a Cepheid SmartCycler® II instrument. In this process, the probe anneals specifically to the template followed by primer extension and amplification. The Pro hMPV+ Assay is based on Taqman chemistry, which utilizes the 5' - 3' exonuclease activity of the Taq polymerase to cleave the probe thus separating the reporter dye from the quencher. This generates an increase in fluorescent signal upon excitation from a light source. With each cycle, additional reporter dye molecules are cleaved from their respective probes, further increasing fluorescent signal. The amount of fluorescence at any given cycle is dependent on the amount of amplification products present at that time. Fluorescent intensity is monitored during each PCR cycle by the SmartCyclerII instrument.

Here's a breakdown of the acceptance criteria and study information for the Prodesse® Pro hMPV®+ Assay, based on the provided document:

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state numerical acceptance criteria in terms of sensitivity, specificity, or predictive values. Instead, the "Verification/Validation Result" column of the "SUBSTANTIAL EQUIVALENCE" table functions as the reported performance, indicating that the modified device continues to meet the performance claims of the predicate device.

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

The document mentions a "retrospective clinical comparison study" (page 2), but does not specify the sample size used for this study. It also does not explicitly state the country of origin. The term "retrospective" indicates that the data was collected prior to the study being designed.

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

The document does not provide information on the number of experts used or their qualifications for establishing ground truth in the clinical comparison study.

4. Adjudication Method for the Test Set

The document does not specify any adjudication method for the test set used in the clinical comparison study.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was done, and the Effect Size of Human Readers Improve with AI vs. Without AI Assistance

This question is not applicable to this device. The Prodesse® Pro hMPV®+ Assay is an in vitro diagnostic test (Real-Time PCR) for qualitative detection of nucleic acid, not an AI-powered diagnostic imaging or interpretation device that would involve human readers or MRMC studies.

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

Yes, the studies described (analytical sensitivity, IC interference, extractor equivalency, and sample stability) and the clinical comparison study effectively demonstrate the standalone performance of the assay as an in vitro diagnostic test. The "algorithm" here is the assay's chemical and enzymatic process, and its performance is evaluated in isolation.

7. The Type of Ground Truth Used

Based on the nature of the device (a diagnostic test for a pathogen), the ground truth for human Metapneumovirus (hMPV) infection would likely be based on clinical diagnosis in conjunction with other laboratory findings, as suggested by the intended use statement: "The detection of hMPV nucleic acid from symptomatic patients aids in the diagnosis of human respiratory hMPV infection if used in conjunction with other clinical and laboratory findings."
For the analytical studies (LOD, interference, etc.), the ground truth would be established by controlled experiments with known concentrations of the target analyte (hMPV nucleic acid) and known interfering substances.

8. The Sample Size for the Training Set

The document does not mention a training set sample size. This is common for predicate-based 510(k) submissions where the device "continues to meet the performance claims" of an already approved device, rather than being a de novo artificial intelligence or machine learning device that requires explicit training data. The development of the original predicate device would have involved internal optimization and validation, but these details are not provided for this submission.

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

As no explicit "training set" is mentioned in the context of this 510(k) submission (due to it being a modification of an existing device), the method for establishing ground truth for a training set (if one were used in the original development) is not described. For the development and optimization of the original assay, ground truth would have been established through methods similar to those described in point 7, involving known positive and negative controls, spiked samples, and potentially clinical samples with confirmed hMPV status.

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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 Pro hMPV™+ Assay is a Real-Time PCR (RT-PCR) in vitro diagnostic test for the qualitative detection of human Metapneumovirus (hMPV) nucleic acid isolated and purified from nasopharyngeal swab (NP) specimens obtained from individuals exhibiting signs and symptoms of acute respiratory infection. This assay targets a highly conserved region of the Nucleocapsid gene of hMPV. The detection of hMPVnucleic acid from symptomatic patients aids in the diagnosis of human respiratory hMPV infection if used in conjunction with other clinical and laboratory findings. This test is not intended to differentiate the four 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.

The Pro hMPV+ Assay enables detection human Metapneumovirus and Internal Control nucleic acid. Nasopharyngeal swab specimens are collected from patients with signs and symptoms of a respiratory infection using a polyester, rayon or nylon tipped swab and placed into viral transport medium.

An Internal Control (IC) is added to each sample prior to nucleic acid isolation to monitor for inhibitors present in the specimens. The isolation and purification of the nucleic acids is performed using either a MagNA Pure LC Instrument (Roche) and the MagNA Pure Total Nucleic Acid Isolation Kit (Roche) or a NucliSENS easyMAGTM System (bioMérieux) and the Automated Magnetic Extraction Reagents (bioMérieux).

The purified nucleic acids are added to Pro hMPV+ Supermix along with enzymes included in the Pro hMPV+ Assay Kit. The Pro hMPV+ Supermix contains oligonucleotide primers and target-specific oligonucleotide probes. The primers are complementary to highly conserved regions of genetic sequences for these respiratory viruses. The probes are dual-labeled with a reporter dye attached to the 51-end and a quencher dye attached to the 3'-end.

Reverse transcription of the RNA in the sample into complementary DNA (cDNA) and subsequent amplification of DNA is performed in a Cepheid SmartCycler® II instrument. In this process, the probe anneals specifically to the template followed by primer extension and amplification. The Pro hMPV+ Assay is based on Taqman chemistry, which utilizes the 5' - 3' exonuclease activity of the Taq polymerase to cleave the probe thus separating the reporter dye from the quencher. This generates an increase in fluorescent signal upon excitation from a light source. additional reporter dye molecules are cleaved from their respective probes, further increasing fluorescent signal. The amount of fluorescence at any given cycle is dependent on the amount of amplification products present at that time, Fluorescent intensity is monitored during each PCR cycle by the SmartCyclerII instrument.

Here's an analysis of the provided information, structured according to your request:

1. Table of Acceptance Criteria and Reported Device Performance

The document compares the "New Pro hMPV+ Assay" (reformulated) to the "Current Pro hMPV+ Assay" (predicate device). The acceptance criteria are implied by the "Percent Positive Agreement" and "Percent Negative Agreement" with confidence intervals. While specific numerical acceptance criteria (e.g., "must be >90%") are not explicitly stated, the reported performance is presented as demonstrating substantial equivalence.

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

• Sample Size (Clinical Comparison): 183 nasopharyngeal swab samples (one sample was excluded from the final analysis, resulting in 182).
• Data Provenance: Retrospective, collected during 2011-2012 from two sites: Milwaukee, WI, and Chicago, IL, USA.

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

The document does not mention the use of experts to establish the ground truth for the clinical comparison study. Instead, the "ground truth" was established by:

• "True" hMPV positives: Defined as any sample that tested positive by the original Pro hMPV+ Assay.
• "True" hMPV negatives: Defined as any sample that tested negative by the original Pro hMPV+ Assay.
• Discrepant Analysis: For samples where the new and original assays disagreed, RT-PCR with hMPV specific primers targeting the hMPV phosphoprotein gene followed by bi-directional genetic sequencing was performed. The document does not specify who performed this analysis or their qualifications, but this would be a laboratory-based method.

4. Adjudication Method for the Test Set

The primary comparison was against the predicate device's results. For discrepancies, a molecular method (RT-PCR followed by bi-directional genetic sequencing) was used to resolve disagreements. This acts as a form of "adjudication" based on a more definitive molecular test, rather than human expert consensus.

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 reported. This study evaluates human reader performance, with or without AI assistance. The described study is a comparison of two in vitro diagnostic (IVD) assays.

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

Yes, the described clinical comparison study is a standalone assessment of the new IVD assay's performance against the predicate IVD assay. There is no human-in-the-loop component mentioned; it evaluates the assay's ability to detect hMPV directly from samples.

7. The Type of Ground Truth Used

The ground truth for the clinical comparison study was multi-faceted:

• Reference standard (initial): The results of the predicate device (original Pro hMPV+ Assay).
• Adjudication/Confirmatory method: For discrepant results, RT-PCR with hMPV specific primers targeting the hMPV phosphoprotein gene followed by bi-directional genetic sequencing was used, which can be considered a more definitive molecular ground truth.

8. The Sample Size for the Training Set

The document does not specify a separate training set. The study describes the re-formulation of an existing assay and its performance evaluation. Diagnostic assays like this typically undergo development and optimization phases (which might involve various "training" or optimization samples), but the clinical comparison details the final performance validation using a test set.

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

As no specific training set is outlined in this document, the method for establishing its ground truth is not provided. The information focuses on the validation of the reformulated assay.

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The Quidel Molecular hMPV assay is a multiplex Real Time RT-PCR assay for the in vitro qualitative detection of human metapneumovirus RNA in nasal and nasopharyngeal swab specimens from patients with signs and symptoms of respiratory infection. This test is intended for use as an aid in the differential diagnosis of human metapneumovirus infections in humans in conjunction with clinical and epidemiological risk factors. This test is not intended to differentiate the four 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 patient management decisions.

The Quidel Molecular 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 the Applied Biosystems 7500 Fast Dx platform. Identification of hMPV occurs by the use of target specific primers and a fluorescent- labeled probe that hybridizes to conserved regions in the RNA dependent RNA polymerase gene of hMPV.

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

Acceptance Criteria and Reported Device Performance

For the clinical study, performance was compared against an FDA-cleared RT-PCR comparator device. While explicit acceptance criteria ("Pass/Fail") are not stated in terms of specific percentages, the reported percentages (Positive Percent Agreement and Negative Percent Agreement) implicitly serve as the achieved performance against which the device's efficacy is judged.

Study Details

1. Sample sizes used for the test set and the data provenance:

   • Clinical Performance Test Set: N=1072 specimens (after excluding invalid results).
     • 742 fresh specimens: Collected for routine respiratory virus testing at thirteen sites across the United States.
     • 374 frozen specimens: Collected and tested at two geographically distinct locations.
   • Data Provenance: United States (multiple sites).
   • Retrospective/Prospective: The clinical study was prospective, conducted during the 2010-2011 respiratory virus season (January to March 2011).

2. Number of experts used to establish the ground truth for the test set and the qualifications of those experts:

   • The ground truth for the clinical performance test set was established by an "FDA Cleared RT-PCR" device (the comparator device, Gen-Probe Prodesse Pro hMPV+ (K082688)).
   • For 2 discrepant specimens that were positive by the Quidel Molecular hMPV assay but negative by the comparator, "sequence analysis" was used to resolve the discrepancy. The number and qualifications of experts for this sequence analysis are not specified in the provided text.

3. Adjudication method for the test set:

   • For the clinical performance study, direct comparison was made against the "FDA Cleared RT-PCR" device.
   • For the two discrepant cases where the Quidel assay was positive and the comparator was negative, "sequence analysis" was used as an adjudication method to determine the true state.

4. 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 submission describes an in vitro diagnostic (IVD) assay where the "reader" is an automated instrument, not a human clinician interpreting images or data for diagnosis. Therefore, improvement of human readers with/without AI assistance is not applicable.

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

   • Yes, the performance characteristics described (analytical and clinical performance) represent the standalone performance of the Quidel Molecular hMPV Assay. It is an automated RT-PCR assay that provides a qualitative result (positive/negative) without human intervention in the interpretation of the primary result from the instrument.

6. The type of ground truth used:

   • Clinical Performance: "FDA Cleared RT-PCR" device (the predicate device) and, for discrepant cases, sequence analysis.
   • Analytical Performance (LoD, Inclusivity, Specificity): Quantified cultures of hMPV strains (TCID50/mL) for LoD and inclusivity, and specified concentrations of viral, bacterial, and yeast strains for specificity.

7. The sample size for the training set:

   • The provided document does not specify a separate training set size for the development of the Quidel Molecular hMPV Assay. This is common for molecular diagnostic kits, where the "training" involves optimizing primer/probe design and assay conditions, rather than training a machine learning algorithm on a specific data set.

8. How the ground truth for the training set was established:

   • As no specific "training set" in the machine learning sense is mentioned, information on how its ground truth was established is not provided. The development of this molecular assay would have involved standard molecular biology techniques, using characterized viral isolates and nucleic acid samples to optimize primer/probe sequences and reaction conditions, rather than a "ground truth" derived from patient data for an algorithm.

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The Pro hMPV+ Assay is a Real Time RT-PCR in vitro diagnostic test for the qualitative detection of human Metapneumovirus (hMPV) nucleic acid isolated and purified from nasopharyngeal swab (NP) specimens obtained from individuals exhibiting signs and symptoms of acute respiratory infection. This assay targets a highly conserved region of the Nucleocapsid gene of hMPV. The detection of hMPV nucleic acid from symptomatic patients aids in the diagnosis of human respiratory hMPV infection if used in conjunction with other clinical and laboratory findings. This test is not intended to differentiate the four 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.

The Pro hMPV+ Assay enables detection human Metapneumovirus and Internal Control nucleic acid. Nasopharyngeal swab specimens are collected from patients with signs and symptoms of a respiratory infection using a polyester, rayon or nylon tipped swab and placed into viral transport medium.

An Internal Control (IC) is added to each sample prior to nucleic acid isolation to monitor for inhibitors present in the specimens. The isolation and purification of the nucleic acids is performed using either a MagNA Pure LC Instrument (Roche) and the MagNA Pure Total Nucleic Acid Isolation Kit (Roche) or a NucliSENS® easyMAG™ System (bioMérieux) and the Automated Magnetic Extraction Reagents (bioMérieux).

The purified nucleic acids are added to Pro hMPV+ Supermix along with enzymes included in the Pro hMPV+ Assay Kit. The Pro hMPV+ Supermix contains oligonucleotide primers and target-specific oligonucleotide probes. The primers are complementary to highly conserved regions of genetic sequences for these respiratory viruses. The probes are dual-labeled with a reporter dye attached to the 5'-end and a quencher dye attached to the 3'-end.

Reverse transcription of the RNA in the sample into complementary DNA (cDNA) and subsequent amplification of DNA is performed in a Cepheid SmartCycler® II instrument. In this process, the probe anneals specifically to the template followed by primer extension and amplification. The Pro hMPV+ Assay is based on Taqman chemistry, which utilizes the 5' - 3' exonuclease activity of the Taq polymerase to cleave the probe thus separating the reporter dye from the quencher. This generates an increase in fluorescent signal upon excitation from a light source. With each cycle, additional reporter dye molecules are cleaved from their respective probes, further increasing fluorescent signal. The amount of fluorescence at any given cycle is dependent on the amount of amplification products present at that time. Fluorescent intensity is monitored during each PCR cycle by the SmartCyclerII instrument.

Here's an analysis of the acceptance criteria and study detailed in the provided 510(k) summary for the Pro hMPV+ Assay:

Acceptance Criteria and Device Performance for Pro hMPV+ Assay

This summary focuses on the clinical performance of the Pro hMPV+ Assay, which is a Real Time RT-PCR in vitro diagnostic test for the qualitative detection of human Metapneumovirus (hMPV).

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state pre-defined acceptance criteria for the clinical performance metrics (Percent Positive Agreement, Percent Negative Agreement). However, the reported performance is presented with 95% Confidence Intervals, which allows for an assessment of the assay's accuracy. For the purpose of this analysis, we will infer the desired performance to be high agreement with the composite reference methods.

2. Sample Size and Data Provenance for the Test Set

• Sample Size: A total of 1275 eligible nasopharyngeal (NP) swab samples were tested and included in the analysis.
• Data Provenance: The study was a prospective study conducted at 4 U.S. clinical laboratories during the 2008 respiratory virus season (January - March). The specimens represented excess NP swab specimens proactively collected from symptomatic individuals suspected of respiratory infection.

3. Number of Experts and Qualifications for Ground Truth Establishment (Test Set)

The ground truth was established using composite reference methods, which involved molecular testing and genetic sequencing, rather than direct expert interpretation of test results. Therefore, the concept of "experts" in the traditional sense (e.g., radiologists) for establishing ground truth doesn't directly apply here.

However, the "experts" involved would be the laboratory personnel performing the molecular (RT-PCR) tests and subsequent genetic sequencing, and those interpreting the sequencing data against the NCBI GenBank database. While no explicit qualifications are given, it can be inferred that these individuals are qualified laboratory professionals experienced in molecular diagnostics and bioinformatics, as they are performing highly specialized and technical analyses for clinical diagnostic purposes.

4. Adjudication Method for the Test Set

The adjudication method for establishing the ground truth was a composite reference standard approach:

• Two independent molecular (RT-PCR) tests for two separate gene targets of hMPV.
• Followed by bidirectional genetic sequencing of those targets.

True hMPV RNA positives were defined as any sample with bidirectional sequencing data meeting pre-defined quality acceptance criteria for one or both gene targets that matched hMPV sequences in the NCBI GenBank database.
True hMPV RNA negatives were defined as any sample tested negative by both comparator methods.

This effectively acts as an internal adjudication process based on multiple, high-specificity molecular methods. There is no mention of a human expert adjudication committee in the traditional sense (e.g., 2+1, 3+1).

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

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not conducted. This device is an in vitro diagnostic test, not an AI-assisted human reader interpretation tool. Therefore, the concept of measuring how much human readers improve with AI vs. without AI assistance is not applicable.

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

Yes, a standalone (algorithm only) performance study was conducted. The "Pro hMPV+ Assay" itself is the algorithm (or diagnostic method) being evaluated. Its performance was assessed directly against the composite reference methods. There is no human-in-the-loop component in its reported diagnostic performance.

7. Type of Ground Truth Used

The type of ground truth used was a composite reference standard based on:

• Molecular diagnostic testing (two independent RT-PCR tests) for different hMPV gene targets.
• Bi-directional genetic sequencing of those targets.
• Comparison of sequencing data to the National Center for Biotechnology Information (NCBI) GenBank database.

This is a highly reliable and objective form of ground truth for viral detection.

8. Sample Size for the Training Set

The document does not provide information regarding a distinct "training set" sample size. For in vitro diagnostic assays like the Pro hMPV+ Assay, the development process typically involves internal validation and optimization studies (which might be analogous to "training"), but specific sample sizes for these internal activities are usually not detailed in 510(k) summaries, which focus on the clinical validation (test set).

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

As no specific "training set" and its sample size were described, the method for establishing its ground truth is also not provided in this document. During assay development, ground truth for optimization and development samples would typically be established using similar highly sensitive and specific methods (e.g., highly characterized positive and negative controls, sequencing, or alternative validated molecular methods).

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