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The CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel is intended for use in realtime 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 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:
• For determination of the subtype of seasonal human influenza A viruses as seasonal A/H1, . A/H3, and/or A/H1pdm09 from viral RNA in upper respiratory tract clinical specimens (including NPS, NS. TS. NA. NW and NPS/TS) and lower respiratory tract specimens (including BAL, BW, TA, sputum and lung tissue) from human patients with signs and symptoms of respiratory infection and/or from viral culture;
• 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 NPS, NS, TS, NA, NW, and NPS/TS) from human patients with signs and symptoms of respiratory infection and/or from viral culture;
• . 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 Virus Real-Time RT-PCR Diagnostic Panel is used in real-time RT-PCR assays (rRT-PCR) on the ABI 7500 Fast Dx Real-Time PCR Instrument. The device 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 CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel 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 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 5' exonuclease 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. The amplification of each target is reflected by a logarithmic increase in fluorescence in comparison to the background signal.

Acceptance Criteria and Device Performance Study for K132508

This submission, K132508, describes modifications to the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel to add the capability to differentiate the two major lineages of influenza B viruses (B/Victoria or B/Yamagata). The predicate device is K130551.

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are primarily demonstrated through analytical performance studies (Limit of Detection, Inclusivity, and Exclusivity) and clinical performance studies (Prospective and Retrospective). The specific quantitative acceptance criteria for percent agreement in clinical studies are implicitly represented by the reported 95% Confidence Intervals.

2. Sample Size and Data Provenance

Test Set for Clinical Performance:

• Prospective Study:
  • Sample Size: 1,002 respiratory specimens initially collected. After exclusions (unknown specimen type, lower respiratory specimen, inconclusive results, technician error), 992 specimens for Invitrogen SuperScript™ analysis and 861 for Quanta qScript™ analysis were used.
  • Data Provenance: Respiratory specimens from patients presenting with influenza-like illness (ILI) from 6 clinical sites in the United States during the 2011-2012 influenza season. This was a prospective collection.
• Retrospective Study:
  • Sample Size: 51 additional specimens initially identified as influenza B positive. The exact breakdown per enzyme kit and lineage is not explicitly stated in combined totals, but tables show 15 samples for VIC (Invitrogen), 32 samples for YAM (Invitrogen), 14 samples for VIC (Quanta), and 29 samples for YAM (Quanta) used in relevant tables.
  • Data Provenance: Routine influenza surveillance samples collected between September 2012 and January 2013 in the United States. These were retrospective samples.

3. Number of Experts and Qualifications for Ground Truth Establishment

The document does not specify the number of experts used to establish the ground truth for the clinical test set. However, the ground truth was established by:

• Bi-directional nucleic acid sequence analysis of a region of the influenza B hemagglutinin (HA) gene on each specimen containing influenza B viral RNA.
• Alignment of sequence data of each sample to B/Victoria and B/Yamagata reference virus sequences of the HA gene.
• Percent homology calculation: A homology exceeding 95% to one lineage reference sequence while showing less than 95% to the other lineage reference sequence confirmed the identification.
• The overall context of the submission indicates this process was conducted by the Centers for Disease Control and Prevention (CDC), implying highly qualified molecular biologists and virologists were involved in this reference sequencing and analysis.

4. Adjudication Method for the Test Set

The document describes the ground truth for the clinical test set being established directly by bi-directional nucleic acid sequence analysis and comparison to reference sequences.

• There is no explicit mention of an adjudication method (e.g., 2+1, 3+1) involving human experts for the interpretation of the sequencing results. The sequencing analysis itself serves as the objective "adjudication" against established phylogenetic criteria (95% homology).

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

There was no Multi-Reader Multi-Case (MRMC) comparative effectiveness study done comparing human readers with AI assistance versus without AI assistance. This device is an in-vitro diagnostic (IVD) assay (RT-PCR panel), not an imaging-based AI system that would typically involve human readers.

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

Yes, a standalone (algorithm only without human-in-the-loop performance) evaluation was performed. The device's performance, as reported in the analytical and clinical studies, represents the direct output of the RT-PCR assay interpreted according to its defined cutoff values, without any explicit human intervention to "interpret" or adjust the device's determination of influenza B lineage. The comparative method for the clinical study (sequencing) also represents a standalone, objective reference standard.

7. Type of Ground Truth Used

The ground truth used for both the prospective and retrospective clinical studies was nucleic acid sequencing (bi-directional sequence analysis of the influenza B hemagglutinin (HA) gene), which is a definitive molecular method for viral lineage determination. The results were confirmed by calculating percent homology to established B/Victoria and B/Yamagata reference virus sequences.

8. Sample Size for the Training Set

The document does not explicitly mention a "training set" in the context of machine learning or AI models. This device is a RT-PCR assay based on pre-designed oligonucleotide primers and probes. Therefore, the concept of a training set for an algorithm is not directly applicable.

Instead, the design of the primers and probes would have been informed by:

• Historical knowledge of influenza B virus genetics: "An alignment of the full length HA sequences from many viruses from both lineages was used to locate a variable region flanked by two conserved regions that could be used to discriminate viruses from the two different lineages." This implies a large dataset of influenza B sequences was used in the initial design and selection of targets for the primers and probes.

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

As noted above, a formal "training set" with ground truth in the AI/ML sense is not described. The "ground truth" equivalent for the design phase of this molecular assay would have been established through extensive phylogenetic analysis and sequence comparison of known influenza B virus strains to identify conserved and variable regions suitable for differential detection. This foundational work would precede the actual development and validation of the RT-PCR panel. The reference viruses B/Yamagata/16/88 and B/Victorial2/87 are explicitly mentioned as representing the two distinct lineages, indicating their role as foundational "ground truth" for lineage classification during assay design.

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The CDC Influenza 2009 A(H1N1)pdm Real-Time RT-PCR Panel (CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel) is intended for use in real-time RT-PCR assays on an Applied Biosystems (ABI) 7500 Fast Dx Real-Time PCR Instrument in conjunction with clinical and epidemiological information:

• For the qualitative detection of influenza virus type A viral RNA from nasopharyngeal swabs (NPS), nasal swabs (NS), nasal aspirates (NA), nasal washes (NW), dual nasopharyngeal / throat swabs (NPS/TS), broncheoalveolar lavage (BAL), tracheal aspirate (TA), and bronchial wash (BW), collected from the respiratory tract of human patients with signs and symptoms of respiratory infection and/or from viral culture.
• For differentiation of 2009 H1N1 influenza virus RNA from nasopharyngeal swabs (NPS), nasal swabs (NS), nasal aspirates (NA), nasal washes (NW) dual nasopharyngeal / throat swabs (NPS/TS), broncheoalveolar lavage (BAL), tracheal aspirate (TA), and bronchial wash (BW) collected from the respiratory tract of human patients with signs and symptoms of respiratory infection and/or from viral culture.
• To provide epidemiologic information for surveillance of the 2009 H1N1 influenza virus.

The CDC Influenza 2009 A(H1N1)pdm Real-time RT-PCR Panel (CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel) is a panel of oligonucleotide primers and dual-labeled hydrolysis (TaqMan®) probes which may be used in real-time RT-PCR (rRT-PCR) assays for the in vitro qualitative detection and characterization of human influenza viruses (RNA) in respiratory specimens from patients presenting with influenza-like illness (ILI). Detection of viral RNA not only aids in the diagnosis of illness caused by seasonal and novel influenza viruses in patients with ILI, but also provides epidemiologic information on influenza and aids in the presumptive laboratory identification of specific novel influenza A viruses. The influenza A primer and probe sets are designed for universal detection of type A influenza viruses, Influenza A subtyping primer and probe sets are designed to specifically detect 2009 A (H1N1) influenza virus. The CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel is an updated version of the assay distributed by CDC. during the 2009 A(H1N1) pandemic strain to qualified laboratories (CDC rRT-PCR Swine Flu Panel G090072) under Emergency Use Authorization (EUA) from the FDA in that it has different sequences of primers and probes that are more specific to the currently circulating 2009 A(H1N1) influenza virus. The CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel includes the following primer and probe sets and positive control: InfA detects all influenza A strains, but does not detect influenza B strains. pdm InfA is specific for 2009 influenza A. pdm H1 is specific for 2009 influenza A, subtype H1. RNase P (RP) detects human RNase P and is used with human clinical specimens to indicate that adequate isolation of nucleic acid resulted from the extraction of the clinical specimen. Inactivated Influenza Typing Panel Real-Time RT-PCR Positive Control is a positive control designed to react with all the primer and probe sets including RNase P.

Device Acceptance Criteria and Study Details: CDC Influenza 2009 A(H1N1)pdm Real-Time RT-PCR Panel

1. Table of Acceptance Criteria and Reported Device Performance

2. Sample Size and Data Provenance (Clinical Performance Test Set)

• Sample Size for Test Set: 1901 total patient specimens.
  • 1191 nasopharyngeal swabs (NPS) and nasal swabs (NS)
  • 50 throat swabs (TS)
  • 519 nasal washes (NW) and nasal aspirates (NA)
  • 99 dual nasopharyngeal / throat swabs (NPS/TS)
  • 42 lower respiratory specimens (broncheoalveolar lavage (BAL), tracheal aspirate (TA), and bronchial wash (BW))
• Data Provenance: Prospective study, supplemented with retrospective specimens. Data collected during the 2009-2010 respiratory virus season (February-April) from 8 U.S. public health laboratories and a Department of Defense (DoD) laboratory. Samples were from individuals symptomatic with influenza-like illness (ILI). Retrospective specimens were used to supplement due to low prevalence of influenza.

3. Number of Experts and Qualifications for Test Set Ground Truth

The document does not explicitly state the "number of experts" used to establish the ground truth in the same way one would delineate expert radiologists for image annotation.

Instead, the ground truth for the clinical performance study was established by standard laboratory methods:

• For InfA (Influenza A detection): Virus culture with Immunofluorescent Antibody (IFA) or Direct Fluorescent Antibody (DFA) was used for screening and identification of influenza type.
• For 2009 H1N1 (subtype detection): Bi-directional sequencing was used for confirmation of 2009 influenza A (H1N1) subtype. (Sequencing was performed only on specimens already identified as positive for influenza A by virus culture).

These methods represent established diagnostic procedures typically performed by trained laboratory personnel, rather than interpretation by "experts" in the context of a consensus reading.

4. Adjudication Method for the Test Set

The document does not describe an explicit "adjudication method" in the context of multiple human readers reconciling differences. The ground truth was established by laboratory methods as described above. If there were discrepancies between initial screening (IFA/DFA) and confirmatory sequencing, the sequencing result would have been the definitive ground truth for subtyping.

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

No, a multi-reader multi-case (MRMC) comparative effectiveness study was not performed. The study evaluates the standalone performance of the RT-PCR panel against laboratory reference methods, not improvement in human reader performance with AI assistance.

6. Standalone (Algorithm Only) Performance Study

Yes, a standalone performance study was done. The entire document focuses on evaluating the analytical and clinical performance of the CDC Influenza 2009 A(H1N1)pdm Real-Time RT-PCR Panel (an algorithm/assay) independent of human interpretation or assistance, against established laboratory reference methods. The results presented for sensitivity, specificity, and agreement are for the device's performance directly.

7. Type of Ground Truth Used

• Clinical Performance Ground Truth:
  • Virus Culture (with IFA/DFA): For the detection of influenza type A (against which the InfA analyte of the device was compared).
  • Bi-directional Sequencing: For the confirmation of 2009 influenza A (H1N1) subtype (against which the pdm InfA and pdm H1 analytes of the device were compared).
• Analytical Performance Ground Truth:
  • Known viral concentrations (EID50/mL, TCID50/mL): For Limit of Detection, Inclusivity, and Exclusivity studies.
  • Known identity of specific influenza and non-influenza organisms: For Inclusivity, Exclusivity, and Reactivity of Non-Influenza Respiratory Viral and Bacterial Pathogens studies.

8. Sample Size for the Training Set

The document does not provide information about a "training set" or its sample size. This type of device (RT-PCR panel) is a diagnostic kit where the primers and probes are designed based on known viral sequences, rather than an AI/ML algorithm that is "trained" on a dataset in the conventional sense. The "development" and "optimization" of such a panel would involve bioinformatic design and iterative testing, not "training" on a sample set like an AI model.

9. How the Ground Truth for the Training Set (if applicable) was Established

As mentioned above, the concept of a "training set" and "ground truth establishment for a training set" as it applies to AI/ML devices is not applicable here. The CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel is a molecular diagnostic assay where the "design" is based on established scientific principles of molecular biology and virology, including viral genome sequencing and primer/probe design. The specificity and sensitivity are intrinsically built into the primer and probe sequences themselves, which are designed to target specific genetic regions of the influenza virus.

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