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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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CDC Human Influenza Real-Time RT-PCR Diagnostic Panel: Influenza A/B Typing Kit (VER 2)
The Influenza A/B Typing 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 qualitative detection of influenza virus type A or B viral RNA in upper respiratory tract clinical specimens (including nasopharyngeal swabs [NPS], nasal 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 epidemiological information for surveillance of circulating influenza viruses.

CDC Human Influenza Real-Time RT-PCR Diagnostic Panel: Influenza A Subtyping Kit (VER 3)
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 [NPS], nasal 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 epidemiological information for surveillance of circulating influenza viruses.

CDC Human Influenza Real-Time RT-PCR Diagnostic Panel: Influenza A/H5 Subtyping Kit (VER 4)
The Influenza A/H5 Subtyping Kit contains reagents and controls of the CDC Human Influenza Virus 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 the presumptive identification of virus in patients who may be infected with influenza A subtype A(H5) (Asian lineage) from viral RNA in human respiratory specimens and viral culture in conjunction with clinical and epidemiological risk factors;
• · To provide epidemiological information for surveillance of circulating influenza viruses.

The CDC Human Influenza Real-Time RT-PCR Diagnostic Panel is used in real-time RT-PCR (rRT-PCR) assays on an in vitro diagnostic real-time PCR system. The panel is configured in four separate kits. Each 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). Oligonucleotide primers and probes for detection of influenza A, influenza B, and 2009 influenza A (swine origin) were selected from highly conserved regions of the matrix (M), non-structural (NS), and nucleoprotein (NP) genes, respectively. Oligonucleotide primers and probes for characterization and differentiation of influenza A(H3) and A(H1)pdm(9 viruses and genetic lineages of influenza B were selected from highly conserved regions of their HA genes. Oligonucleotide primers and probes to detect the human RNase P gene (RP) in control samples and clinical specimens is also included in the panel.

Here's a breakdown of the acceptance criteria and study information for the CDC Human Influenza Virus Real-time RT-PCR Diagnostic Panel, based on the provided text:

Acceptance Criteria and Reported Device Performance

The acceptance criteria are primarily related to analytical sensitivity (Limit of Detection - LOD) and inclusivity for various influenza A strains, and analytical specificity (cross-reactivity and exclusivity). The clinical performance evaluation also serves as a form of acceptance criteria for positive and negative agreement with previously characterized samples.

Study Details:

2. Sample sizes used for the test set and the data provenance (e.g., country of origin of the data, retrospective or prospective)

• LOD Equivalency & Confirmation:

  • Sample Size: Varies by virus strain and specific assay. For LOD equivalency, triplicate samples of serial dilutions were tested. For LOD confirmation, 20 individually extracted samples were tested for each target.
  • Data Provenance: Virus strains are identified by name and origin (e.g., A/Michigan/45/2015, A/Illinois/20/2018, A/Hong Kong/4801/2014, A/Abu Dhabi/240/2018, A/duck/Vietnam/NCVD-1544/2012, A/duck/Vietnam/NCVD-17A231/2016). Specific country of origin for all strains is not explicitly stated but implied from nomenclature.
  • Retrospective/Prospective: Experimental, controlled laboratory studies using characterized virus stocks.

• Inclusivity:

  • Sample Size: 10 influenza A(H1N1)pdm09 viruses and 24 influenza A viruses of various types/subtypes. Each virus sample was tested in triplicate.
  • Data Provenance: Virus strains represented temporal, geographic, and genetic diversity (e.g., A/Florida/81/2018, A/Alaska/35/2018, A/Switzerland/8060/2017).
  • Retrospective/Prospective: Experimental, controlled laboratory studies using characterized virus stocks.

• Analytical Specificity (Cross-Reactivity & Exclusivity):

  • Sample Size: Cross-reactivity: Various influenza viruses (e.g., A/Perth/16/2009, B/Maryland/15/2016) tested in triplicate. Exclusivity: 35 organisms (16 viruses, 18 bacteria, 1 yeast) tested.
  • Data Provenance: Organisms are identified by strain name.
  • Retrospective/Prospective: Experimental, controlled laboratory studies using characterized stocks of various organisms.

• Clinical Performance Evaluation:

  • Sample Size:
    • Modified InfA Assay: 62 positive (35 A(H1N1)pdm09, 27 A(H3N2)) and 50 negative residual human respiratory clinical specimens.
    • Modified pdmInfA and pdmH1 Assays: 35 positive (for A(H1N1)pdm09) and 50 negative residual human respiratory clinical specimens.
  • Data Provenance: Residual human respiratory clinical specimens collected from patients during previous influenza seasons in the United States (2011-12 and 2013-14).
  • Retrospective/Prospective: Retrospective.

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 to establish ground truth. For the analytical studies (LOD, inclusivity, specificity), the ground truth is based on the known identity and titer of the cultured virus or bacterial/yeast strains. For the clinical performance evaluation, the ground truth for "positive" or "negative" status was established by prior testing (comparator) with the FDA-cleared CDC Human Influenza Real-Time RT-PCR Diagnostic Panel (K190302), which implicitly would have been validated using established laboratory methods or expert consensus in its own clearance process.

4. Adjudication method (e.g., 2+1, 3+1, none) for the test set

No explicit adjudication method (like 2+1 or 3+1 consensus) is described. The analytical studies often cite "number of positive replicates out of three total replicates tested," implying a direct comparison to the expected outcome from the known sample. For clinical studies, the comparator is the already FDA-cleared predicate device.

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 device is an in vitro diagnostic real-time RT-PCR diagnostic panel, not an AI-assisted diagnostic tool that humans interpret. There are no "human readers" in the context of interpreting the device's output, nor is there a multi-reader multi-case study described.

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

Yes, the studies described are standalone because the device itself is a diagnostic assay (a collection of reagents and controls) that produces a result (detection/characterization of viral RNA) through a real-time RT-PCR instrument. While laboratory personnel operate the instrument and interpret the output, the core performance studies evaluate the assay's ability to detect the target without human intervention influencing the assay's chemical and enzymatic reactions. The term "algorithm" is not directly applicable in the same way as with AI software, but the assay's 'logic' for detection is intrinsic to its design.

7. The type of ground truth used (expert consensus, pathology, outcomes data, etc.)

• For Analytical Studies (LOD, Inclusivity, Specificity): The ground truth is based on known, characterized viral and microbial strains with established titers (e.g., TCID50/mL or EID50/mL) or concentrations (CFU/mL, ng/µL). This is a highly controlled laboratory ground truth.
• For Clinical Performance Evaluation: The ground truth was established by prior testing with the FDA-cleared CDC Human Influenza Real-Time RT-PCR Diagnostic Panel (K190302), acting as the comparator or reference method for the collected residual human respiratory clinical specimens.

8. The sample size for the training set

The document does not explicitly describe a separate "training set" in the context of machine learning or AI. This device is a RT-PCR based assay, and its development would typically involve empirical optimization and validation against a variety of known strains and clinical samples rather than a formal training set for an AI model.

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

As there is no explicit "training set" described for an AI model, this question is not directly applicable. For the development and optimization of the RT-PCR assays, ground truth for evaluating probe and primer design would have been established through a combination of sequencing data to identify conserved regions and target specificity, and testing against known, characterized viral isolates/strains to ensure desired reactivity.

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The Influenza A/B Typing 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 qualitative detection of influenza virus type A or B viral RNA in upper respiratory tract clinical specimens (including nasopharyngeal swabs [NPS], 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 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 B Lineage Genotyping 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 ) assays on an in vitro diagnostic realtime PCR instrument that has been FDA-cleared for use with this kit in conjunction with clinical and epidemiological information:
· For the determination of the genetic lineage of human influenza B viruses as B/Victoria or B/Yamagata lineage 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]) 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/H5 Subtyping Kit contains reagents and controls of the CDC Human Influenza Virus RT-PCR Diagnostic Panel and is intended for use in real-time RT-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 the presumptive identification of virus in patients who may be infected with influenza A subtype A(H5) (Asian lineage) from viral RNA in human respiratory specimens and viral culture in conjunction with clinical and epidemiological risk factors;
· To provide epidemiologic information for surveillance of circulating influenza viruses.

The CDC Human Influenza Real-Time RT-PCR Diagnostic Panel is used in real-time RT-PCR (rRT-PCR) assays on an in vitro diagnostic real-time PCR system. The panel is configured in four separate kits. Each 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). Oligonucleotide primers and probes for detection of influenza A, influenza B, and 2009 influenza A (swine origin) were selected from highly conserved regions of the matrix (M), non-structural (NS), and nucleoprotein (NP) genes, respectively. Oligonucleotide primers and probes for characterization and differentiation of influenza A(H3) and A(H1)pdm(9 viruses and genetic lineages of influenza B were selected from highly conserved regions of their HA genes. Oligonucleotide primers and probes to detect the human RNase P gene (RP) in control samples and clinical specimens is also included in the panel.

Here's a breakdown of the acceptance criteria and study details based on the provided text:

1. Table of Acceptance Criteria and Reported Device Performance:

The document focuses on demonstrating substantial equivalence of the modified device (with new PCR instruments) to a predicate device. The key acceptance criteria are related to analytical sensitivity (Limit of Detection or LOD) and reproducibility, and clinical agreement.

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

• Analytical Sensitivity (LOD):

  • For each virus type and dilution: Triplicate samples were tested. The testing involved multiple influenza viruses (e.g., A/H3N2, A/H1N1pdm09, B/Victoria, B/Yamagata, A/H5N8) and two enzyme systems (SuperScript and qScript) across the original instrument and the two new instruments. It's difficult to give a single "sample size" number but it involved a substantial number of replicates across various conditions to establish LOD.
  • Data Provenance: The samples were "characterized influenza viruses of known egg infectious dose 50% titer," diluted in BPL-treated A549 cells in viral transport medium (VTM). This indicates laboratory-prepared, controlled samples.

• Analytical Precision (Reproducibility):

  • Sample Size: A blinded panel of 5 contrived samples (moderate positive A/H3N2, moderate positive B/Victoria, negative, low positive A/H3N2, low positive B/Victoria). Each sample was tested by two analysts at each of three sites, over five different days. This means for each contrived sample, there were $3 \text{ sites} \times 2 \text{ analysts} \times 5 \text{ days} = 30$ results. Across the 5 samples this totals $5 \text{ samples} \times 30 \text{ tests/sample} = 150$ tests for the QSDx and 150 for the QMDx.
  • Data Provenance: Contrived samples (BPL-treated A549 cells in VTM, spiked with BPL-treated influenza A(H3N2) or B/Victoria virus).

• Carryover and Cross-contamination:

  • Sample Size: An alternating pattern of high positive (HP) and negative (N) contrived samples. There were 5 high positive samples and 5 negative samples tested across 5 runs. The tables show 5 sets of HP samples and 5 sets of N samples for each assay (InfA, H5a, H5b, RP). For each assay, this means $5 \text{ HP samples} \times 5 \text{ runs} = 25$ tests and $5 \text{ N samples} \times 5 \text{ runs} = 25$ tests.
  • Data Provenance: Contrived samples (BPL-inactivated influenza A/gyrfalcon/41088-6/2014 (H5N8) in A549 cells).

• Clinical Performance Evaluation:

  • Sample Size: A total of 50 clinical specimens and 10 contrived samples with influenza A(H5) were used as positive samples (totaling 60 positive samples if the contrived A(H5) are counted separate from the 50 general clinical positives). And 50 negative specimens. So, a total of 100 retrospective samples (50 positive, 50 negative) were evaluated against the QSDx and QMDx independently.
  • Data Provenance:
    • Retrospective clinical specimens: Collected during the 2013-2014 influenza seasons. (Country of origin not specified, but given the CDC involvement, likely U.S.)
    • Contrived samples: The 10 A(H5) samples were "prepared with BPL-inactivated influenza A(H5) virus in a suspension of human A549 cells and virus transport medium."

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 the number of experts or their specific qualifications for establishing ground truth for the test set (clinical performance evaluation).

• For the analytical studies (LOD, reproducibility, carryover), the ground truth was established by the known characteristics of the prepared viral stocks and dilutions.
• For the clinical performance, the ground truth was established by the "previously determined" results using the FDA-cleared Applied Biosystems™ 7500 Fast Dx. This implies that the predicate device serves as the ground truth. It does not state that independent experts re-adjudicated these samples, but rather that the previously established results from a gold-standard method were used.

4. Adjudication Method for the Test Set:

No explicit adjudication method (e.g., 2+1, 3+1) is described for the clinical test set. The clinical samples were "previously determined to be positive using the Applied Biosystems™ 7500 Fast Dx." This implies the predicate device's results were considered the reference standard for comparison.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done, What Was the Effect Size of How Much Human Readers Improve with AI vs Without AI Assistance:

This device is an in-vitro diagnostic (IVD) RT-PCR kit, not an AI-assisted diagnostic imaging device that involves human "readers" or AI assistance to humans. Therefore, an MRMC comparative effectiveness study involving human readers and AI assistance is not applicable and was not performed.

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

The entire study focuses on the standalone performance of the device (RT-PCR kit with its associated instruments). The device is an automated molecular diagnostic assay, not an AI algorithm. Its performance is evaluated objectively based on its ability to detect and characterize viral RNA. While human analysts perform the procedure and interpret the output, the core 'performance' refers to the analytical and clinical accuracy of the molecular reactions and instrument readings.

7. The Type of Ground Truth Used:

• Analytical Studies (LOD, Reproducibility, Carryover): The ground truth was based on known concentrations of characterized viral stocks (EID50/mL titer) and the specific composition of contrived samples (known to be positive or negative for certain influenza strains). This is a form of laboratory-established ground truth.
• Clinical Performance Evaluation: The ground truth for the clinical specimens was derived from results previously obtained using the FDA-cleared Applied Biosystems™ 7500 Fast Dx. This acts as a reference standard from a legally marketed and established device. For the A(H5) samples, it was based on the known composition of the contrived samples.

8. The Sample Size for the Training Set:

The document describes an evaluation study for substantial equivalence of a molecular diagnostic kit with new instrument platforms. It does not mention a "training set" in the context of machine learning. The "training" for such a molecular kit would involve the extensive R&D and optimization of the primer/probe sets and PCR conditions during the device's development, not a data-based training set for an AI algorithm.

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

As mentioned above, there is no explicit "training set" in the machine learning sense described in this document. The ground truth for the development and optimization of such a diagnostic panel typically involves:

• Known viral isolates/strains: Using cultured viruses with confirmed identity and titer.
• Sequencing data: To design highly specific and sensitive primers/probes targeting conserved regions of the viral genome.
• Clinical validation: Testing against a large panel of clinical samples, where the true infection status (ground truth) is established through gold-standard methods (e.g., viral culture, sequencing, or other highly sensitive and specific molecular tests) performed by expert laboratories.

The document briefly states: "Oligonucleotide primers and probes for detection of influenza A, influenza B, and 2009 influenza A (swine origin) were selected from highly conserved regions of the matrix (M), non-structural (NS), and nucleoprotein (NP) genes, respectively. Oligonucleotide primers and probes for characterization and differentiation of influenza A(H3) and A(H1)pdm(9 viruses and genetic lineages of influenza B were selected from highly conserved regions of their HA genes." This suggests an established ground truth based on genomic sequencing and bioinformatic analysis to design the assay.

Ask a specific question about this device

The Influenza A/B Typing 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 qualitative detection of influenza virus type A or B viral RNA in upper respiratory tract clinical specimens (including nasopharyngeal swabs [NPS], nasal 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 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 nasopharyngeal swabs [NPS], nasal 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 B Lineage Genotyping 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 the determination of the genetic lineage of human influenza B viruses as B/ Victoria or B/Yamagata lineage from viral RNA in upper respiratory tract clinical specimens (including nasopharyngeal swabs [NPS], nasal swabs [NS], throat swabs [TS], nasal aspirates [NA], nasal washes [NW] and dual nasopharyngeal/ throat swabs [NPS/TS]) 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/H5 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 (tRT-PCR) assays on an Applied Biosystems (ABI) 7500 Fast Dx Real-Time PCR instrument in conjunction with clinical and epidemiological information:

• For the presumptive identification of virus in patients who may be infected with influenza A subtype A(H5) (Asian lineage) from viral RNA in human respiratory specimens and viral culture in conjunction with clinical and epidemiological risk factors;
• To provide epidemiologic information for surveillance of circulating influenza viruses.

The CDC Human Influenza Real-Time RT-PCR Diagnostic Panel is used in real-time RT-PCR (rRT-PCR) assays on the Applied Biosystems® (ABI) 7500 Fast Dx Real-time PCR system. The panel is configured in four separate kits. Each 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). Oligonucleotide primers and probes for detection of influenza A, influenza B, and 2009 influenza A (swine origin) were selected from highly conserved regions of the matrix (M), non-structural (NS), and nucleoprotein (NP) genes, respectively. Oligonucleotide primers and probes for characterization of influenza A(H3) and A(H1)pdm09 viruses and genetic lineages of influenza B were selected from highly conserved regions of their HA genes. Oligonucleotide primers and probes to detect the human RNase P gene (RP) in control samples and clinical specimens is also included in the panel.

This document describes the performance evaluation of a modified CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel, specifically focusing on the addition of new nucleic acid isolation options. The study aims to demonstrate that the modified device is substantially equivalent to its predicate.

Here's an analysis based on your request:

Acceptance Criteria and Reported Device Performance

The acceptance criteria are implicitly defined by the demonstration of equivalence to a cleared predicate device. This equivalence is shown through specific performance metrics: Limit of Detection (LOD) and Analytical Precision (Reproducibility), and Clinical Performance.

Table of Acceptance Criteria and Reported Device Performance:

Additional Information:

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

   • LOD Equivalency: For each investigational instrument/kit combination, a single characterized influenza A(H3N2) virus (A/Hong Kong/4801/2014) was serially diluted. Triplicate samples of each dilution were tested for both the cleared and investigational methods. The exact number of dilutions is not explicitly stated as a fixed sample size, but the tables show 5 dilution points for each test. This is an analytical study, not directly using human patient samples.
   • Reproducibility: A blinded panel (contrived samples) containing a moderate positive, a low positive (near LOD), and a negative sample was used. For each of the three instrument/method combinations, the sample panel was tested 5 times by two different analysts at three separate sites over 5 different days. This means 5 samples (3 test specimens, plus internal controls likely) x 5 days x 2 analysts x 3 sites = 150 test events for each instrument/method. The specific agreement percentages shown in the tables are for 30 samples (presumably 10 runs per site over 5 days by 2 analysts, combined for totals of 30 for each category, e.g., A(H3) Moderate). Data provenance is not explicitly stated as country of origin, but it is an internal study of the CDC's diagnostic panel. The samples for reproducibility were contrived.
   • Clinical Performance: A total of 60 retrospective clinical specimens were used for each investigational instrument/kit combination: 30 positive for influenza A(H3) and 30 negative. The specimens were collected during the 2011-2012 and 2013-2014 influenza seasons. Data provenance is internal ("study was performed internally") using retrospective specimens, likely from the U.S. (given CDC's location).

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

   • Analytical studies (LOD and Reproducibility): Ground truth was established by precise laboratory preparation of known viral concentrations and by a reference method (the cleared predicate method and the CDC's established influenza real-time RT-PCR diagnostic panel). Human experts were involved in performing the tests and interpreting results (e.g., "All users, analysts, 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"). However, the ground truth itself is based on the inherent properties of the prepared samples and the established performance of the predicate device, not on expert consensus of clinical cases.
   • Clinical Performance: The ground truth for the retrospective clinical specimens was their prior determination as positive or negative for influenza A(H3) virus. The document does not specify how this prior determination was made (e.g., by culture, another PCR, or an expert committee). It implies that the "comparator" (Roche MagNA Pure Compact instrument using the RNA Isolation Kit) served as the reference standard for this equivalency study.

3. Adjudication method (e.g. 2+1, 3+1, none) for the test set:
   Not applicable (N/A). This study evaluates a laboratory diagnostic test (RT-PCR) with objective results (positive/negative based on PCR signal) and quantitative metrics (Ct values, EID50/mL). It does not involve human interpretation of images or clinical assessments requiring adjudication.

4. If a multi-reader multi-case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance:
   N/A. This is a study of an in vitro diagnostic (IVD) device (RT-PCR), not an AI-based imaging or interpretation tool. It does not involve human readers interpreting cases or AI assistance.

5. If a standalone (i.e. algorithm only without human-in-the-loop performance) was done:
   This is an IVD device. Its performance is inherent in the device itself (reagents, instrumentation, assay design). The tests performed (LOD, reproducibility, clinical equivalency) are standalone evaluations of the analytical and clinical performance of the new nucleic acid extraction methods in conjunction with the existing PCR panel. While human analysts operate the device, the "performance" refers to the device's output, not human interpretation.

6. The type of ground truth used (expert consensus, pathology, outcomes data, etc.):

   • Analytical Studies (LOD & Reproducibility): "Spiked" or "contrived" samples with known concentrations of influenza virus.
   • Clinical Performance: Previously determined positivity/negativity of retrospective clinical specimens, with the results from the predicate device serving as the comparator/reference.

7. The sample size for the training set:
   N/A. This is a 510(k) submission for a device modification, focusing on testing the performance of added components (nucleic acid extraction systems) within an already established diagnostic panel. There is no mention of a "training set" in the context of machine learning or algorithm development. The "training" described in the document refers to human analysts being trained to use the device.

8. How the ground truth for the training set was established:
   N/A, as there is no machine learning training set in this context.

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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.

• BW: 1/1 (100.0% PPA, 95% CI: 20.7-100.0)
• NPS, NS: 34/35 (97.1% PPA, 95% CI: 85.5-99.5)
• NW: 4/4 (100.0% PPA, 95% CI: 51.0-100.0)
• TS: 2/2 (100.0% PPA, 95% CI: 34.2-100.0)
  Quanta qScript™:
• BW: 1/1 (100.0% PPA, 95% CI: 20.7-100.0)
• NPS, NS: 33/33 (100.0% PPA, 95% CI: 89.6-100.0)
• NW: 4/4 (100.0% PPA, 95% CI: 51.0-100.0)
• TS: 2/2 (100.0% PPA, 95% CI: 34.2-100.0) |
  | Clinical Performance - A(H3) Positive Agreement | High positive agreement with previously positive clinical samples (e.g., >95% PPA with tight CI). | Invitrogen SuperScript™:
• NA: 1/1 (100.0% PPA, 95% CI: 20.7-100.0)
• NPS, NS: 30/30 (100.0% PPA, 95% CI: 88.7-100.0)
• NW: 1/1 (100.0% PPA, 95% CI: 20.7-100.0)
  Quanta qScript™:
• NA: 1/1 (100.0% PPA, 95% CI: 20.7-100.0)
• NPS, NS: 30/30 (100.0% PPA, 95% CI: 88.7-100.0)
• NW: 1/1 (100.0% PPA, 95% CI: 20.7-100.0) |
  | Clinical Performance - A(H1)pdm09 Negative Agreement | High negative agreement with previously negative clinical samples (e.g., >95% NPA with tight CI). | Invitrogen SuperScript™:
• NPS: 53/53 (100.00% NPA, 95% CI: 93.2-100.0)
  Quanta qScript™:
• NPS: 52/52 (100.00% NPA, 95% CI: 93.1-100.0) |
  | Clinical Performance - A(H3) Negative Agreement | High negative agreement with previously negative clinical samples (e.g., >95% NPA with tight CI). | Invitrogen SuperScript™:
• NPS: 29/29 (100.00% NPA, 95% CI: 88.3-100.0)
  Quanta qScript™:
• NPS: 28/28 (100.00% NPA, 95% CI: 87.9-100.0) |

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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