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The Prodesse® ProFluTM+ Assay is a multiplex Real-Time PCR (RT-PCR) in vitro diagnostic test for the rapid and qualitative detection and discrimination of 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 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 differentiation of Influenza A Virus, Influenza B Virus, Respiratory Syncytial Virus (RSV) (Types A and B), and Universal Internal Control nucleic acids. Nasopharyngeal swab specimens are collected from symptomatic patients using a polyester, rayon or nylon tipped swab and place 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 Influenza B/RSV Mix along with enzymes included in the ProFlu+ Assay Kit. The 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.

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 ProFlu+ 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 SmartCycler II instrument.

The provided text is related to a 510(k) premarket notification for a medical device called Prodesse® ProFlu™+ Assay. This assay is an in vitro diagnostic test for the rapid and qualitative detection and discrimination of certain influenza and respiratory syncytial viruses. The document describes the device, its intended use, and substantial equivalence to predicate devices. It also mentions a study performed to test the reactivity of the assay to an emerging strain of Influenza A.

However, the provided document does not contain a table of acceptance criteria and reported device performance in the typical format requested for clinical studies (e.g., sensitivity, specificity, accuracy with confidence intervals). It also does not detail the study design elements such as sample size for the test set, data provenance, number and qualifications of experts, adjudication methods, MRMC studies, standalone performance, ground truth type, training set details, or how training ground truth was established, as these are typically found in more comprehensive clinical study reports or summaries, not a 510(k) clearance letter and its associated summary.

The document mainly focuses on the regulatory aspects of the device, its components, and a specific study conducted to address the reactivity of the assay to a new strain of influenza A to update the product's instructions for use. It outlines the analytical performance regarding the detection of this new strain.

Therefore, many of the requested points cannot be extracted directly from the given text.

Here's an attempt to answer based only on the provided information, noting what isn't available:

Acceptance Criteria and Study for Prodesse® ProFlu™+ Assay

The document provided details a 510(k) premarket notification for the Prodesse® ProFlu™+ Assay. While it references a study for a modified device, it does not explicitly state "acceptance criteria" in the format of numerical thresholds for clinical performance metrics (e.g., sensitivity, specificity) for the overall device. Instead, it describes a reactivity study for a specific emerging strain of Influenza A, aimed at updating the Instructions for Use.

The implied acceptance criterion for this specific reactivity study appears to be the ability of the assay to detect the new strain.

1. Table of Acceptance Criteria and Reported Device Performance

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

• Sample Size: Not explicitly stated for this particular reactivity study. The phrase "a study was performed to test the reactivity... to an emerging strain" suggests a focused analytical study rather than a large clinical test set. The concentration mentioned (2x10¹ TCID50/mL) implies testing was done on dilutions of a cultured virus.
• Data Provenance: Not specified (e.g., country of origin, retrospective/prospective). This was an analytical study performed on cultured virus.

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

• Not applicable/Not provided. For an analytical reactivity study, the "ground truth" would be the known presence and concentration of the cultured virus, not expert interpretation of clinical samples.

4. Adjudication method for the test set

• Not applicable/Not provided. This was an analytical reactivity study, not a clinical study requiring adjudication of expert readings.

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 assay (a lab test), not an AI-assisted diagnostic tool that involves human readers for interpretation of images or other complex data.

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

• The information provided refers to an in vitro diagnostic (IVD) assay. IVDs inherently operate in a "standalone" manner in the sense that the assay itself generates a result (e.g., positive/negative, concentration). The results are then interpreted by laboratory personnel and clinicians. The study described is an analytical test of the assay's capability to detect a specific viral strain.

7. The type of ground truth used

• For the specific reactivity study mentioned, the ground truth was the known presence and concentration of a cultured viral strain (Influenza A, A/New York/1/2015 H3N2), quantified by TCID50/mL.

8. The sample size for the training set

• Not applicable/Not provided. This document describes a modification to an existing device and a reactivity study, not the development or training of a machine learning model.

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

• Not applicable/Not provided. As above, this is not a machine learning model; thus, a "training set" in that context is not relevant. The device's underlying chemistry and primer/probe design (described in the "Product Description") would have been developed based on known viral genetic sequences, but this is a different concept from a "training set" for an algorithm.

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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 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 ProFlu+™ Assay is a multiplex Real Time 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. It is recommended that negative RSV results be confirmed by culture.

Performance characteristics for Influenza A Virus were established when Influenza A/H3 and A/H1 were the predominant Influenza A viruses in circulation. 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 (RSV) (Types A and B), and Internal Control. Nasopharyngeal swab specimens are collected from symptomatic patients using a polyester, rayon or nylon tipped swab and place 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 ProFlu+ Supermix along with enzymes included in the ProFlu+ Detection Kit. The ProFlu+ 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 ProFlu+ 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 instrument.

This document describes the ProFlu+ Assay, a multiplex Real Time 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. However, the provided text does not contain the information requested in your prompt regarding acceptance criteria, specific study details, sample sizes, ground truth establishment, expert qualifications, or comparative effectiveness study results.

The document primarily focuses on:

• Identification of the device: ProFlu+ Assay
• Contact information: Prodesse, Inc.
• Predicate device: K081030 - ProFlu+ Assay, Prodesse, Inc. and K091667 - ID-Tag Respiratory Virus Panel, Luminex Molecular Diagnostics, Inc.
• Intended Use: Aid in differential diagnosis of Influenza A, Influenza B, and RSV viral infections from nasopharyngeal swab specimens from symptomatic patients. Explicitly states it's not for Influenza C, and negative results for RSV should be confirmed by culture.
• Product Description: Details the methodology (multiplex Real Time RT-PCR, use of internal control, MagNA Pure LC Instrument or NucliSENS® easyMAGTM System for nucleic acid isolation, Cepheid SmartCycler® II for RT-PCR, Taqman chemistry).
• Substantial Equivalence: Notes that the intended use remains the same, and the device is reactive to the 2009 H1N1 Influenza Virus.
• FDA Clearance: Provides formal communication from the FDA regarding the 510(k) clearance (K092500).

Therefore, I cannot fulfill your request for the specific details outlined in your prompt based on the provided text. The document does not include a table of acceptance criteria, reported device performance metrics against such criteria, sample sizes for test/training sets, ground truth methods, expert details, or information on MRMC studies.

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The ProFlu+™ Assay is a multiplex Real Time 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. It is recommended that negative RSV results be confirmed by culture.

Performance characteristics for Influenza A Virus were established when Influenza A/H3 and A/H1 were the predominant Influenza A viruses in circulation. When other Influenza A viruses are emerging, performance characteristics may vary.

If infections 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 (RSV) (Types A and B), and Internal Control. Nasopharyngeal swab specimens are collected from symptomatic patients using a polyester, rayon or nylon tipped swab and place 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 ProFlu+ Supermix along with enzymes included in the ProFlu+ Detection Kit. The ProFlu+ 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 ProFlu+ 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 instrument.

Here's an analysis of the ProFlu+ Assay's acceptance criteria and the study data provided, structured according to your request:

Acceptance Criteria and Device Performance

The acceptance criteria are not explicitly stated in numerical thresholds in the provided document. However, the intent is to demonstrate substantial equivalence to predicate devices, implying that the performance should be comparable or superior. The reported device performance is presented in terms of Sensitivity and Specificity with 95% Confidence Intervals.

Table 1: Acceptance Criteria (Implied) and Reported Device Performance (Prospective Study)

Table 2: Acceptance Criteria (Implied) and Reported Device Performance (Retrospective Study)

Study Details:

1. Sample Sizes Used for the Test Set and Data Provenance:

   • Prospective Study Test Set: 891 nasopharyngeal (NP) swab samples. After excluding 5 unresolved samples, 826 samples were used in the analysis (Note: The sum of total samples in individual analyte tables is 826).
   • Retrospective Study Test Set: Not explicitly stated as a total, but individual analyte tables sum to 60 samples for Influenza A, 60 samples for Influenza B, and 60 samples for RSV (likely the same set of 60 samples analyzed for all three).
   • Data Provenance:
     • Prospective Study: Conducted at 3 U.S. clinical laboratories during the 2006-2007 respiratory virus season (February - April).
     • Retrospective Study: Conducted at 1 U.S. site during the 2006-2007 respiratory virus season (February - April).
     • The samples were "collected for routine influenza or RSV testing by each site," indicating real-world clinical samples.

2. 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 of experts or their qualifications for establishing the initial reference method (rapid culture/DFA).
   • The ground truth for discrepant analysis was established using "RT-PCR with virus specific primers obtained from literature followed by sequencing." This implies a molecular biology expert, but no specific number or qualifications are given.

3. Adjudication Method for the Test Set:

   • The primary reference method was rapid culture (shell vial) followed by direct fluorescent antibody (DFA) screening and identification.
   • For samples where the ProFlu+ Assay and the reference method (culture/DFA) disagreed, discrepant analysis using RT-PCR with virus-specific primers followed by sequencing was performed. This acts as a tie-breaker or a higher-tier reference method for discordant results.

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

   • No. The study described is a direct comparison of the ProFlu+ assay against a reference method (culture/DFA and sequencing), not a multi-reader multi-case study comparing human readers with and without AI assistance. The ProFlu+ Assay is an in vitro diagnostic test, not an AI-assisted interpretation tool for human readers.

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

   • Yes, the performance data presented (Sensitivity, Specificity) represent the standalone performance of the ProFlu+ Assay diagnostic test itself, processing the samples and providing results without human interpretation influencing the diagnostic outcome beyond standard laboratory procedures (e.g., sample handling, instrument operation).

6. The Type of Ground Truth Used:

   • The primary ground truth for the test set was established by a combination of methods:
     • Rapid culture (shell vial) and direct fluorescent antibody (DFA).
     • For discrepant results, the ground truth was re-established using RT-PCR with virus-specific primers followed by sequencing (which can be considered molecular pathology/genetic confirmation).

7. The Sample Size for the Training Set:

   • The document does not provide details about a specific "training set" for the ProFlu+ Assay. This assay is a diagnostic test based on molecular biology principles (RT-PCR), not a machine learning or AI model that typically requires a separate training set. The "design" and "optimization" of the primers and probes would be done during assay development, but not in the same sense as training a predictive algorithm on labeled datasets.

8. How the Ground Truth for the Training Set Was Established:

   • As noted above, the concept of a "training set" with ground truth in the context of an RT-PCR diagnostic assay like ProFlu+ is not directly applicable in the same way it would be for an AI algorithm. The development of the assay (e.g., selection of primer/probe sequences) would rely on known viral genetic sequences and established molecular biology techniques, rather than a "ground truth" derived from patient samples for algorithm training.

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The ProFlu+™ Assay is a multiplex Real Time 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.

A negative test is presumptive and it is recommended these results be confirmed by cell culture. 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.

Performance characteristics for Influenza A Virus were established when Influenza A/H3 and A/H1 were the predominant Influenza A viruses in circulation. When other Influenza A viruses are emerging, performance characteristics may vary.

If infections with a novel Influenza A virus are 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 (RSV) (Types A and B), and Internal Control. Nasopharyngeal swab specimens are collected from symptomatic patients 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 a MagNA Pure LC Instrument (Roche) and the MagNA Pure Total Nucleic Acid Isolation Kit (Roche).

The purified nucleic acids are added to ProFlu+ Supermix along with enzymes included in the ProFlu+ Detection Kit. The ProFlu+ 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 (see table below).

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 ProFlu+ 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 SmartCycler instrument.

Here's an analysis of the acceptance criteria and study details for the ProFlu+ Assay, based on the provided text:

Acceptance Criteria and Device Performance

The acceptance criteria are not explicitly stated in a formalized table within the provided text. However, they can be inferred from the reported sensitivity and specificity values. The study reports the following performance for the ProFlu+ Assay:

Table 1: ProFlu+ Assay Performance (Inferred Acceptance Criteria vs. Reported Performance)

Reproducibility (Overall Percent Agreement):

• Implied Acceptance Criteria: High (e.g., >95%)
• Reported Device Performance: 98% (96% - 99% CI)

Study Details

2. Sample Size and Data Provenance

• Sample Size for Test Set:
  • Prospective Study: 826 nasopharyngeal (NP) swab samples. A total of 891 samples were initially tested, but 5 unresolved samples were excluded.
  • Retrospective Study: 60 samples.
• Data Provenance:
  • Prospective Study: 3 U.S. clinical laboratories (prospective collection during February - April 2007 respiratory virus season).
  • Retrospective Study: 1 U.S. site.

3. Number of Experts and Qualifications for Ground Truth

The document does not explicitly state the number of "experts" (e.g., pathologists, radiologists) in the conventional sense for oncology or imaging studies. For this in vitro diagnostic (IVD) device:

• The "experts" can be considered the laboratory personnel performing the reference methods. The study was conducted at 3 U.S. clinical laboratories and 1 U.S. site, implying multiple trained technicians/scientists.
• Qualifications: While not explicitly detailed, it is assumed these individuals were qualified laboratory professionals experienced in performing rapid culture (shell vial) and direct fluorescent antibody (DFA) screening and identification for respiratory viruses, as these were the reference methods.

4. Adjudication Method for the Test Set

The adjudication method used for discrepant results was:

• Discrepant Analysis: For samples where ProFlu+ Assay results and culture results disagreed, RT-PCR with virus-specific primers (obtained from literature) followed by sequencing was performed. This served as the definitive arbiter for these cases.
• For the 23 DFA Respiratory Virus Screen positive samples that had too few cells for specific identification, genetic sequencing analysis was used to confirm the specific virus or to confirm negativity for Influenza A, Influenza B, and RSV.

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

• No, an MRMC comparative effectiveness study was not done. This is an in vitro diagnostic device for laboratory analysis, not an imaging device requiring human reader interpretation in a clinical setting in the way MRMC studies are typically performed. The "readers" here are the automated instruments and laboratory technicians, whose performance is assessed through reproducibility and accuracy against reference methods.

6. Standalone (Algorithm Only) Performance

• Yes, a standalone performance study was done. The entire clinical performance section (both prospective and retrospective studies) evaluates the ProFlu+ Assay as a standalone device, comparing its results directly against reference methods. There is no mention of a "human-in-the-loop" component for its primary diagnostic function.

7. Type of Ground Truth Used

The ground truth for the clinical performance studies was established using a combination of:

• Reference Methods:
  • Rapid culture (shell vial)
  • Direct fluorescent antibody (DFA) screening and identification
• Discrepant Analysis/Confirmation:
  • RT-PCR with virus-specific primers followed by sequencing (for resolving disagreements between ProFlu+ and reference methods, and for confirming DFA screen positives with insufficient cells).

Essentially, the ground truth was a combination of established laboratory methods, with molecular sequencing as the definitive arbiter for ambiguous or conflicting results.

8. Sample Size for the Training Set

• The document does not specify a sample size for a training set. This type of IVD (multiplex Real-Time RT-PCR assay) is developed based on pre-defined molecular targets and amplification conditions. While there would have been extensive assay development and optimization, the concept of a "training set" in the machine learning sense is not directly applicable or explicitly described for an RT-PCR assay in this context. The study focuses on clinical validation of the developed assay.

9. How Ground Truth for Training Set Was Established

• As a "training set" is not explicitly mentioned or relevant in the context of conventional machine learning for this specific device (a molecular diagnostic assay), the method for establishing ground truth for it is not applicable or described in the document. The assay's design relies on known genetic sequences of the target viruses, and its performance is validated clinically.

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