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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], 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.

Performance characteristics for influenza were established during a season when seasonal influenza viruses A(H1N1) and A(H3N2) were the predominant influenza A viruses in circulation and during a season when the A(H1N1)pdm09 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 or co-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.

CDC Human Influenza Real-Time RT-PCR Diagnostic Panel: Influenza A Subtyping Kit (VER 4)

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.

Performance characteristics for influenza were established during a season when seasonal influenza viruses A(H1N1) and A(H3N2) were the predominant influenza A viruses in circulation and during a season when the A(H1N1)pdm09 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 or co-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.

CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel: Influenza B Lineage Genotyping Kit (VER 1.1 and 2)

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 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 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.

Performance characteristics for influenza B lineage genotyping were established during a season when influenza B/Victoria and B/Yamagata lineages were in circulation.

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 or co-infection with other viruses. The agent detected may not be the definite cause of disease.

CDC Human Influenza Virus 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 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 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, conjunctival swabs, and viral culture in conjunction with clinical and epidemiological risk factors;

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

Performance characteristics for influenza were established during a season when seasonal influenza viruses A(H1N1) and A(H3N2) were the predominant influenza A viruses in circulation and during a season when the A(H1N1)pdm09 influenza virus was the predominant influenza A virus in circulation. Performance characteristics may vary with other emerging influenza A viruses.

Testing with the influenza H5a and H5b primer and probe sets should not be performed unless the patient meets the most current U.S.Department of Health and Human Services (DHHS) clinical and epidemiologic criteria for testing suspect A(H5) specimens. The definitive identification of influenza A(H5) (Asian lineage) either directly from patient specimens or from virus cultures requires additional laboratory testing, along with clinical and epidemiological assessment in consultation with national influenza surveillance experts.

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 or co-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.

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 instrument that has been FDA-cleared for use with the CDC device. 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) or from viral culture.

Oligonucleotide primers and probes for detection of influenza A, influenza B, and influenza A of 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)pdm09 viruses, avian influenza A(H5) viruses, and genetic lineages of influenza B were selected from highly conserved regions of their hemagglutinin (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.

The provided document, K243274: CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel, is an FDA 510(k) clearance letter. This document primarily describes the device, its intended use, and confirms its substantial equivalence to a previously cleared predicate device (K243931). Critically, it states that no analytical or clinical testing was performed for this specific modification, as the submission was to add a Predetermined Change Control Plan (PCCP).

Therefore, the document does not contain the detailed study results, acceptance criteria, sample sizes, ground truth establishment, or expert involvement for the original validation of the device's performance. It explicitly states that "The performance characteristics of the CDC Flu rRT-PCR Dx Panel... were previously established and remain the same as the predicate device (K243931)."

As such, I cannot provide the specific information requested in the prompt based on the provided text alone. The prompt asks for details of "the device" meeting acceptance criteria, and this document pertains to a modification that did not involve re-evaluating performance.

However, I can interpret what would typically be sought for such an analysis in the context of an in vitro diagnostic (IVD) PCR panel like the one described. I will outline what the acceptance criteria and the study that proves the device meets them would likely entail for an IVD, but with the explicit understanding that the provided document does not contain these details.

Based on the provided document, the device's performance characteristics were "previously established" for the predicate device (K243931) and are simply carried over here as "remaining the same." The current submission (K243274) is for adding a Predetermined Change Control Plan (PCCP) and states that "No analytical testing was performed for this modification" and "No clinical testing was performed for this modification."

Therefore, the detailed information regarding acceptance criteria, reported performance, sample sizes, expert involvement, and ground truth establishment for the original validation of this device is NOT present in the provided text. The tables and descriptions below represent what would typically be expected for an FDA cleared RT-PCR diagnostic panel, assuming the original studies were conducted to industry standards, but are not extracted directly from the given document.

Acceptance Criteria and Device Performance (Hypothetical for an RT-PCR IVD)

For a real-time RT-PCR diagnostic panel like the CDC Human Influenza Virus Panel, acceptance criteria and performance would typically focus on analytical sensitivity (Limit of Detection - LoD), analytical specificity (cross-reactivity, inclusivity), and clinical performance (sensitivity, specificity).

1. Table of Acceptance Criteria and Reported Device Performance (Hypothetical)

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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 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 evidemiological 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 epidemiological information for surveillance of circulating influenza viruses.

CDC Human Influenza Real-Time RT-PCR Diagnostic Panel: Influenza A Subtyping Kit (VER 4)

The Influenza A 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 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 epidemiological information for surveillance of circulating influenza viruses.

CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel: Influenza B Lineage Genotyping Kit (VER 1.1 and 2)

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 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.

CDC Human Influenza Virus 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, conjunctival swabs, 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 consists of four real-time RT-PCR (rRT-PCR) assays used on IVD-labeled real-time PCR instruments that has been FDA-cleared for use with this device. The panel is configured in four separate kits: Influenza A/B Typing kit, Influenza A/H5 Subtyping kit, and Influenza B Genotyping kit. 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 of influenza virus RNA in respiratory and conjunctival specimens from patients presenting with influenza-like illness (IL). 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)pdm09 viruses, genetic lineages of influenza A(H5) viruses 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 are also included in the panel.

The provided text describes the acceptance criteria and study proving the device meets those criteria for the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel: Influenza A/H5 Subtyping Kit (VER 4), specifically concerning the addition of conjunctival swabs as a specimen type.

Here’s a breakdown of the requested information based on the provided text:

1. Table of acceptance criteria and the reported device performance:

The document does not explicitly present a table of acceptance criteria in terms of specific quantitative thresholds (e.g., minimum sensitivity/specificity percentages) for clinical performance. Instead, it describes an evaluation based on demonstrating successful detection capabilities and similarity to a predicate device. The performance is reported descriptively based on the clinical study results.

Reported Device Performance (for Influenza A/H5 Subtyping Kit (VER 4) on conjunctival swabs):

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

• Clinical Test Set Sample Size:
  • 46 total cases (44 confirmed, 2 probable) initially considered.
  • 35 confirmed cases were assessed for performance due to exclusion of 9 cases where not all necessary assay reagents were used.
  • 2 probable cases were assessed.
  • The document also specifies the number of clinical specimens available for testing: e.g., 32 confirmed cases had subject-matched paired URT and CS specimens, 3 confirmed cases had only CS specimens, 1 probable case had subject-matched paired URT and CS specimens, and 1 probable case had only a CS specimen.
• Data Provenance:
  • Country of Origin: US (from public health investigations in the US).
  • Retrospective or Prospective: The data was collected from specimens from March to November 2024 as part of public health investigations into outbreaks. This indicates a retrospective collection of existing specimens from human cases.

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

• The ground truth for confirmed/probable cases of A/H5N1 was established based on "the criteria described in the current Council of State and Territory Epidemiologists (CSTE) document 24-ID-09." The document does not specify the number of individual experts or their qualifications directly. This refers to established public health guidelines rather than individual expert consensus.

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

• The ground truth was established by adherence to existing CSTE criteria, not through a human reader adjudication process of the device's output. The device results were compared against these established "confirmed or probable case status."

5. If a multi reader multi case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance:

• No, an MRMC comparative effectiveness study was not done. This document describes the validation of a laboratory diagnostic kit (RT-PCR) with specific specimen types and does not involve human readers interpreting images or AI assistance.

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

• Yes, this is effectively a standalone performance study. The device itself is an RT-PCR diagnostic panel. The "performance" described is that of the kit (reagents and controls run on a PCR instrument) in detecting viral RNA, without human interpretation of raw data beyond reading the instrument's output based on established cut-offs (e.g., Ct values). It's an "algorithm only" in the sense that the test results in a definitive positive, negative, or inconclusive outcome based on chemical reactions and signal detection.

7. The type of ground truth used:

• The ground truth for the clinical performance evaluation was based on "confirmed or probable case status for each case on the criteria described in the current Council of State and Territory Epidemiologists (CSTE) document 24-ID-09." This can be categorized as outcomes data/epidemiological criteria as determined by public health authorities.

8. The sample size for the training set:

• The document describes the validation of a diagnostic panel (PCR-based), not an AI algorithm. Therefore, there is no specific "training set" in the machine learning sense. The device's components (primers, probes) and their performance characteristics are established through analytical studies (e.g., LoD, interfering substances) and clinical validation with patient samples. The development of such a kit is based on extensive prior knowledge of influenza virus genetics and PCR technology, but not a "training set" in the context of supervised learning for AI.

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

• As there is no "training set" in the AI sense, this question is not applicable to the device described. The "ground truth" for the analytical development of the RT-PCR panel would be based on well-characterized viral samples (e.g., BPL inactivated virus with known concentrations) and established laboratory techniques for nucleic acid detection.

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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.

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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 [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.

Performance characteristics for influenza B lineage genotyping were established during a season when influenza B/ Victoria and B/Yamagata lineages were in circulation.

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.

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 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 included in the Influenza B Lineage Genotyping Kit for detection of influenza B and the two major genetic lineages of influenza B were selected from highly conserved regions of the non-structural (NS) and HA genes, respectively. Oligonucleotide primers and probes to detect the human RNase P gene (RP) in control samples and clinical specimens are also included in the kit.

Here's a summary of the acceptance criteria and study details for the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel: Influenza B Lineage Genotyping Kit, based on the provided document:

This document describes a 510(k) premarket notification for modifications to an existing influenza B lineage genotyping kit (K172091). The changes primarily address recent evolutionary changes in circulating influenza B viruses that could impact the reactivity of the current kit, and the evaluation of an alternative fluorescent hydrolysis probe quencher chemistry (ZEN™). The study aims to demonstrate that the modified device remains substantially equivalent to the predicate device.

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are generally demonstrated through 100% agreement or high percentage sensitivity/specificity with a clear lower bound for the 95% Confidence Interval (CI).

Influenza B Lineage Genotyping Assay (VER 1.1 and VER 2 VIC & YAM assays): All assays demonstrated similar reactivity with apparent LOD endpoints within one 5-fold dilution of each other for various B/Victoria and B/Yamagata strains across both enzyme systems (Invitrogen Superscript™ and Quanta qScript™). For example, LOD for B/Maryland/15/2016 (VER 1.1 & VER 2) was 10^1.7 EID50/mL for both enzymes. |
| Analytical Sensitivity (LOD Confirmation) | Lowest virus concentration where InfB and VIC or InfB and YAM primer and probe sets demonstrated ≥ 95% detection. | Demonstrated LODs for B/Maryland/15/2016 (Victoria) were 10^1.7 EID50/mL for both VER 1.1 and VER 2 with both enzyme systems. Demonstrated LODs for B/Texas/81/2016 (Yamagata) were 10^2.2 EID50/mL (VER 1.1 & VER 2 with Invitrogen) and 10^1.5 EID50/mL (VER 1.1 & VER 2 with Quanta). |
| Analytical Sensitivity (Inclusivity) | 100% positivity (3 out of 3 replicates) for the tested strains at or near the established LOD. | B/Victoria (VER 1.1 & VER 2): All 10 tested B/Victoria strains showed 3/3 positive replicates (100% inclusivity) at or near their LOD for both VER 1.1 and VER 2, across both enzyme systems.
B/Yamagata (VER 1.1 & VER 2): All 10 tested B/Yamagata strains showed 3/3 positive replicates (100% inclusivity) at or near their LOD for both VER 1.1 and VER 2, across both enzyme systems. |
| Analytical Specificity (Cross-Reactivity) | No cross-reactivity detected with the opposite influenza B lineage. | No cross-reactivity was detected with either VER 1.1 or VER 2 VIC and YAM primer/probe sets when tested against 10 high-titer B/Yamagata lineage viruses (for VIC assays) and 10 high-titer B/Victoria lineage viruses (for YAM assays), across both enzyme systems. (Results consistently showed '-') |
| Analytical Specificity (Exclusivity with Influenza A Viruses) | No cross-reactivity detected with various influenza A viruses. | No cross-reactivity was detected with either VER 1.1 or VER 2 VIC and YAM assays when tested against 8 high-titer Influenza A viruses of various human and animal subtypes, across both enzyme systems. (Results consistently showed '-') |
| Analytical Specificity (Exclusivity with Non-Influenza Respiratory Pathogens) | No cross-reactivity detected with common non-influenza respiratory pathogens or flora. | No cross-reactivity was detected with VER 2 VIC and YAM assays when tested against 36 organisms (16 viruses, 19 bacteria, and 1 yeast) at high titers, across both enzyme systems. (Results consistently showed '-') |
| Clinical Performance (Retrospective InfB Assay) | 100% positive and negative agreement with the cleared oligonucleotide probe (BHQ quencher). | 100.0% Positive Agreement (95% CI: 88.7-100.0) for 30/30 positive samples. 100.0% Negative Agreement (95% CI: 92.9-100.0) for 50/50 negative samples. (Both enzyme systems showed identical results). |
| Clinical Performance (VER 2 B/Victoria Prospective Study) | High sensitivity and specificity, typically aiming for >90% sensitivity and >95% specificity with a tight 95% CI. | Sensitivity: 100.0% (5/5 positives) (95% CI: 56.6 – 100.0).
Specificity: 100.0% (574/574 negatives) (95% CI: 99.3 – 100.0). (Note: Low number of positives impacts CI width for sensitivity). |
| Clinical Performance (VER 2 B/Yamagata Prospective Study) | High sensitivity and specificity, typically aiming for >90% sensitivity and >95% specificity with a tight 95% CI. | Sensitivity: 100.0% (38/38 positives) (95% CI: 90.8 – 100.0).
Specificity: 99.8% (540/541 negatives) (95% CI: 99.0 – 100.0). |
| Clinical Performance (Retrospective VER 1.1 & VER 2 VIC & YAM assays) | 100% positive and negative agreement with the FDA-cleared Influenza B Lineage Genotyping Kit. | VIC VER 1.1: 100.0% Positive Agreement (30/30) (95% CI: 88.7 - 100.0); 100.0% Negative Agreement (157/157) (95% CI: 97.6 - 100.0).
YAM VER 1.1: 100.0% Positive Agreement (96/96) (95% CI: 96.2 - 100.0); 100.0% Negative Agreement (91/91) (95% CI: 96.0 - 100.0).
VIC VER 2: 100.0% Positive Agreement (30/30) (95% CI: 88.7 - 100.0); 100.0% Negative Agreement (157/157) (95% CI: 97.6 - 100.0).
YAM VER 2: 100.0% Positive Agreement (96/96) (95% CI: 96.2 - 100.0); 100.0% Negative Agreement (91/91) (95% CI: 96.0 - 100.0). |

2. Sample Size for the Test Set and Data Provenance

Test Set Sample Sizes:

• Analytical Sensitivity (LOD Equivalency & Inclusivity): Varied. Typically, 3 replicates per dilution/strain. For LOD confirmation, 20 extraction replicates per dilution.
• Analytical Specificity (Cross-Reactivity & Exclusivity): For cross-reactivity, 10 B/Yamagata strains for VIC assays and 10 B/Victoria strains for YAM assays. For exclusivity with Influenza A, 8 Influenza A strains. For exclusivity with non-influenza pathogens, 36 organisms (16 viruses, 19 bacteria, 1 yeast). All tested with both enzyme systems.
• Clinical Performance (InfB Assay Retrospective): 30 positive and 50 negative upper respiratory tract clinical samples.
• Clinical Performance (VER 2 Prospective Study):
  • Total collected: 592 samples.
  • Analyzed: 579 upper respiratory tract clinical specimens.
  • B/Victoria: 5 positive, 574 negative.
  • B/Yamagata: 38 positive, 541 negative.
• Clinical Performance (VER 1.1 & VER 2 Retrospective Study):
  • 126 specimens positive for either influenza B/Victoria or B/Yamagata viruses.
  • 61 specimens negative for influenza B viruses.
  • (An additional 4 lung tissue specimens were tested for informational purposes but not included in performance calculations).

Data Provenance:

• Analytical studies: Laboratory-characterized virus stocks and reference strains (e.g., CDC repositories, ATCC).
• Clinical Performance (InfB Assay Retrospective): Retrospective clinical samples collected during the 2011-2012 influenza season.
• Clinical Performance (VER 2 Prospective Study): Prospective clinical investigation conducted at 3 U.S. public health laboratories using upper respiratory tract specimens collected during the 2016-2017 influenza season.
• Clinical Performance (VER 1.1 & VER 2 Retrospective Study): Retrospective upper respiratory tract clinical samples collected during the 2017 and 2017-2018 influenza seasons.

Country of Origin: Primarily the United States (CDC, U.S. public health laboratories) with some virus strains noted as originating from various international locations (e.g., Hong Kong, Bolivia, Laos, Michigan, etc.) for inclusivity and exclusivity testing, reflecting global diversity.

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

The ground truth for the test sets (both analytical and clinical) appears to be established by molecular diagnostic methods using an FDA-cleared predicate device, the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel: Influenza B Lineage Genotyping Kit (K172091), and/or by using well-characterized virus stocks with known titers (EID50/mL or TCID50/mL).

For clinical samples, they were "previously determined to be positive or negative for influenza B virus" or "determined to be positive for influenza B/Victoria or B/Yamagata viruses using the FDA-cleared Influenza B Lineage Genotyping Kit."

The document does not explicitly mention the use of a specific "number of experts" (e.g., clinicians, virologists, or laboratory professionals) for establishing the individual patient-level clinical ground truth for the test sets in the same way one might describe image interpretation by radiologists. Instead, the ground truth is primarily based on the results of the already FDA-cleared predicate diagnostic test or characterized reference materials. The "competent instructor" trained individuals (users, analysts, and those reporting results) on how to perform and interpret the device results.

4. Adjudication Method for the Test Set

The document does not describe an adjudication method for conflicting results in the test set, such as a 2+1 or 3+1 system. This is typical for molecular diagnostic studies where the comparison is generally made against a well-established reference method (the predicate device or characterized stocks). Any discrepancies would likely be investigated to determine the cause (e.g., retesting, review of sample integrity, or confirmation by alternative gold standard) rather than being resolved by expert consensus in a traditional adjudication panel format. For example, in the LOD studies, "3 out of 3 replicates" or "≥ 95% detection" criteria are used, indicating a reliance on consistent assay performance rather than expert adjudication.

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

No, a multi-reader multi-case (MRMC) comparative effectiveness study was not explicitly described. MRMC studies are typically performed for imaging devices or other diagnostics where human interpretation is a key component and the effect of AI assistance on human performance is being evaluated. This submission is for a molecular diagnostic (RT-PCR kit) where the output is a quantitative measurement (Ct values) and qualitative result (positive/negative, lineage determination) interpreted directly by the instrument software or a trained individual following predefined rules, not a subjective interpretation by multiple human readers.

Therefore, there is no mention of an effect size of how much human readers improve with AI vs. without AI assistance.

6. Standalone (Algorithm Only) Performance

Yes, the studies presented primarily represent standalone performance of the device (or its modified components). The analytical performance evaluations (sensitivity, inclusivity, specificity, exclusivity) directly assess the algorithm's (reagent kit's) ability to detect and differentiate influenza B lineages from viral RNA. The clinical performance studies compare the results of the investigational device directly against the FDA-cleared predicate device on clinical samples. While human technicians perform the laboratory procedures, the "algorithm" here refers to the specific RT-PCR assay chemistry, probes, and primer sets that yield a result, which is then interpreted according to established cut-offs/rules, not an AI algorithm that assists human interpretation.

7. Type of Ground Truth Used

The ground truth used for the studies is a combination of:

• Reference standard/Predicate device: For clinical samples, the ground truth was largely established by the "FDA-cleared CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel: Influenza B Lineage Genotyping Kit."
• Characterized virus stocks: For analytical studies (LOD, inclusivity, cross-reactivity, exclusivity), the ground truth was established using well-characterized influenza virus stocks and other respiratory pathogens with known strain designations, subtypes, and titers (EID50/mL, TCID50/mL, CFU/mL, or ng/µL). This implies a high level of confidence in the identity and concentration of the biological material.

8. Sample Size for the Training Set

The document does not explicitly state a sample size for a "training set" in the context of an algorithm's development. This is because the device described is a molecular diagnostic kit (RT-PCR) with specific primers and probes designed based on biological knowledge of viral sequences, rather than a machine learning or artificial intelligence algorithm that requires a distinct training phase with labeled data.

The "modifications" were "primarily to address recent evolutionary changes in circulating influenza B viruses that may impact the reactivity of the current Influenza B Lineage Genotyping Kit. Two design approaches were evaluated that address specific genetic mutations in the targeted hemagglutinin (HA) gene..." This indicates a design process driven by biological knowledge and engineering based on viral genetics, not data-driven machine learning training.

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

Since there's no explicitly defined "training set" for an AI algorithm in the provided text, the concept of establishing ground truth for it doesn't directly apply. The design of the new VER 1.1 and VER 2 assays was based on scientific understanding of circulating influenza B virus evolutionary changes and specific genetic mutations in the HA gene. This informed the selection of the oligonucleotide primers and probes. The efficacy of these new designs was then evaluated using the analytical and clinical studies described in the rest of the document, where the ground truth was established as detailed in point 7.

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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 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.

Ask a specific question about this device

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.

Ask a specific question about this device

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 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/HS (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.

Performance characteristics for influenza were established during a seasonal influenza viruses A/H1 and A/ H3 were the predominant influenza A viruses in circulation and during a season when the A/H1pdm09 influenza virus was the predominant influenza A virus in circulation. Performance characteristics may vary with other emerging influenza A viruses.

Testing with the influenza H5a and H5b primer and probe sets should not be performed unless the most current U.S. Department of Health and Human Services (DHHS) clinical and epidemiologic criteria for testing suspect A/ H5 specimens. The definitive identification of influenza A/H5 (Asian lineage) either directly from patient or from virus cultures requires additional laboratory testing, along with clinical and epidemiological assessment in consultation with national influenza surveillance experts.

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 a state or local health department for testing. Viral culture should not be attempted unless a BSL 3+ 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/H5 Subtyping Kit is a real-time RT-PCR) assay that utilizes the Applied Biosystems® (ABI) 7500 Fast Dx Realtime PCR system. The Influenza A/H5 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 viruses (InfA) were selected from highly conserved regions of the matrix (M) protein. Oligonucleotide primers and probes for characterization of avian influenza A/H5 (Asian lineage) viruses (H5a and H5b) were selected from highly conserved regions of their HA genes. The Influenza A/H5 Subtyping kit also contains primers and probes to detect the human RNase P gene (RP) in control samples and clinical specimens.

Here's an analysis of the provided text regarding the acceptance criteria and the study that proves the device meets the acceptance criteria:

Device Name: CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel, Influenza A/H5 Subtyping Kit (VER 3)
K Number: K153148

1. Table of Acceptance Criteria and Reported Device Performance

The document describes the analytical and clinical performance studies, but directly stated "acceptance criteria" for each metric are not explicitly listed in a formatted table with pass/fail thresholds. Instead, the results are presented as the device's performance, implying these values met internal criteria for submission and approval. Inferring from the nature of diagnostic device validation, these would typically be high percentages for sensitivity and specificity.

Based on the provided data, here's a table summarizing the observed performance:

• Invitrogen SuperScript™: 10^3.8 EID50/mL
• Quanta qScript™: 10^2.4 EID50/mL
• A/duck/Vietnam/NCVD-1544/2012 (A/H5N1):
  • Invitrogen SuperScript™: 10^3.1 EID50/mL
  • Quanta qScript™: 10^3.1 EID50/mL
• A/gyrfalcon/Washington/41088-6/2014 (A/H5N8):
  • Invitrogen SuperScript™: 10^3.35 EID50/mL
  • Quanta qScript™: 10^3.35 EID50/mL
    Reported as the lowest concentration where InfA and both H5a/H5b showed uniform detection. |
    | Analytical Inclusivity | Detection of all listed A/H5 (Asian lineage) isolates representative of different geographic locations and phylogenetic clades, at or near the established LOD. | - Reactive with all 15 tested H5 (Asian lineage) isolates (all 3/3 positive for InfA, H5a, and H5b) with both enzyme systems.
• Predicted to be reactive with A/Bangladesh/3222/2011 (in silico analysis). |
  | Analytical Specificity (Cross-Reactivity) | No detection of common human influenza A (non-H5), human influenza B, swine influenza, avian influenza (non-H5), canine influenza, or equine influenza viruses when tested at high titers. | - InfA assay detected all tested human influenza A and various animal influenza A strains (as expected for an InfA primer/probe set).
• H5a and H5b assays showed no cross-reactivity with human influenza A (non-H5), human influenza B, most animal influenza A (non-H5), and specifically did not detect A/duck/Singapore-Q/F119-3/1997 (H5N3) when tested with Invitrogen SuperScript™. (Note: The H5a/H5b assays did detect A/duck/Singapore-Q/F119-3/1997 H5N3, which is an H5 subtype, albeit not specified as Asian lineage. This is an expected positive result for H5 detection.) |
  | Analytical Specificity (Exclusivity) | No detection (no false positives) of common non-influenza human respiratory viruses, respiratory bacteria, and commensal organisms of the human respiratory tract when tested at high concentrations. | - No detection (all negative) for all 18 bacteria, 1 yeast, and 16 non-influenza human respiratory viruses tested with Invitrogen SuperScript™ for InfA, H5a, and H5b assays. |
  | Clinical Performance (Positive Agreement) | High Positive Agreement (e.g., >90% with a reasonable lower bound for 95% CI). | - Quanta qScript™: 100% (95% CI: 92.9 – 100.00) (50/50)
• Invitrogen SuperScript™: 88.0% (95% CI: 76.2 - 94.4) (44/50) |
  | Clinical Performance (Negative Agreement) | High Negative Agreement (e.g., >90% with a reasonable lower bound for 95% CI). | - Quanta qScript™: 100% (95% CI: 94.4 – 100.00) (65/65)
• Invitrogen SuperScript™: 100% (95% CI: 94.4 - 100.00) (65/65) |

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

• Analytical Sensitivity (LOD):

  • Test Set Sample Size: 20 replicates for each enzyme kit (Invitrogen SuperScript™ and Quanta qScript™) for the highest virus dilution for each of the three A/H5 strains to confirm LOD. Initial testing used 3 replicates.
  • Data Provenance: Laboratory-generated (characterized viruses).

• Analytical Inclusivity:

  • Test Set Sample Size: 3 replicates per virus isolate for 15 A/H5 (Asian lineage) viruses for both enzyme systems. One virus was tested in silico. Total real wet-lab tests: 15 viruses * 3 replicates * 3 assays (InfA, H5a, H5b) * 2 enzyme systems = 270 individual reactions.
  • Data Provenance: Laboratory-generated (characterized viruses representing different geographic locations and phylogenetic clades).

• Analytical Specificity (Cross-Reactivity):

  • Test Set Sample Size: 3 replicates per virus for 14 human and animal influenza A and influenza B viruses tested. Total: 14 viruses * 3 replicates * 3 assays (InfA, H5a, H5b) = 126 individual reactions.
  • Data Provenance: Laboratory-generated (characterized viruses).

• Analytical Specificity (Exclusivity):

  • Test Set Sample Size: A single test for each of 35 non-influenza organisms (16 viruses, 18 bacteria, 1 yeast). Total: 35 organisms * 3 assays (InfA, H5a, H5b) = 105 individual reactions.
  • Data Provenance: Laboratory-generated (propagated, titered, and characterized organisms).

• Clinical Performance Evaluation:

  • Test Set Sample Size:
    • Positive Contrived Samples: 50 samples.
    • Negative Specimens: 65 specimens.
  • Data Provenance:
    • Positive Contrived Samples: Laboratory-generated (grown virus added to A549 cell suspension).
    • Negative Specimens: Obtained from a clinical study conducted during the 2011-2012 influenza season. This suggests the negative specimens were retrospective clinical samples (human respiratory specimens), originating likely from the US where CDC studies are typically conducted.

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

The document does not explicitly state the number or qualifications of experts used to establish the ground truth for the test set.

• For analytical studies (LOD, Inclusivity, Specificity), the ground truth is established by the known characteristics of the laboratory-controlled and characterized viral and bacterial stocks. This does not typically involve human expert consensus in the same way clinical diagnostics do.
• For the clinical performance evaluation:
  • The "positive contrived samples" have their ground truth by definition (known virus added).
  • The "negative specimens" were previously tested as negative for influenza A by the CDC Human Influenza rRT-PCR Diagnostic Panel. The ground truth for these would have been established by the predicate device's results, which were likely interpreted by trained laboratory personnel, rather than experts in a concensus panel for the purpose of this study.

4. Adjudication Method for the Test Set

The document does not describe an adjudication method for the test set.

• For analytical studies, the results are typically quantitative (e.g., Cq values) or qualitative (positive/negative call) based on predetermined assay thresholds. There's no indication of a need for expert adjudication of these raw results.
• For clinical performance, the comparison is made against the known status of contrived samples or the prior result from a predicate device. An adjudication process (like 2+1, 3+1, etc.) is typically used when the ground truth itself is uncertain or to resolve discrepancies between readers/tests, which is not stated as part of this study design.

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.

• MRMC studies are typically used to compare the performance of human readers (e.g., radiologists, pathologists) with and without AI assistance or against another diagnostic method, often looking at reader agreement and diagnostic accuracy.
• This submission describes a RT-PCR diagnostic panel, which is an in vitro diagnostic (IVD) device. The performance is determined by the assay's ability to detect viral RNA, not by human reader interpretation of complex images or clinical data in the same way an MRMC study would apply. Therefore, the concept of "how much human readers improve with AI vs without AI assistance" is not applicable here.

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

Yes, the studies described are essentially standalone performance evaluations of the device (assay) itself.

• The analytical studies (LOD, Inclusivity, Specificity, Exclusivity) inherently assess the algorithm/reagent's ability to detect targets or avoid off-target detection.
• The 'clinical performance evaluation' also tests the device's ability to correctly identify positive and negative contrived/clinical samples.
• While a human laboratory technologist performs the PCR steps and interprets the output (e.g., reads Cq values and applies the interpretation algorithm to call positive/negative), the study evaluates the device's performance characteristics given those outputs, not the technologist's diagnostic ability with or without the device. The data presented is the direct output of the device's reaction, not a human interpretation layer being separately assessed for its impact.

7. The Type of Ground Truth Used

• Analytical Studies (LOD, Inclusivity, Cross-Reactivity, Exclusivity):
  • Known Characteristics of Propagated and Characterized Viruses/Organisms: For these studies, the ground truth is established by the known identity, titer, and genetic makeup of the laboratory-prepared viral and bacterial stocks. For example, an A/H5N1 virus is known to be A/H5N1 because it was characterized as such.
• Clinical Performance Evaluation:
  • Contrived Samples: The ground truth for positive samples was established by the known addition of virus to a cell suspension.
  • Negative Specimens: The ground truth for negative specimens was established by their prior categorization as "negative for influenza A" using the CDC Human Influenza rRT-PCR Diagnostic Panel (likely the predicate device or a similar established method) in a previous clinical study. This is a form of comparator method ground truth.

8. The Sample Size for the Training Set

The document does not explicitly mention a training set or its sample size.

• For IVD assays like RT-PCR, the development process (which would involve "training" or optimization) typically focuses on primer/probe design, reaction conditions, and calibration using initial lab-generated samples. The 'training' in this context is implicit in the assay development and refinement, rather than a distinct, formal training set for a machine learning algorithm.
• The studies described are for validation (testing), not for development or training.

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

As no explicit training set is mentioned (see point 8), the document does not describe how ground truth for a training set was established.

• If an implicit 'training' phase (assay development) occurred, the ground truth would have been established through well-characterized laboratory reference materials and known viral isolates, similar to the ground truth for the analytical validation studies.

Ask a specific question about this device

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 (rRT-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 Influenza A/H5 Subtyping Kit contains components of the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel that is used in real-time RT-PCR (rRT-PCR) assays on the ABI 7500 Fast Dx Real-Time PCR Instrument. The Influenza A/H5 Subtyping Kit consists of oligonucleotide primers and dual-labeled hydrolysis (TaqMan®) probes to be used in rRT-PCR for the in vitro qualitative detection and characterization of human influenza viruses from viral RNA in respiratory specimens from patients presenting with influenza-like illness (ILI).

The Influenza A/H5 Subtyping Kit is based on technology which is used in many molecular diagnostic assays. rRT-PCR assays are one-tube assays that first reverse-transcribe specific regions of RNA into cDNA copies. The cDNA then serves as a template for a polymerase chain reaction that utilizes a thermocyclic heating and cooling of the reaction to logarithmically amplify a specific region of DNA. The probe anneals to a specific internal target sequence located between the target loci of the forward and reverse primers. During the extension phase of the PCR cycle, the 5' nuclease activity of Tag polymerase degrades any probe molecules hybridized to amplified target sequence, causing the reporter dye to separate from the quencher dye, and generating a fluorescent signal. With each cycle, additional reporter dye molecules are cleaved from their respective probes, increasing the fluorescence intensity. Fluorescence intensity is monitored at each PCR cycle. Amplification of targets is reflected by logarithmic increase in fluorescence over time in comparison to the background signal.

This document is a 510(k) premarket notification from the FDA, asserting substantial equivalence for the "CDC Human Influenza Virus Real-time RT-PCR Diagnostic Panel, Influenza A/H5 Subtyping Kit." The document does not contain acceptance criteria or a study proving device performance against such criteria. Instead, it compares the proposed device to a predicate device (K132508) and concludes that the changes are primarily for labeling and do not alter the device's design, technological attributes, or intended use.

Therefore, I cannot provide the requested information from this document. The document explicitly states:

• "The changes proposed to the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel subject to this special 510(k) are for labeling purposes only and will not alter the technological attributes of the device." (Page 6)
• "The changes proposed to the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel do not alter the device's design or technological attributes. In addition, the indications for use and intended use of the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel will remain the same." (Page 9)

This type of 510(k) submission (a Special 510(k)) is used for modifications to a manufacturer's own legally marketed device where the modification does not affect the device's fundamental scientific technology or its intended use. As such, it relies on the previous clearance of the predicate device (K132508) for performance data rather than presenting new performance studies against acceptance criteria.

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