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The Influenza A Subtyping Kit contains reagents and controls of the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel and is intended for use in real-time RT-PCR (rRT-PCR) assays on an in vitro diagnostic real-time PCR instrument that has been FDA-cleared for use with this kit in conjunction with clinical and epidemiological information:

· For determination of the subtype of seasonal human influenza A viruses as seasonal A(H3) and/or A(H1)pdm09 from viral RNA in upper respiratory tract clinical specimens (including nasopharyngeal swabs [NS], throat swabs [TS], nasal aspirates [NA], nasal washes [NW] and dual nasopharyngeal/throat swabs [NPS/TS]) and lower respiratory tract specimens (including bronchoalveolar lavage [BAL], bronchial wash [BW], tracheal aspirate [TA], sputum, and lung tissue) from human patients with signs and symptoms of respiratory infection and/or from viral culture;

· To provide epidemiologic information for surveillance of circulating influenza viruses.

The Influenza A Subtyping Kit contains components of the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel that is used in rRT-PCR assays on an FDA-cleared in vitro diagnostic real-time PCR instrument. The primer and probe sets contained in the Influenza A Subtyping Kit are designed for the detection and characterization of influenza type A viruses that infect humans.

The Influenza A Subtyping Kit consists of oligonucleotide primers and dual-labeled hydrolysis (TagMan®) probes and controls. which may be used in rRT-PCR assays for the in vitro qualitative detection and characterization of the human influenza virus RNA in respiratory specimens from patients presenting with influenza-like illness (IL). The oligonucleotide primers and probes for detection of Influenza A and 2009 Influenza A (swine origin) were selected from highly conserved regions of the matrix (M), and the nucleoprotein (NP), respectively. Oligonucleotide primers and probes for characterization and differentiation of seasonal influenza A(H3) and A(H1)pdm09 viruses were selected from highly conserved regions of their respective HA genes. Detection of viral RNA not only aids in the diagnosis of illness caused by seasonal. newly emerging, and novel influenza viruses in patients with ILI, but also provides epidemiological and surveillance information on influenza and aids in the presumptive laboratory identification of specific novel influenza A viruses.

Here's a breakdown of the acceptance criteria and the study proving the device meets them, based on the provided text:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are implied through the performance metrics evaluated and compared to the predicate device and NGS as a gold standard. The core idea is that the modified assay should perform equivalently or better than the previous version, especially for strains with the 3' mutation.

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

• Sample Size (Clinical Evaluation):
  • Positive Specimen Panel: 60 influenza A(H3) specimens (30 with 3' mutation, 30 without 3' mutation).
  • Negative Specimen Panel: 60 negative specimens (from symptomatic patients known to be positive for influenza H1N1).
  • Total Clinical Test Set: 120 specimens (60 positive, 60 negative).
• Data Provenance: Retrospective study. Clinical specimens were collected from patients during previous influenza seasons in the United States.

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

Not applicable. The ground truth for the clinical test set was established using Next Generation Sequencing (NGS) and direct clinical specimen classification, not through expert consensus of visual or diagnostic interpretation.

4. Adjudication Method for the Test Set

Not applicable. The ground truth for the clinical test set was established via Next Generation Sequencing (NGS), which directly determines the genetic identity of the virus, rather than a consensus among human reviewers.

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 real-time RT-PCR diagnostic panel, not an AI-assisted diagnostic tool that would involve human readers interpreting AI output.

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

Yes, the performance evaluation in this submission is of the standalone device (algorithm only). The RT-PCR assay itself provides the result, without human interpretation of the assay's primary output (e.g., a visual scan of a reaction). The results are considered definitive from the machine output.

7. The Type of Ground Truth Used

• Analytical Performance: The ground truth for analytical sensitivity (LoD) and inclusivity studies was based on known, quantified viral stocks (EID50/mL or ID50/mL) of specific influenza strains.
• Clinical Performance: The ground truth for positive and negative clinical specimens was established using Next Generation Sequencing (NGS) as the comparator assay. This method directly confirms the identity and specific genetic characteristics (e.g., presence of 3' mutation) of the influenza virus in the samples.

8. The Sample Size for the Training Set

The document does not explicitly state a "training set" in the context of device development. This is a modification to an existing RT-PCR assay, and the "development" or "training" of such a device primarily involves bioinformatic analysis, primer/probe design adjustments, and analytical testing with known isolates. The "in-silico analysis" described (Process 1: assessment of primers against global H3N2 sequence information from GISAID EpiFlu database; Process 2: BLAST against NCBI nr/nt database) serves a similar function to a training set for algorithm-based devices by informing the optimal primer and probe sequences. No numerical sample size is provided for these bioinformatic databases.

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

As noted above, for an RT-PCR assay, the "training set" concept is different from an AI/ML context. The ground truth for informing the primer/probe design (which is effectively the "training" data for the assay's specificity and sensitivity for detecting target sequences) was established through:

• GISAID EpiFlu database: This database contains comprehensive, publicly shared influenza sequence information, used to assess potential primer and probe sets against known H3N2 sequences and calculate nucleotide mismatches. The sequences in this database are derived from laboratories globally undergoing influenza surveillance.
• NCBI BLAST+ against the nr/nt database: This is a vast database of non-redundant nucleotide sequences, against which primer sequences were compared to confirm inclusivity for H3 Influenza virus HA segments and exclusivity against non-target sequences.

This information is based on established genetic sequences, which serve as the fundamental "ground truth" for designing molecular diagnostic assays.

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The Influenza A Subtyping Kit contains reagents and controls of the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel and is intended for use in real-time RT-PCR (rRT-PCR) assays on an Applied Biosystems (ABI) 7500 Fast Dx Real-Time PCR instrument in conjunction with clinical and epidemiological information:

· For determination of the subtype of seasonal human influenza A viruses as seasonal A(H3) and/or A(H1)pdm09 from viral RNA in upper respiratory tract clinical specimens (including nasopharyneed swabs [NS], throat swabs [TS], nasal aspirates [NA], nasal washes [NW] and dual nasopharyngeal/throat swabs [NPS/TS]) and lower respiratory tract specimens (including bronchoalveolar lavage [BAL], bronchial wash [BW], tracheal aspirate [TA], sputum, and lung tissue) from human patients with signs and symptoms of respiratory infection and/or from viral culture;

· To provide epidemiologic information for surveillance of circulating influenza viruses.

Performance characteristics for influenza were established during a seasonal influenza viruses A(HIN1) and A(H3N2) were the predominant influenza A viruses in circulation and during a season when the A(H1N) Jodm09 influenza virus was the predominant influenza A virus in circulation. Performance characteristics may vary with other emerging influenza A viruses.

Negative results do not preclude influenza virus infection and should not be used as the sole basis for treatment or other patient management decisions. Conversely, positive results do not rule out bacterial infection with other viruses. The agent detected may not be the definite cause of disease.

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 unless a BSL 3E facility is available to receive and culture specimens.

All users, and any person reporting results from use of this device should be trained to perform and interpret the results from this procedure by a competent instructor prior to use. CDC Influenza Division will limit the distribution of this device to only those users who have successfully completed a training course provided by CDC instructors or designees.

The CDC Human Influenza Real-Time RT-PCR Diagnostic Panel Influenza A Subtyping Kit is a realtime RT-PCR (rRT-PCR) assay that utilizes the Applied Biosystems® (ABI) 7500 Fast Dx Real-time PCR system. The Influenza A Subtyping Kit consists of oligonucleotide primers, fluorescently labeled hydrolysis probes, and controls which are used in rRT-PCR assays for the in vitro qualitative detection and characterization of influenza virus RNA in respiratory specimens from patients presenting with influenza-like illness (ILI). The oligonucleotide primers and probes for detection of influenza A and 2009 influenza A (swine origin) were selected from highly conserved regions of the matrix (M), and the nucleoprotein (NP), respectively. Oligonucleotide primers and probes for characterization and differentiation of influenza A(H3) and A(H1)pdm09 viruses were selected from highly conserved regions of their HA genes. The Influenza A Subtyping kit also contains primers and probes to detect the human RNase P gene (RP) in control samples and clinical specimens.

This document describes the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel, Influenza A Subtyping Kit (VER 2), which is intended for the qualitative detection and characterization of influenza virus RNA. The document outlines changes from a predicate device (K140851) including modifications to the pdmH1 assay and the removal of the H1 assay.

Here's an analysis of the acceptance criteria and supporting studies as presented in the document:

1. Table of Acceptance Criteria and Reported Device Performance

The document primarily focuses on demonstrating the analytical and clinical performance of the modified pdmH1 assay and the H3 assay. While explicit acceptance criteria (e.g., "PPA must be >X%") are not formalized in a table of the document, the studies demonstrate a high level of performance. Based on the results, implicit acceptance criteria would involve:

• Analytical Sensitivity (LOD): Consistent detection at low viral concentrations for A(H1)pdm09 and A(H3) assays using both enzyme kits.
• Inclusivity: Successful detection of diverse A(H1)pdm09 strains.
• Clinical Performance (Positive Agreement): High percentage of agreement with previously positive clinical samples for A(H1)pdm09 and A(H3).
• Clinical Performance (Negative Agreement): High percentage of agreement with previously negative clinical samples for influenza A for both pdm and H3 assays.

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

• Analytical Sensitivity (LOD):
  • pdmH1 assay: For each of the two A(H1)pdm09 strains tested, 20 replicates of the highest virus dilution were used to confirm LOD.
  • H3 assay: A range finding study used 3 replicates per dilution for the LOD equivalency, but the confirmatory LOD for H3 is not explicitly stated in the same manner as pdmH1. However, the study concludes equivalency based on consistent positive results for 3/3 replicates across a range of dilutions.
• Inclusivity Testing: Ten (10) A(H1)pdm09 strains were tested, with each strain tested in triplicate.
• Cross-Reactivity Testing: Five (5) influenza A(H1) virus strains were tested, each in triplicate.
• Clinical Performance Evaluation:
  • Positive A(H1)pdm09: A total of 42 retrospective clinical samples previously determined positive for A(H1)pdm09 were evaluated. The breakdown by specimen type for reported results is 1 BW, 35 NPS/NS, 4 NW, and 2 TS for Invitrogen; and 1 BW, 33 NPS/NS, 4 NW, and 2 TS for Quanta.
  • Positive A(H3): A total of 32 retrospective clinical samples previously determined positive for A(H3) were evaluated. The breakdown by specimen type is 1 NA, 30 NPS/NS, and 1 NW for both enzyme systems.
  • Negative for Influenza A (pdm assays): A total of 53 retrospective clinical samples (NPS) previously determined negative for influenza A were tested for pdm assays.
  • Negative for Influenza A (H3 assay): A total of 30 retrospective clinical samples (NPS) previously determined negative for influenza A were tested for H3 assay.
• Data Provenance: Retrospective clinical samples collected during the 2011-2012, 2013-2014, and 2015-2016 influenza seasons. The country of origin is not explicitly stated but implied to be the US given the CDC context.

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

The document does not explicitly state the number or qualifications of experts used to establish the ground truth for the clinical test set. The ground truth for clinical samples was based on prior determination using the "CDC Human Influenza rRT-PCR Diagnostic Panel" and, for the 2015-2016 samples, confirmation by genetic sequence analysis. This implies the use of laboratory professionals and possibly virologists or epidemiologists, but specific details on their expertise are not provided.

4. Adjudication Method for the Test Set

The document does not describe an explicit adjudication method for the test set results. For the analytical studies, replicates are used, and the standard reporting of "X/X (+)" indicates all replicates were positive. For clinical samples, results were compared against prior determinations and genetic sequencing, suggesting these served as the reference standards rather than a separate adjudication process.

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 device is a diagnostic assay (RT-PCR kit), not an imaging or interpretation device that would typically involve multiple human readers. The study focuses on the analytical and clinical performance of the assay itself.

6. If a Standalone (Algorithm Only Without Human-in-the-Loop Performance) Was Done

Yes, the studies conducted are standalone performance evaluations of the diagnostic assay. The device is a "Real-Time RT-PCR Diagnostic Panel," meaning the "algorithm" is the biochemical and molecular process of the assay itself, followed by data interpretation based on established thresholds (e.g., Ct values). The results presented are the output of this assay system.

7. The Type of Ground Truth Used

• Analytical Studies (LOD, Inclusivity, Cross-Reactivity): Ground truth was established using characterized viruses of known titers (ID50/mL or EID50/mL).
• Clinical Performance Studies:
  • For positive samples: Ground truth was based on previous positive determination with the "CDC Human Influenza rRT-PCR Diagnostic Panel" and, for some 2015-2016 samples, confirmed by genetic sequence analysis.
  • For negative samples: Ground truth was based on previous negative determination for influenza A with the "CDC Human Influenza rRT-PCR Diagnostic Panel."

8. The Sample Size for the Training Set

The document does not explicitly describe a "training set" in the context of machine learning, as this is a molecular diagnostic assay. The development of the oligonucleotide primers and probes would involve extensive bioinformatics and laboratory work, but these are not referred to as a "training set" in the presented material. The "characterization" of the device involves testing its performance with known inputs rather than training an algorithm.

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

As no explicit "training set" for a machine learning algorithm is described, this question is not directly applicable. The "ground truth" for the development of the assay components (primers and probes) would have been established through molecular biology techniques, genomic sequencing, and epidemiological data on circulating influenza strains to identify conserved regions and ensure specificity. The document mentions that the primers and probes were "selected from highly conserved regions" of the viral genes, implying this foundational work.

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