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The Prodesse® ProParaflu®+ Assay is a multiplex Real-Time PCR (RT-PCR) in vitro diagnostic test for the qualitative detection and discrimination of Parainfluenza 1 Virus, Parainfluenza 2 Virus and Parainfluenza 3 Virus (HPIV-1. HPIV-2 and HPIV-3) nucleic acids isolated and purified from nasopharvngeal (NP) swab specimens obtained from individuals exhibiting signs and symptoms of respiratory tract infections. This Assay targets the conserved regions of the Hemagglutinin-Neuraminidase (HN) gene of HPIV-1, HPIV-3, respectively. The detection and discrimination of HPIV-1, HPIV-2 and HPIV-3 nucleic acids from symptomatic patients aid in the diagnosis of human respiratory tract parainfluenza infections if used in conjunction with other clinical and laboratory findings. This test is not intended to detect Parainfluenza 4a or Parainfluenza 4b Viruses.

Negative test results are presumptive and should be confirmed by cell culture. Negative results do not preclude Parainfluenza 1, 2 or 3 virus infections and should not be used as the sole basis for treatment or other management decisions.

The ProParaflu+ Assay enables detection and differentiation of Parainfluenza 1 Virus. Parainfluenza 2 Virus, Parainfluenza 3 Virus 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 ProParaflu+ Supermix along with enzymes included in the ProParaflu+ Assay Kit. The ProParaflu+ 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 ProParaflu+ 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 dve 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 SmartCycler II instrument.

The provided document describes a special 510(k) submission for the Gen-Probe Prodesse, Inc. Prodesse® ProParaflu®+ Assay (K132238). This submission focuses on modifications to an existing device (predicate device K091053, ProParaflu 101+ Assay) rather than a completely new device. Therefore, the details provided about acceptance criteria and study designs are predominantly related to demonstrating substantially equivalent performance with the modifications, rather than establishing initial performance for a novel diagnostic.

Here's an analysis of the provided information:

1. Table of Acceptance Criteria and Reported Device Performance

The document doesn't explicitly state numerical "acceptance criteria" in the format of a threshold to be met. Instead, it describes the objective of the verification/validation studies for the modified device: to ensure that the modifications did not negatively impact the device's ability to detect target organisms at the limit of detection or change its clinical performance. The reported performance is framed as meeting these objectives and demonstrating substantial equivalence to the previous device.

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

• Sample Size for Test Set: The document mentions a "retrospective clinical comparison study" for the UIC modification but does not specify the sample size for this study or any other test sets.
• Data Provenance: The document states "nasopharyngeal (NP) swab specimens obtained from individuals exhibiting signs and symptoms of respiratory tract infections." The country of origin is not specified but is implied to be within the scope of where Gen-Probe Prodesse, Inc. operates (Waukesha, WI, USA, suggests data from the USA). The clinical comparison study is explicitly stated to be retrospective.

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

This information is not provided in the document. As this is an in vitro diagnostic device, the ground truth is typically established by other laboratory methods rather than expert interpretation of images or other subjective data.

4. Adjudication Method for the Test Set

This information is not provided in the document. Given that it's an in vitro diagnostic test, the concept of expert adjudication in the same way it applies to image analysis might not be directly relevant. The "ground truth" would likely be determined by a different gold standard assay or cell culture, not a consensus of human reviewers of the device's output.

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

An MRMC study is not applicable and was not done. This device is an in vitro diagnostic for detecting viral nucleic acids, not an imaging device requiring human reader interpretation.

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

The device is an in vitro diagnostic Real-Time PCR assay. Its operation is inherently "standalone" in the sense that the assay itself generates a result (presence/absence of viral nucleic acid) based on the biochemical reaction and instrument detection. There isn't a "human-in-the-loop" component in the interpretation of the RT-PCR output itself, though a human performs the test and interprets the final qualitative result (positive/negative) from the instrument's readout. The performance studies (Analytical Sensitivity, IC Interference, Extractor Equivalency, Sample Stability, and the retrospective clinical comparison) demonstrate this standalone performance.

7. Type of Ground Truth Used

The document mentions that negative test results are presumptive and should be confirmed by cell culture. This indicates that cell culture is considered a gold standard or a primary method for confirming negative findings, and likely forms part of the "ground truth" for clinical evaluations. For positive results, the ground truth would typically be established by clinical diagnosis and/or comparison to a known highly sensitive and specific comparator assay or other reference methods in the clinical comparison study.

8. Sample Size for the Training Set

The document does not provide any information about a training set. As this is a molecular diagnostic assay using primers and probes targeting specific gene sequences, the "training" aspect is built into the assay design (selecting highly conserved regions) rather than a machine learning training paradigm with a specific dataset.

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

Since there is no mention of a "training set" in the context of machine learning, this question is not applicable. The "ground truth" for the assay's design (e.g., confirming the suitability of the chosen gene targets and primer/probe sequences) would have been established through bioinformatics analysis and empirical testing with characterized viral isolates.

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The Prodesse® ProFAST + Assay is a multiplex Real Time RT-PCR in vitro diagnostic test for the qualitative detection and discrimination of seasonal Influenza A/H1, seasonal Influenza A/H3 and 2009 H1N1 Influenza viral nucleic acids isolated and purified from nasopharyngeal (NP) swab specimens from human patients with signs and symptoms of respiratory infection in conjunction with clinical and epidemiological risk factors. This Assay targets conserved regions of the Hemagglutinin (HA) gene for seasonal Influenza A/H1, seasonal Influenza A/H3 and 2009 H1N1 Influenza Virus, respectively. This Assay is not intended to detect Influenza B or Influenza C Viruses.

A negative ProFAST+ Assay result is a presumptive negative result for Influenza A. These results should be confirmed by an FDA cleared nucleic acid-based test (NAT) detecting Influenza A.

Negative results do not preclude Influenza virus infection and should not be used as the sole basis for treatment or other patient management decisions.

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 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 Prodesse® ProFAST + Assay is a multiplex Real Time RT-PCR in vitro diagnostic test for the qualitative detection and discrimination of seasonal Influenza A/H1, seasonal Influenza A/H3 and 2009 H1N1 Influenza viral nucleic acids isolated and purified from nasopharyngeal (NP) swab specimens from human patients with signs and symptoms of respiratory infection in conjunction with clinical and epidemiological risk factors. This Assay targets conserved regions of the Hemagglutinin (HA) gene for seasonal Influenza A/H1, seasonal Influenza A/H3 and 2009 H1N1 Influenza Virus, respectively. This Assay is not intended to detect Influenza B or Influenza C Viruses.

The ProFAST+ Assay enables detection and discrimination of Influenza A Virus subtypes: seasonal A/H1, seasonal A/H3, and 2009 H1N1 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 ProFAST+ Supermix along with enzymes included in the ProFAST+ Assay Kit. The ProFAST+ Supermix contains oligonucleotide primers and targetspecific oligonucleotide probes. The primers are complementary to highly conserved regions of the Hemagglutinin (HA) gene for seasonal influenza A/H1, seasonal influenza A/H3 and 2009 H1N1 Influenza Virus. 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 ProFAST+ Assay is based on Tagman 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 SmartCycler II instrument.

The provided text describes a special 510(k) submission for the Prodesse® ProFAST®+ Assay, primarily focusing on modifications to the internal control and positive control, and an additional reactivity claim for H3N2v. The submission argues for substantial equivalence to a predicate device (K101855, ProFAST 101+ Assay).

Crucially, the document does not present acceptance criteria or detailed results from a study that "proves the device meets the acceptance criteria" in the format of a typical clinical validation study. Instead, it focuses on demonstrating that modifications did not negatively impact performance compared to the previously cleared predicate device.

Here's an attempt to extract the requested information, noting where details are missing based on the provided text:

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state quantitative acceptance criteria (e.g., minimum sensitivity, specificity, or agreement percentages) for a clinical performance study of the modified device. Instead, it refers to the previous performance claims of the ProFAST+ Assay (the predicate device for the modifications) and states that the modified assay "continues to meet the performance claims."

The closest to "reported device performance" are the results of the verification/validation studies for the modifications:

Note: The document explicitly states that "the performance characteristics of this device with clinical specimens that are positive for H3N2v influenza virus have not been established." This means for H3N2v, only analytical reactivity was shown, not clinical performance.

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

• Sample Size for Test Set: The document mentions a "retrospective clinical comparison study" for the UIC modification but does not provide the sample size used in this study.
• Data Provenance: The document states "clinical comparison study," implying human patient samples were used. The term "retrospective" indicates that these samples were collected in the past. The country of origin is not specified but is implicitly the US given the FDA submission.

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

Not provided. The nature of the ground truth (e.g., a reference method like viral culture or another FDA-cleared NAT) is not detailed, nor is the number or qualifications of experts, if any, involved in establishing it. It's likely the "ground truth" for the clinical comparison study would have been established by the reference method against which the predicate device's original performance claims were made.

4. Adjudication Method for the Test Set

Not provided. Given that this appears to be a comparison study against a historical reference or predicate, an adjudication method might not have been
explicitly described in this type of submission.

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

Not applicable. This device is an in vitro diagnostic (IVD) RT-PCR assay for detecting viral nucleic acids, not an AI-assisted diagnostic tool that would be used by "human readers" in the sense of image interpretation. Therefore, an MRMC study with human readers and AI assistance is not relevant to this device. The "reader" here is the instrument interpreting PCR amplification curves.

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

Yes, the device is inherently a standalone algorithm/assay without human-in-the-loop performance influencing its primary result. It provides a qualitative (positive/negative) detection and discrimination of influenza A subtypes. The "retrospective clinical comparison study" would represent the standalone performance of the modified assay.

7. The Type of Ground Truth Used (expert consensus, pathology, outcomes data, etc.)

Not explicitly stated in the provided text. For RT-PCR assays, the ground truth for clinical studies is typically established by:

• A "gold standard" laboratory method (e.g., viral culture if available and sensitive enough, or a highly sensitive and specific FDA-cleared reference molecular test).
• A composite reference method combining multiple tests or clinical findings.

Given it's a "clinical comparison study," it implies comparison to established clinical diagnoses or reference lab results, but the specifics are absent.

8. The Sample Size for the Training Set

Not applicable/Not provided. This is an RT-PCR assay, not a machine learning or AI algorithm that requires a "training set" in the conventional sense. The "training" for such a device involves assay optimization and analytical validation using characterized samples (e.g., contrived samples with known viral concentrations, characterized clinical samples) to establish parameters like limit of detection, linearity, and specificity. The document refers to "Analytical Sensitivity, IC Interference, Extractor Equivalency, and Sample Stability studies," which utilize such characterized samples, but a specific "training set sample size" as per AI/ML terminology is not relevant here.

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

Not applicable/Not provided in the AI/ML context. For analytical studies, the "ground truth" (e.g., viral presence and concentration) is established by using characterized stocks, reference materials, or quantified clinical samples whose status is independently verified (e.g., by culture, sequencing, or quantitative PCR methods).

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The Prodesse® ProAdeno®+ Assay is a multiplex Real Time PCR in vitro diagnostic test for the qualitative detection of human Adenovirus (HAdV) DNA isolated and purified from nasopharyngeal (NP) swab specimens obtained from individuals exhibiting signs and symptoms of acute respiratory infection. This test is intended for use to aid in the diagnosis of HAdV infections in humans in conjunction with other clinical and laboratory findings. The test detects, but does not differentiate, serotypes 1-51.

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

The ProAdeno+ Assay enables detection of human adenovirus 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 ProAdeno+ Supermix included in the ProAdeno+ Assay Kit. The ProAdeno+ Supermix contains oligonucleotide primers, target-specific oligonucleotide probes and a Taq DNA polymerase. The primers are complementary to highly conserved regions of the HAdV hexon gene. The probes are dual-labeled with a reporter dye attached to the 5-end and a quencher dye attached to the 3'-end.

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 ProAdeno+ Assay is based on Tagman 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 SmartCycler II instrument.

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

Acceptance Criteria and Device Performance

The provided document describes a Special 510(k) Submission, which means the device (Prodesse® ProAdeno®+ Assay) is a modification of an already cleared device (K102952, ProAdeno™+ Assay). Therefore, the primary acceptance criteria revolve around demonstrating that the modifications did not negatively impact the performance established for the predicate device and that the device continues to meet the performance claims.

The acceptance criteria are implied by the "Potential Impact of Modification" and the "Verification/Validation Result" sections. The "Verification/Validation Result" column details how the device performance was shown to meet these implicit criteria.

Study Details

The submission focuses on proving substantial equivalence after minor modifications to an already cleared device. Therefore, the studies conducted are primarily verification and validation studies to confirm that the modifications did not alter the fundamental scientific technology or performance.

2. Sample Sizes and Data Provenance

• Test Set Sample Size:
  • Analytical Sensitivity, IC Interference, Extractor Equivalency, and Sample Stability studies: No specific sample sizes are provided in the document for these analytical studies.
  • Retrospective Clinical Comparison Study: No specific sample size for the clinical comparison study is provided.
  • Positive Control Effectiveness Study: No specific sample size is provided.
• Data Provenance:
  • The "retrospective clinical comparison study" indicates retrospective data.
  • The document does not specify the country of origin of the data for any of the studies.

3. Number of Experts and Qualifications for Ground Truth

• The document describes an in vitro diagnostic test (nucleic acid amplification assay). For such tests, "ground truth" is typically established through reference methods, gold standard assays (like sequencing or culture in some contexts), or clinical diagnosis. It does not typically involve human expert readers in the same way as imaging or pathology devices.
• Therefore, the concepts of "number of experts" and "qualifications of those experts" for establishing ground truth are not applicable in this context.

4. Adjudication Method

• Adjudication methods (e.g., 2+1, 3+1) are primarily relevant for studies involving human interpretation (e.g., radiology reads).
• Given this is an in vitro diagnostic (IVD) assay, and the ground truth is established analytically or through reference methods, an adjudication method in the human-reader sense is not applicable.

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

• No, an MRMC comparative effectiveness study was not done.
• MRMC studies are typically for devices that assist human interpretation (e.g., CAD systems for radiologists). The Prodesse® ProAdeno®+ Assay is a standalone diagnostic test for detecting viral DNA, not an assistive tool for human readers.

6. Standalone Performance Study

• Yes, a standalone performance was done for the device. The entire submission is about the performance of the algorithm/assay itself in detecting Human Adenovirus (HAdV) DNA.
• The "Analytical Sensitivity, IC Interference, Extractor Equivalency, and Sample Stability studies" and the "retrospective clinical comparison study" all pertain to the standalone performance of the Prodesse® ProAdeno®+ Assay.

7. Type of Ground Truth Used

• For the analytical studies (Analytical Sensitivity, IC Interference, etc.), the ground truth would typically be established by known concentrations of target nucleic acid or reference methods for determining presence/absence of inhibitors or specific analytes.
• For the "retrospective clinical comparison study," the ground truth for HAdV infection in patient samples would likely have been established by a predicate device (ProAdeno™+ Assay) or another validated reference method for HAdV detection, but the document specifically states the comparison was to show the modified assay "continues to meet the performance claims for the current ProAdeno+ Assay," implying the predicate device's expected results served as a comparator for clinical performance. The details of the "gold standard" for the initial predicate device are not in this document.

8. Sample Size for the Training Set

• The document describes an assay (Real Time PCR in vitro diagnostic test), not a machine learning or AI-based device that requires a "training set" in the conventional sense.
• Therefore, the concept of a "training set sample size" is not applicable. The assay's performance is based on its chemical and biological reactions, not on data-driven learning.

9. How Ground Truth for the Training Set Was Established

• As explained above, there is no "training set" for this type of device.

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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 Prodesse ProFlu™+ Assay is a multiplex Real-Time PCR (RT-PCR) in vitro diagnostic test for the rapid and qualitative detection and discrimination of Influenza A Virus. Influenza B Virus, and Respiratory Syncytial Virus (RSV) nucleic acids isolated and purified from nasopharyngeal (NP) swab specimens obtained from symptomatic patients. This test is intended for use to aid in the differential diagnosis of Influenza A. Influenza B and RSV viral infections in humans and is not intended to detect Influenza C.

Negative results do not preclude influenza or RSV virus infection and should not be used as the sole basis for treatment or other 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 and A/H1 were the predominant Influenza A viruses in circulation (2006 - 2007 respiratory season). Performance characteristics for Influenza A were confirmed when Influenza A/H1, Influenza A/H3, and Influenza A/2009 H1N1 were the predominant Influenza A viruses in circulation (2008 and 2009). When other Influenza A viruses are emerging, performance characteristics may vary.

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 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 ProFlu+ Assay enables detection and discrimination of Influenza A Virus, Influenza B Virus, RSV and universal 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 Purc 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 Influenza B/RSV Mix along with enzymes included in the ProFlu+ Assay Kit. The Influenza A/Influenza B/RSV Mix 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 ProFity- Assay is based on Tagman 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.

The provided document describes a 510(k) premarket notification for a modified in vitro diagnostic device, the Prodesse ProFlu™+ Assay. As such, the information typically associated with acceptance criteria and a detailed study proving device performance against those criteria in the context of AI/ML or image processing devices is not present. This document focuses on demonstrating substantial equivalence to a predicate device, rather than proving performance against specific acceptance criteria with detailed statistical results.

However, I can extract the relevant information based on the prompt's request, interpreting "acceptance criteria" as the claimed performance or non-inferiority that the modification verification studies aimed to confirm.

Here's a breakdown of the requested information, acknowledging the limitations of a 510(k) submission for a molecular diagnostic device:

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state numerical "acceptance criteria" in the format typically seen for sensitivity/specificity for algorithms. Instead, the modifications were verified to ensure the fundamental scientific technology and clinical performance remained unchanged from the predicate device. The "acceptance criteria" were met if these aspects were confirmed.

• Clinical performance remains consistent with the current ProFlu+ Assay. | - The UIC did not affect the ability of the ProFlu+ Assay to detect target organisms at the limit of detection (LOD) as evinced by results of Analytical Sensitivity, IC Interference, Extractor Equivalency, and Sample Stability studies.
• A retrospective clinical comparison study demonstrated the modified ProFlu+ Assay with UIC continues to meet the performance claims for the current ProFlu+ Assay. |
  | Modified Positive Controls | - Stability claims are met.
• Continued ability to monitor for global assay failures. | - Stability studies demonstrated current stability claims are met.
• Clinical validation of the modified positive controls demonstrated their continued ability to monitor for global assay failures. |
  | Influenza A H3N2v and H7N9 Reactivity | - Ability to detect specific strains (A/Indiana/10/2011 (H3N2v) and A/Anhui/1/2013 (H7N9)). | - Results of the Reactivity Study demonstrated the ability of the ProFlu+ Assay to detect A/Indiana/10/2011 (H3N2v) and A/Anhui/1/2013 (H7N9) nucleic acids at concentrations near the limit of detection of the assay. |
  | Increased Freeze-Thaw Cycles (M-MLV RT & RNase II) | - Assay performance is not affected by 10 freeze-thaw cycles. | - Stability studies demonstrated that ProFlu+ Assay performance was not affected when the MMLV Reverse Transcriptase and the RNase Inhibitor II underwent 10 freeze-thaw cycles. |

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

The document mentions a "retrospective clinical comparison study" for the UIC impact. However, the specific sample size used for this test set is not provided. The geographic provenance (e.g., country of origin) of the data is also not specified. The study is described as "retrospective."

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

For this type of molecular diagnostic device, ground truth is typically established by laboratory testing methods (e.g., gold standard PCR, sequencing, or culture) rather than expert human interpretation of images or clinical data, especially for a retrospective study focused on assay performance. Therefore, the concept of "number of experts" and their "qualifications" for establishing ground truth as one might consider for imaging devices does not directly apply in this context. The "ground truth" would be the result of a reference laboratory method.

4. Adjudication Method for the Test Set

Given that ground truth is likely based on objective laboratory methods, an "adjudication method" involving multiple human readers (e.g., 2+1, 3+1) is not applicable here.

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 done. This type of study is relevant for evaluating the impact of AI assistance on human reader performance, which is not applicable to a non-AI molecular diagnostic assay validation.

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

The ProFlu™+ Assay is a molecular diagnostic test. The entire assay system (reagents, instrumentation, and protocol) constitutes the "algorithm" in a broad sense. The verification studies assess the performance of this system directly. Therefore, the "standalone" performance is what was evaluated in the analytical and clinical studies described, as there isn't a separate "human-in-the-loop" component in the direct interpretation of the PCR results for this device. The user performs the test and interprets the results based on predefined thresholds, but the core detection is algorithmic.

7. The Type of Ground Truth Used

The ground truth for the verification studies would likely be established through:

• Reference molecular methods: Such as a validated laboratory-developed test (LDT), sequencing, or other nucleic acid amplification tests (NAATs) that are considered the gold standard for detecting the target viruses.
• Viral culture: For confirmation of viable virus.
• Analytical spiking: For analytical sensitivity and reactivity studies, where known concentrations of target nucleic acids are used.

While the document doesn't explicitly state the exact "ground truth" method for the clinical comparison, for a molecular diagnostic, it would invariably involve a highly accurate reference laboratory test.

8. The Sample Size for the Training Set

This submission describes modifications to an existing device and its verification, not the development of a de novo algorithm requiring a "training set" in the context of machine learning. Therefore, the concept of a "training set" does not apply here. The initial development of the predicate ProFlu+ Assay would have involved studies to establish its design parameters.

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

As noted in point 8, the concept of a "training set" is not relevant to this type of device modification submission.

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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 Prodesse® ProGastro SSCS Assav is a multiplex real time PCR in vitro diagnostic test for the qualitative detection and differentiation of Salmonella, Shigella, and Campylobacter (C. jejimi and C. coli only, undifferentiated) nucleic acids and Shiga Toxin 1 (stxl) and Shiga Toxin 2 (stx2) genes. Shiga toxin producing E. coli (STEC) typically harbor one or both genes that encode for Shiga Toxins I and 2. Nucleic acids are isolated and purified from preserved stool specimens obtained from symptomatic patients exhibiting signs and symptoms of gastroenteritis. This test is intended for use, in conjunction with clinical presentation and epidemiological risk factors, as an aid in the differential diagnosis of Salmonella, Shigella, Campylobacter jejuni/Campylobacter coli, and STEC infections in humans.

The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Positive results do not rule out co-infection with other organisms that are are not detected by this test, and may not be the sole or definitive cause of patient illness. Negative ProGastro SSCS Assay results in the setting of clinical illness compatible with gastroenteritis may be due to infection by pathogens that are not detected by this test or non-infectious causes such as ulcerative colitis, irritable bowel syndrome, or Crohn's disease.

The ProGastro SSCS Assay enables detection and differentiation of Salmonella, Shigella, Campylobacter (C. jejuni and C. coli only, undifferentiated) and an Internal Control in the SSC Mix and Shiga Toxin Producing E. coli (STEC, stx1 and stx2 differentiated) and an Internal Control in the STEC Mix.

An overview of the procedure is as follows:

• 1. Collect raw stool specimens from symptomatic patients and place into Cary Blair Transport Medium or ParaPak C&S (C&S) Transport Medium .
• 2. Add the Gastro RNA/DNA Internal Control (GIC) to every sample to monitor for inhibitors present in the specimens.
• 3. Perform isolation and purification of nucleic acids using a NucliSENS easyMAG System and the Automated Magnetic Extraction Reagents (bioMérieux).
• 4. Add purified nucleic acids to the SSC Mix included in the ProGastro SSCS Assay Kit. The SSC Mix contains target-specific oligonucleotide primers and probes for detection of Salmonella, Shigella, and Campylobacter (C. jejuni and C. coli only). The primers and probes are complementary to highly conserved regions of genetic sequences for these organisms. The probes are dual-labeled with a reporter dye and a quencher (see table below).
• 5. Add purified nucleic acids to the STEC Mix included in the ProGastro SSCS Assay Kit. The STEC Mix contains target-specific oligonucleotide primers and probes for detection of Shiga Toxin 1 and 2 genes (stxl and stx2). The primers and probes are complementary to highly conserved regions of these genes. The probes are dual-labeled with a reporter dye and a quencher (see table below).
• 6. Perform amplification of DNA in a Cepheid SmartCycler II instrument. In this process, the probe anneals specifically to the template followed by primer extension and amplification. The ProGastro SSCS Assay is based on Tagman reagent chemistry, which utilizes the 5' - 3' exonuclease activity of 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 real-time instrument.

Here's a breakdown of the acceptance criteria and study information for the ProGastro SSCS Assay:

The document provided details the clinical performance and analytical performance (reproducibility and precision) of the ProGastro SSCS Assay. The primary acceptance criteria for clinical performance are presented as Sensitivity, Specificity, Positive Percent Agreement (PPA), and Negative Percent Agreement (NPA). For analytical performance, it focuses on the agreement with expected results and the Coefficient of Variation (CV) for Ct values.

1. Table of Acceptance Criteria and Reported Device Performance

Clinical Performance (Prospective Study):

Clinical Performance (Retrospective Study):

Reproducibility (Across 3 Sites, 2 Operators/Site, 5 Days - Total 90 runs per condition):

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The ProFlu™+ Assay is a multiplex Real-Time PCR (RT-PCR) in vitro diagnostic test for the rapid and qualitative detection and discrimination of Influenza A Virus, Influenza B Virus, and Respiratory Syncytial Virus (RSV) nucleic acids isolated and purified from nasopharyngeal (NP) swab specimens obtained from symptomatic patients. This test is intended for use to aid in the differential diagnosis of Influenza A, Influenza B and RSV viral infections in humans and is not intended to detect Influenza C.

Negative results do not preclude influenza or RSV virus infection and should not be used as the sole basis for treatment or other management decisions. Conversely, positive results do not ruleout 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 and A/H1 were the predominant Influenza A viruses in circulation (2006 - 2007 respiratory season). Performance characteristics for Influenza A were confirmed when Influenza A/H1, Influenza A/H3, and Influenza A/2009 H1N1 were the predominant Influenza A viruses in circulation (2008 and 2009). When other Influenza A viruses are emerging, performance characteristics may vary.

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 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 ProFlu+ Assay enables detection and differentiation of Influenza A Virus. Influenza B Virus, Respiratory Syncytial Virus (Types A and B), and Internal Control.

An overview of the procedure is as follows:

1. Collect nasopharyngeal swab specimens from symptomatic patients using a polyester, rayon or nylon tipped swab and place into viral transport medium.
2. Add an Internal Control (IC) to every sample to monitor for inhibitors present in the specimens.
3. Perform isolation and purification of nucleic acids using a MagNA Pure LC System (Roche) and the MagNA Pure Total Nucleic Acid Isolation Kit (Roche) or a NucliSENS easyMAG System (bioMérieux) and the Automated Magnetic Extraction Reagents (bioMérieux).
4. Add purified nucleic acids to Influenza A/Influenza B/RSV Mix along with enzymes included in the ProFlu+ Detection Kit. The Influenza A/Influenza B/RSV Mix 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 and a quencher (see table below).
5. Perform reverse transcription of RNA into complementary DNA (cDNA) and subsequent amplification of DNA in a Cepheid SmartCycler II instrument. In this process, the probe anneals specifically to the template followed by primer extension and amplification. The ProFlu+ Assay is based on Taqman reagent 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 realtime instrument.

Here's a breakdown of the acceptance criteria and the study details for the Gen-Probe Prodesse, Inc. ProFlu+ Assay, based on the provided text:

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state pre-defined acceptance criteria in terms of numerical thresholds for Agreement percentages. However, the study aims to demonstrate substantial equivalence by showing high agreement rates with the original ProFlu+ Assay. The reported performance is presented as Percent Positive Agreement (PPA) and Percent Negative Agreement (PNA) with 95% Confidence Intervals (CI).

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

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

The document does not explicitly mention "experts" in the traditional sense (e.g., radiologists, pathologists) for establishing ground truth for individual cases. The ground truth for the comparison study was based on a reference standard as described below.

4. Adjudication Method for the Test Set

The primary reference for the comparison study was the original ProFlu+ Assay.

• "True" influenza A, influenza B or RSV positives were considered as any sample that tested positive for the respective analyte by the original ProFlu+ Assay.
• "True" Influenza A, Influenza B or RSV negatives were considered as any sample that tested negative by the original ProFlu+ Assay.

For discordant results (where the "New" ProFlu+ Assay differed from the "Current" ProFlu+ Assay), bidirectional sequencing was used as an adjudicator:

• For Influenza A: One sample was negative by the current ProFlu+ but positive by the new ProFlu+. Sequencing confirmed it was positive for Influenza A.
• For RSV: Two samples were negative by the current ProFlu+ but positive by the new ProFlu+. Sequencing confirmed they were positive for RSV.

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

This is not applicable. The ProFlu+ Assay is an in vitro diagnostic test (a lab assay), not an AI-powered image analysis or diagnostic tool that assists human readers. Therefore, an MRMC study or an assessment of human reader improvement with AI assistance would not be conducted for this type of device.

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

This is not applicable in the context of "algorithm only" as typically referred to for AI products. The ProFlu+ Assay is a PCR-based diagnostic test, and its performance is evaluated as a standalone test in the laboratory, without direct human cognitive input into the result generation process itself. The "New ProFlu+ Assay" was compared directly against the "Current ProFlu+ Assay" as standalone tests.

7. The Type of Ground Truth Used

The ground truth for the comparison study was established using a combination of a pre-existing validated assay (the original ProFlu+ Assay) as the primary reference, and bidirectional sequencing for resolving discordant results.

Specifically:

• Initial determination: The results from the original ProFlu+ Assay served as the provisional "true" positive or negative status.
• Adjudication for discrepant results: Bidirectional sequencing (targeting a different gene or region of the same gene) was used to confirm the presence of the virus in samples where the new and original assays disagreed. This sequencing acts as a higher-level confirmatory test.

8. The Sample Size for the Training Set

The document does not explicitly describe a "training set" in the context of machine learning (AI). This device is a diagnostic assay, not an AI model requiring a training phase with labeled data in the same way. The "reformulated supermix" implies a development and optimization process, but the specific data used for that internal development (if analogous to training data) is not detailed. The numbers provided (233 samples per analyte) refer to the clinical comparison study data.

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

As noted above, the concept of a "training set" with ground truth in the AI/machine learning sense is not directly applicable here. The assay's development (including the reformulated supermix) would have involved extensive R&D and validation using characterized samples, but this information is not provided in a format that aligns with AI training set descriptions.

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The ProAdeno™+ Assay is a multiplex Real Time PCR in vitro diagnostic test for the qualitative detection of human Adenovirus (HAdV) DNA isolated and purified from nasopharyngeal (NP) swab specimens obtained from individuals exhibiting signs and symptoms of acute respiratory infection. This test is intended for use to aid in the diagnosis of HAdV infections in humans in conjunction with other clinical and laboratory findings. The test detects, but does not differentiate, serotypes 1-51.

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

The ProAdeno+ Assay enables detection of human adenovirus and an Universal Internal Control.

An overview of the procedure is as follows:

1. Collect nasopharyngeal swab specimens from symptomatic patients using a polyester, rayon or nylon tipped swab and place into viral transport medium.
2. Add Universal Internal Control (UIC) to every sample to monitor for inhibitors present in the specimens.
3. Perform isolation and purification of nucleic acids using a MagNA Pure LC System (Roche) and the MagNA Pure Total Nucleic Acid Isolation Kit (Roche) or a NucliSENS® easyMAG™ System (bioMérieux) and the Automated Magnetic Extraction Reagents (bioMérieux).
4. Add purified nucleic acids to ProAdeno+ Supermix included in the ProAdeno+ Assay Kit. The ProAdeno+ Mix contains oligonucleotide primers, target-specific oligonucleotide probes, and a Taq DNA polymerase. The primers are complementary to a highly conserved region of human adenovirus. The probes are dual-labeled with a reporter dye attached to the 5'-end and a quencher dye attached to the 3'-end (see table below).
5. Perform amplification of DNA in a Cepheid SmartCycler® II instrument. In this process, the probe anneals specifically to the template followed by primer extension and amplification. The ProAdeno+ Assay is based on Tagman reagent 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 real-time instrument.

The provided text describes the clinical performance and reproducibility testing for the ProAdeno™+ Assay. Here's a breakdown of the requested information:

Acceptance Criteria and Reported Device Performance

The document does not explicitly state pre-defined acceptance criteria values for sensitivity and specificity. However, the reported performance is presented in a table. For reproducibility, the overall percent agreement is stated.

Note: The 95% CI for Sensitivity and Specificity appear to have a typo in the provided text (87.1% - 94.3% combined for both). Assuming the first value is for sensitivity and the second for specificity, the corrected representation would be:

• Sensitivity: 97.5% (95% CI: 87.1% - 94.3%)
• Specificity: 95.6% (95% CI: 94.3% - 96.7%)

However, the CI values are given as a range, which makes this interpretation potentially problematic. Without further clarification from the original document, the exact intended 95% CI for sensitivity and specificity remains ambiguous as presented. For the purpose of this analysis, I will retain the values as they appear in the source text, acknowledging the potential discrepancy in the CI range.

Study Details

2. Sample size used for the test set and the data provenance

• Sample Size: 1167 nasopharyngeal (NP) swab samples were initially tested. One sample was excluded due to unresolved results, leading to a final test set of 1166 samples for the clinical performance analysis.
• Data Provenance: The study was a prospective study conducted at 4 U.S. clinical laboratories from October 2009 to August 2010. Samples were NP swab specimens collected for routine respiratory viral testing by each site.

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

The document does not specify the number or qualifications of experts used for establishing the ground truth. It states that the reference method was "rapid culture (shell vial) followed by direct fluorescent antibody (DFA) screening and identification." Discrepant analysis for samples in disagreement was performed using "PCR primers obtained from literature followed by sequencing." This implies laboratory-based methods for ground truth, rather than expert human interpretation of images or other subjective data.

4. Adjudication method for the test set

The document describes a form of discrepant analysis. For samples where the ProAdeno+ Assay and the initial reference method (culture/DFA) disagreed, further testing was performed using PCR primers obtained from literature followed by sequencing. This served as the adjudicating method to establish the true status of discrepant samples.

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 done. The study evaluates the performance of a diagnostic assay (ProAdeno+ Assay) against a reference laboratory method, not human readers with or without AI assistance.

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

Yes, this was a standalone performance study. The ProAdeno+ Assay is an in vitro diagnostic test, and its performance was evaluated directly against a reference method without human interpretation as part of the primary outcome.

7. The type of ground truth used

The ground truth was established using a combination of laboratory methods:

• Primary reference method: Rapid culture (shell vial) followed by direct fluorescent antibody (DFA) screening and identification.
• Discrepant analysis ground truth: PCR primers obtained from literature followed by sequencing.

8. The sample size for the training set

The document does not specify a separate training set. The descriptions provided are for the clinical performance evaluation and reproducibility assessment. This is typical for a diagnostic test validation, where the focus is on evaluating the device's performance characteristics in a clinical setting rather than training a machine learning model.

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

As no separate training set is mentioned or described, the method for establishing its ground truth is not applicable from the provided text.

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The ProFAST™+ Assay is a multiplex Real Time RT-PCR in vitro diagnostic test for the qualitative detection and discrimination of seasonal Influenza A/H1, seasonal Influenza A/H3 and 2009 H1N1 Influenza viral nucleic acids isolated and purified from nasopharyngeal (NP) swab specimens from human patients with signs and symptoms of respiratory infection in conjunction with clinical and epidemiological risk factors. This assay targets conserved regions of the Hemagglutinin (HA) gene for seasonal Influenza A/H1, seasonal Influenza A/H3 and 2009 H1N1 Influenza Virus, respectively. This assay is not intended to detect Influenza B or Influenza C Viruses.

A negative ProFAST+ Assay result is a presumptive negative result for Influenza A. These results should be confirmed by an FDA cleared nucleic acid-based test (NAT) detecting Influenza A.

Negative results do not preclude Influenza virus infection and should not be used as the sole basis for treatment or other patient management decisions.

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 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 ProFAST+ Assay enables detection and discrimination of Influenza A Virus subtypes: seasonal H1, seasonal H3, and 2009 H1N1.

An overview of the procedure is as follows:

1. Collect nasopharyngeal swab specimens from symptomatic patients using a polyester, ravon, or nylon tipped swab and place into viral transport medium (refer to Materials Required but not Provided section of this Instruction for Use).
2. Add an Internal Control (IC) to every sample to monitor for inhibitors present in the specimens.
3. Perform isolation and purification of nucleic acids using 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).
4. Add purified nucleic acids to the ProFAST+ Supermix along with enzymes included in the ProFAST+ kit. The ProFAST+ Supermix contains target-specific oligonucleotide primers and probes. The primers are complementary to conserved regions of the Hemagglutinin (HA) gene for seasonal influenza A/H1, seasonal influenza A/H3 and 2009 H1N1 Influenza Virus (swine-origin), respectively. The probes are dual-labeled with a reporter dye and a quencher dye (see table below).
5. Perform reverse transcription of RNA into complementary DNA (cDNA) and subsequent amplification of DNA in a Cepheid SmartCycler II instrument. In this process, the probe anneals specifically to the template followed by primer extension and amplification. The ProFAST+ 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 real-time instrument.

Here's a summary of the acceptance criteria and study details for the ProFAST+ Assay based on the provided document:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are implied by the "Positive Percent Agreement" and "Negative Percent Agreement" values, along with their 95% Confidence Intervals. For the reproducibility study, the acceptance criterion is an "overall percent agreement with the expected result" of 99.7%.

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

• Prospective Study:
  • Sample Size: 842 eligible prospective NP swab specimens (initially 874, but 32 were excluded).
  • Data Provenance: U.S. clinical laboratories (4 sites), collected between December 2009 and May 2010. Also included prospectively collected archived samples (from January-March, 2008, February-March, 2009, and October-November, 2009) due to absence of seasonal influenza during the 2009-2010 winter season. These represented "excess, remnants of nasopharyngeal (NP) swab 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."
• Retrospective Study:
  • Sample Size: 160 retrospective nasopharyngeal (NP) swab samples.
  • Data Provenance: Two clinical sites, collected from January-March, 2008, January-November 2009, and March 2010.

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

The document does not specify the number or qualifications of experts involved in establishing the ground truth. The ground truth was established by a "composite comparator/reference method."

4. Adjudication Method for the Test Set

The document does not describe an explicit adjudication method between human experts, as the ground truth was established by a composite of laboratory tests rather than expert consensus on interpretation. It mentions that samples were "considered as any sample that was tested positive for Influenza A by the ProFlu+ Assay, and had bidirectional sequencing data meeting pre-defined quality acceptance criteria."

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 performed. This is a diagnostic test (RT-PCR assay) intended for standalone use, not an imaging device requiring human reader interpretation, so this type of study would not be applicable.

6. If a Standalone Study Was Done

Yes, a standalone study (algorithm/device-only performance without human-in-the-loop performance) was done. The ProFAST+ Assay's performance was compared directly against a composite reference method. The clinical performance data (Positive and Negative Percent Agreements) reflect the standalone performance of the device.

7. The Type of Ground Truth Used

The ground truth used was a composite comparator/reference method consisting of:

• FDA cleared ProFlu+ Assay (another nucleic acid-based test for Influenza A).
• Individual well-characterized Influenza A subtype-specific RT-PCR assays.
• Bi-directional sequencing (targeting different regions of the hemagglutinin gene than the ProFAST+ Assay).

"True" positives required:

• Positive for Influenza A by ProFlu+ Assay
• And bidirectional sequencing data meeting pre-defined quality acceptance criteria, matching seasonal A/H1, A/H3, or A/2009 H1N1 sequences in the NCBI GenBank database with acceptable E-values.

"True" negatives required:

• Negative for Influenza A by ProFlu+ Assay
• OR positive for Influenza A by ProFlu+ Assay but negative by the respective Influenza A subtype-specific RT-PCR assay followed by bi-directional sequencing.

8. The Sample Size for the Training Set

The document does not explicitly state the sample size used for a training set. The clinical performance studies describe the evaluation of the device using prospective and retrospective samples, implying these are test sets rather than training sets. Typically for an in vitro diagnostic (IVD) assay like this, development and optimization (which might use internal "training-like" datasets) are usually completed before the formal clinical validation described in the 510(k) summary.

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

Since a distinct "training set" is not explicitly mentioned as part of this premarket notification validation, the method for establishing its ground truth is not detailed. For typical IVD development, internal positive and negative controls, spiked samples, and characterized clinical samples would be used during development and optimization phases.

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