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.

Device Description

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.

AI/ML Overview

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.

Performance Metric	Acceptance Criteria (Implied)	Reported Device Performance (CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel, Influenza A Subtyping Kit (VER 2))
Analytical Sensitivity (LOD) - pdmH1 assay A/West Virginia/01/2016	Consistent detection (≥ 95% of replicates positive) at specified viral concentrations.	Invitrogen SuperScript™: 100.9 ID50/mLQuanta qScript™: 100.9 ID50/mLConfirmed by testing 20 replicates, with ≥95% positive.
Analytical Sensitivity (LOD) - pdmH1 assay A/California/07/2009	Consistent detection (≥ 95% of replicates positive) at specified viral concentrations.	Invitrogen SuperScript™: 103.1 ID50/mLQuanta qScript™: 103.8 ID50/mLConfirmed by testing 20 replicates, with ≥95% positive.
Analytical Sensitivity (LOD) - H3 assay A/Hong Kong/4801/2014 Equivalency	Equivalency in LOD between cleared H3 BHQ probe and H3 ZEN probe.	All 3/3 replicates positive for both H3 IVD BHQ and H3 ZEN probes across all dilutions ($10^{4.9}$ to $10^{0.9}$ EID50/mL) for both enzyme systems (Invitrogen Superscript™ and Quanta qScript™).
Inclusivity (for 10 A(H1)pdm09 strains)	Reactive with all tested A(H1)pdm09 isolates at or near LOD.	The kit was reactive with all 10 tested H1N1pdm09 isolates (3/3 positives for InfA, pdmInfA, and pdmH1 assays with both enzyme systems).
Clinical Performance - A(H1)pdm09 Positive Agreement	High positive agreement with previously positive clinical samples (e.g., >95% PPA with tight CI).	Invitrogen SuperScript™: - 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.

Summary

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Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-0002

Centers for Disease Control and Prevention (CDC) Yon Yu, Pharm.D. Associate Director for Regulatory Affairs 1600 Clifton Road, MS-C18 Atlanta, GA 30329-4027

Re: K161556

Trade/Device Name: CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel. Influenza A Subtyping Kit Regulation Number: 21 CFR 866.3980 Regulation Name: Respiratory viral panel multiplex nucleic acid assay Regulatory Class: II Product Code: OZE, NSU, NXD, OEP, OQW Dated: June 3, 2016 Received: June 6, 2016

Dear Dr. Yu:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration. listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

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If you desire specific advice for your device on our labeling regulations (21 CFR Parts 801 and 809), please contact the Division of Industry and Consumer Education at its toll-free number (800) 638 2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to

http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

You may obtain other general information on your responsibilities under the Act from the Division of Industry and Consumer Education at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/Resourcesfor You/Industry/default.htm.

Sincerely yours,

Steven R. Gitterman -S

for Uwe Scherf, M.Sc., Ph.D. Director Division of Microbiology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K161556

Device Name

CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel Influenza A Subtyping Kit

Indications for Use (Describe)

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 (ABD) 7500 Fast Dx Real-Time PCR instrument in conjunction with clinical and epidemiological information:

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

Type of Use (Select one or both, as applicable)

X Prescription Use (Part 21 CFR 801 Subpart D)

Over-The-Counter Use (21 CFR 801 Subpart C)

CONTINUE ON A SEPARATE PAGE IF NEEDED.

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This section applies only to requirements of the Paperwork Reduction Act of 1995.

DO NOT SEND YOUR COMPLETED FORM TO THE PRA STAFF EMAIL ADDRESS BELOW.

The burden time for this collection of information is estimated to average 79 hours per response, including the time to review instructions, search existing data sources, gather and maintain the data needed and complete and review the collection of information. Send comments regarding this burden estimate or any other aspect of this information collection, including suggestions for reducing this burden, to:

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"An agency may not conduct or sponsor, and a person is not required to respond to, a collection of information unless it displays a currently valid OMB number."

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8. 510(k) Summary

I. GENERAL INFORMATION

Submitter: Centers for Disease Control and Prevention 1600 Clifton Road, NE Atlanta, GA 30333

Contact Person: CDR Yon Yu, Pharm. D. Associate Director for Regulatory Affairs Office of the Director National Center for Emerging and Zoonotic Infectious Diseases Centers for Disease Control and Prevention 1600 Clifton Road, MS-C18 Atlanta, GA 30329-4027 Phone: 404-639-3046 Fax: 404-639-1275 Email: fkb8@cdc.gov

Date Prepared: June 3, 2016

II. DEVICE INFORMATION

Proprietary Name:	CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel,Influenza A Subtyping Kit (VER 2)
Common Name:	Influenza A Subtyping Kit
Regulation Section:	866.3980-Respiratory viral panel multiplex nucleic acid assay
Subsequent RegulationSections:	866.3332-Reagents for detection of specific novel influenza Aviruses862.2570-Instrumentation for clinical multiplex systems
Device Classification:	Class II
Product Code:	OZE
Subsequent Product Codes:	NSU, NXD, OEP, OQW
Panel:	Microbiology

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III. PREDICATE DEVICE

CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel, Influenza A Subtyping Kit (K140851)

DEVICE DESCRIPTION IV.

V. INTENDED USE

. 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.

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.

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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. 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.

VI. TECHNOLOGICAL CHARACTERISTICS

CDC has identified nucleotide substitutions in the HA gene segment in a small proportion of currently circulating influenza A(H1N1)pdm09 viruses belonging to the 6B.1 genetic clade. The nucleotide substitutions are located in the targeted regions of the reverse primer and the probe of the pdmH1 assay and may cause aberrant reactivity of the pdmH1 assay included in the Influenza A Subtyping Kit. CDC is modifying the pdmH1 assay to improve reactivity with the 6B.1 clade. The performance of the remaining assays in the Influenza A Subtyping Kit (InfA, H3, pdmInfA, and RP) has remained the same and CDC is not modifying these assays. The predicate pdmH1 assay consists of two primers and one probe. The modified pdmH1 assay targets the HA gene at the same location and includes minor nucleotide changes to the reverse primer and the probe. The oligonucleotide sequence of the forward pdmH1 primer remains the same.

CDC is removing the H1 assay from the Influenza A Subtyping Kit. The influenza A(H1) viruses targeted by this assay are no longer circulating and thus a diagnostic assay to detect these viruses is unnecessary. Performance of the remaining assays in the Influenza A Subtyping Kit was verified with analytical performance studies.

In addition to evaluating the modified oligonucleotide primers and probes, CDC has evaluated the ZENT™ Double-Quenched Probe technology (manufactured by Integrated DNA Technologies) as an alternate fluorescent hydrolysis probe quencher chemistry. The Influenza A Subtyping Kit assays contain ZENTM double quenched probes (ZEN probes) that include an internal ZENTM quencher located nine nucleotides away from the 5' FAM reporter dye in addition to an Iowa Black® FQ quencher (IABkFQ) at the 3' end of the probe.

VII. SUBSTANTIAL EQUIVALENCE COMPARISON

The CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel, Influenza A Subtyping Kit (K140851) will serve as the predicate for the intended change. See the following table for a detailed comparison.

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Device Comparison

	CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel Diagnostic Panel,Influenza A Subtyping Kit (K140851)	CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel Diagnostic Panel,Influenza A Subtyping Kit (VER 2)
	The Influenza A Subtyping Kit contains reagentsand controls of the CDC Human Influenza VirusReal-Time RT-PCR Diagnostic Panel and isintended for use in real-time RT-PCR (rRT-PCR)assays on an Applied Biosystems (ABI) 7500 FastDx Real-Time PCR instrument in conjunction withclinical and epidemiological information:For determination of the subtype of seasonalhuman influenza A viruses as seasonal A/H1,A/H3, and/or A/H1pdm09 from viral RNA inupper respiratory tract clinical specimens(including nasopharyngeal swabs [NPS],nasal swabs [NS], throat swabs [TS], nasalaspirates [NA], nasal washes [NW] and dualnasopharyngeal/throat swabs [NPS/TS]) andlower respiratory tract specimens (includingbronchoalveolar lavage [BAL], bronchialwash [BW], tracheal aspirate [TA], sputum,and lung tissue) from human patients withsigns and symptoms of respiratory infectionand/or from viral culture; To provide epidemiologic information forsurveillance of circulating influenza viruses.	Minor changes to the intended use were made toreflect the current WHO nomenclature of influenzaviruses:The Influenza A Subtyping Kit contains reagentsand controls of the CDC Human Influenza VirusReal-Time RT-PCR Diagnostic Panel and isintended for use in real-time RT-PCR (rRT-PCR)assays on an Applied Biosystems (ABI) 7500 FastDx Real-Time PCR instrument in conjunction withclinical and epidemiological information:For determination of the subtype of seasonalhuman influenza A viruses as seasonalA(H3), and/or A(H1)pdm09 from viral RNAin upper respiratory tract clinical specimens(including nasopharyngeal swabs [NPS],nasal swabs [NS], throat swabs [TS], nasalaspirates [NA], nasal washes [NW] and dualnasopharyngeal/throat swabs [NPS/TS]) andlower respiratory tract specimens (includingbronchoalveolar lavage [BAL], bronchialwash [BW], tracheal aspirate [TA], sputum,and lung tissue) from human patients withsigns and symptoms of respiratory infectionand/or from viral culture;
Intended Use	Performance characteristics for influenza wereestablished during a season when seasonal influenzaviruses A/H1 and A/H3 were the predominantinfluenza A viruses in circulation and during aseason when the A/H1pdm09 influenza virus wasthe predominant influenza A virus in circulation.Performance characteristics may vary with otheremerging influenza A viruses.Negative results do not preclude influenza virusinfection and should not be used as the sole basis fortreatment or other patient management decisions.Conversely, positive results do not rule out bacterialinfection or co-infection with other viruses. Theagent detected may not be the definite cause ofdisease.If infection with a novel influenza A virus issuspected based on current clinical andepidemiological screening criteria recommended bypublic health authorities, specimens should becollected with appropriate infection controlprecautions for novel virulent influenza viruses andsent to state or local health department for testing.Viral culture should not be attempted unless a BSL3+ facility is available to receive and culturespecimens.	To provide epidemiologic information forsurveillance of circulating influenza viruses. Performance characteristics for influenza wereestablished during a season when seasonal influenzaviruses A(H1N1) and A(H3N2) were thepredominant influenza A viruses in circulation andduring a season when the A(H1N1)pdm09 influenzavirus was the predominant influenza A virus incirculation. Performance characteristics may varywith other emerging influenza A viruses.Negative results do not preclude influenza virusinfection and should not be used as the sole basis fortreatment or other patient management decisions.Conversely, positive results do not rule out bacterialinfection or co-infection with other viruses. Theagent detected may not be the definite cause ofdisease.If infection with a novel influenza A virus issuspected based on current clinical andepidemiological screening criteria recommended bypublic health authorities, specimens should becollected with appropriate infection controlprecautions for novel virulent influenza viruses andsent to state or local health department for testing.Viral culture should not be attempted unless a BSL3+ facility is available to receive and culturespecimens.
	All users, analysts, and any person reporting results from use of this device should betrained to perform and interpret the results from this procedure by a competentinstructor prior to use. CDC Influenza Division will limit the distribution of this deviceto only those users who have successfully completed a training course provided byCDC instructors or designees.	All users, analysts, and any person reporting results from use of this device should betrained to perform and interpret the results from this procedure by a competentinstructor prior to use. CDC Influenza Division will limit the distribution of this deviceto only those users who have successfully completed a training course provided byCDC instructors or designees.
OrganismDetected	Universal influenza A viruses (animal and human),Swine-origin influenza A viruses, Influenza Asubtypes: seasonal A(H1), A(H3), A(H1)pdm09	Universal influenza A viruses (animal and human),Swine-origin influenza A viruses, Influenza Asubtypes: seasonal A(H3) and A(H1)pdm09
Specimen Types	Nasopharyngeal swabs, nasal swabs, throat swabs,nasal aspirates, nasal washes and dualnasopharyngeal/throat swabs, bronchoalveolarlavages, bronchial aspirates, bronchial washes,tracheal aspirates, sputum, and lung tissue.	Same
Nucleic AcidExtraction	Yes	Same
ExtractionMethod	• QIAamp® DSP Viral RNA Mini Kit, Qiagen• MagNA Pure Compact -Nucleic Acid Isolation Kit I,Roche• MagNA Pure Compact - RNA Isolation Kit, Roche• MagNA Pure LC - Total Nucleic Acid Kit, Roche• Qiagen QIAcube - QIAamp® DSP Viral RNA MiniKit, Qiagen• NucliSENS® easyMAG®, bioMerieux	Same
Enzyme MasterMix	Invitrogen SuperScript™ III Platinum® One-StepQuantitative RT-PCR Kit (with or without ROX)ORQuanta BioSciences qScript™ One-Step qRT-PCRKit, Low ROX	Same
PCR Technology	Real-Time RT-PCR	Same
RequiredInstrumentation	Applied Biosystems 7500 Fast Dx Real-Time PCRInstrument with SDS software version 1.4	Same
ProbeQuenchingMolecule	Black Hole Quencher Probe® (BHQ-1)	ZEN™ Double-Quenched Probe (InfA, H3,pdmInfA, pdmH1, and RP assays)ORBlack Hole Quencher Probe® (InfA, H3, pdmInfA,pdmH1, and RP assays)
Oligonucleotides	H1 Assay-Targets a region of the HA geneH3 assay-Targets a region of the HA genepdmH1 assay-Targets a region of the HA geneInfA assay-Targets a conserved region of thematrix gene in Influenza A virusespdmInfA-Targets a conserved region of thenucleoprotein gene in Influenza A(H1N1)pdm09viruses	Gene targets of the oligonucleotide assays are thesame as the predicate; minor changes to the pdmH1oligonucleotide sequences have been made; the H1assay is not included in this version of theInfluenza A Subtyping Kit

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VIII. ANALYTICAL PERFORMANCE EVALUATION

Analytical Sensitivity - Limit of Detection Study (LOD)

Analytical sensitivity of the pdmH1 assay of the Influenza A Subtyping Kit was demonstrated by determining the LOD using Quanta qScript™ and Invitrogen SuperScript™ enzyme kits. Characterized viruses of known 50% infectious dose titers (ID50/mL) were extracted, and the RNA

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was serially diluted and tested (n=3 replicates) in order to determine an apparent endpoint range. The LOD for each primer and probe set was confirmed by testing extraction replicates (n=20) of the highest virus dilution where ≥ 95% of all replicates tested positive. Virus dilutions were prepared in virus transport medium containing human A549 cells to emulate clinical specimen matrix. The lowest concentration where the InfA, pdmInfA, and pdmH1 primer and probe sets demonstrated uniform detection was reported as the LOD. The results are summarized in the table below.

Influenza VirusTested	Influenza Strain Designation	LOD (ID50/mL)
		InvitrogenSuperScriptTM	Quanta qScriptTM
A(H1)pdm09	A/West Virginia/01/2016	100.9	100.9
	A/California/07/2009	103.1	103.8

	pdmH1 Assay LOD Summary

A range finding study was also performed for the unmodified H3 assay to demonstrate LOD equivalency between the currently cleared H3 BHQ probe and the H3 ZEN probe. Characterized virus of a known 50% egg infectious dose titer (EID50/mL) was extracted, and the RNA was serially diluted and tested (n=3 replicates) in order to determine an apparent endpoint range.

Titer (EID50/mL)	Invitrogen Superscript™		Quanta qScript™
	H3 IVD BHQ	H3 ZEN	H3 IVD BHQ	H3 ZEN
$10^{4.9}$	3/3(+)	3/3(+)	3/3(+)	3/3(+)
$10^{3.9}$	3/3(+)	3/3(+)	3/3(+)	3/3(+)
$10^{2.9}$	3/3(+)	3/3(+)	3/3(+)	3/3(+)
$10^{1.9}$	3/3(+)	3/3(+)	3/3(+)	3/3(+)
$10^{0.9}$	3/3(+)	3/3(+)	3/3(+)	3/3(+)
$10^{-0.1}$	-	-	-	-
$10^{-1.1}$	-	-	-	-

A/Hong Kong/4801/2014 LOD Equivalency Data for H3 assay

Analytical Sensitivity - Inclusivity Testing

Inclusivity testing was conducted to demonstrate the capability of the oligonucleotide primers and probes in the Influenza A Subtyping Kit to detect strains of influenza A(H1)pdm09 viruses representative of different geographic locations and phylogenetic clades. Inclusivity testing was performed with ten representative H1pdm09 viruses at or near the established LOD. The viruses were grown to high titer, harvested, and serially diluted to near the LOD of the assays. The diluted influenza A(H1)pdm09 viruses were extracted and tested in triplicate with the InfA, and pdmH1 assays to demonstrate reactivity. Inclusivity of the Influenza A Subtyping Kit was evaluated

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with both enzyme systems (i.e., Invitrogen SuperScript™ and Quanta qScript™) and one cleared extraction method. The Influenza A Subtyping Kit was reactive with all H1pdm09 isolates that were tested. The inclusivity results are summarized in the table below.

Influenza VirusStrain Identification	Subtype	ID50/mL	InvitrogenSuperScript™			Quanta qScript™
			InfA	pdmInfA	pdmH1	InfA	pdmInfA	pdmH1
A/California/04/2009	A(H1N1)pdm09	102.9	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)
A/California/07/2009	A(H1N1)pdm09	103.5	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)
A/Colorado/14/2012	A(H1N1)pdm09	101.1	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)
A/Florida/27/2011	A(H1N1)pdm09	101.9	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)
A/Florida/62/2014	A(H1N1)pdm09	102.2	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)
A/Maryland/13/2012	A(H1N1)pdm09	101.0	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)
A/Minnesota/03/2011	A(H1N1)pdm09	103.9	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)
A/North Carolina/4/2014	A(H1N1)pdm09	103.3	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)
A/Utah/13/2016	A(H1N1)pdm09	101.5	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)
A/Washington/24/2012	A(H1N1)pdm09	102.5	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)

Inclusivity Results of the Influenza A Subtyping Kit

Analytical Specificity - Cross-Reactivity

Cross-reactivity of the Influenza A Subtyping Kit was evaluated by testing influenza A(H1) virus strains representing diverse geographic locations and different sources. Samples were tested in triplicate using RNA extracted from high titer preparations of viruses (≥ 106 EID50/mL). Crossreactivity testing of the Influenza A Subtyping Kit was evaluated with both enzyme systems (i.e., Invitrogen SuperScript™ and Quanta qScript™) and one cleared extraction method. The results are shown in the following table.

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Strain Designation	Subtype	ID50/mL	InvitrogenSuperScript™			Quanta qScript™
			InfA	pdmInfA	pdmH1	InfA	pdmInfA	pdmH1
A/Brisbane/59/07	A(H1N1)	108.4	(+)3/3	-	-	(+)3/3	-	-
A/Hawaii/15/2001	A(H1N1)	108.1	(+)3/3	-	-	(+)3/3	-	-
A/Iowa/1/2006	A(H1N1v)	108.2	(+)3/3	(+)3/3	(+)3/3	(+)3/3	(+)3/3	(+)3/3
A/Texas/14/2008	A(H1N1v)	108.3	(+)3/3	(+)3/3	(+)3/3	(+)3/3	(+)3/3	(+)3/3
A/Ohio/09/2015	A(H1N1v)	107.7	(+)3/3	(+)3/3	(+)3/3	(+)3/3	(+)3/3	(+)3/3
A/Minnesota/19/2011	A(H1N2v)	107.1	(+)3/3	(+)3/3	-	(+)3/3	(+)3/3	-

Influenza Viruses for Cross-Reactivity Testing

CLINICAL PERFORMANCE EVALUATION IX.

The clinical performance of oligonucleotide primer and probe sets of the Influenza A Subtyping Kit were evaluated using retrospective clinical samples collected during the 2011-2012 and 2013-2014 influenza seasons. Clinical samples from the 2015-2016 influenza season that produced aberrant results with the currently cleared pdmH1 assay and were confirmed by genetic sequence analysis were also tested to validate reactivity with the updated assay. A total of forty-two samples that were previously determined to be positive with A(H1)pdm09 influenza virus were evaluated with the InfA. pdmInfA, and pdmH1 assays. Samples that yielded inconclusive results were excluded from the analysis. A total of thirty-two samples that were previously determined to be positive with A(H3) influenza virus were evaluated with the InfA and H3 assays. Clinical testing was performed with both enzymes approved for use with the kit and one of the currently cleared extraction methods. The results are summarized in the tables below.

Retrospective Positive Clinical Study Results-A(H1)pdm09 Comparison

	Invitrogen SuperScript™		Quanta qScript™
Specimen Type	# ofPositives	% PositiveAgreement (95%CI)	# ofPositives	% PositiveAgreement (95%CI)
BW	1/1	100.0 (20.7-100.0)	1/1	100.0 (20.7-100.0)
NPS, NS	34/35	97.1 (85.5-99.5)	33/33	100.0 (89.6-100.0)
NW	4/4	100.0 (51.0-100.0)	4/4	100.0 (51.0-100.0)
TS	2/2	100.00 (34.2-100.0)	2/2	100.00 (34.2-100.0)

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	Invitrogen SuperScript™		Quanta qScript™
Specimen Type	# ofPositives	% PositiveAgreement (95%CI)	# ofPositives	% PositiveAgreement (95% CI)
NA	1/1	100.0 (20.7-100.0)	1/1	100.0 (20.7-100.0)
NPS, NS	30/30	100.0 (88.7-100.0)	30/30	100.00 (88.7-100.00)
NW	1/1	100.0 (20.7-100.0)	1/1	100.0 (20.7-100.0)

Retrospective Positive Clinical Study Results-A(H3) Comparison

The Influenza A Subtyping Kit oligonucleotide primer and probe sets were also evaluated in specimens that tested negative for influenza A with the CDC Human Influenza rRT-PCR Diagnostic Panel and were obtained from a clinical study conducted during the 2011-2012 influenza season. The specimens that tested negative for influenza A with the currently cleared CDC Human Influenza rRT-PCR Diagnostic Panel were utilized as the comparator for the updated Influenza A Subtyping Kit. For the pdm assays a total of fifty-three samples were tested and for the H3 assay a total of thirty samples were tested. Samples that yielded inconclusive results were excluded from the analysis. The results are summarized in the tables below.

Retrospective Negative Clinical Study Results-A(H1)pdm09 Comparison

		Invitrogen SuperScript™	Quanta qScript™
Specimen Type	# ofNegatives1	% NegativeAgreement (95%CI)	# ofNegatives1	% NegativeAgreement (95%CI)
NPS	53/53	100.00 (93.2-100.0)	52/52	100.00 (93.1-100.0)

1 Proportion of negative samples correctly identified versus the comparator.

Retrospective Negative Clinical Study Results-A(H3) Comparison

		Invitrogen SuperScript™		Quanta qScript™
Specimen Type	# of Negatives1	% Negative Agreement (95% CI)	# of Negatives1	% Negative Agreement (95% CI)
NPS	29/29	100.00 (88.3-100.0)	28/28	100.00 (87.9-100.0)

1Proportion of negative samples correctly identified versus the comparator.

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X. CONCLUSION

The modification of the CDC Human Influenza Virus rRT-PCR Diagnostic Panel, Influenza A Subtyping Kit to ensure comprehensive detection of influenza A(H1)pdm09 viruses does not substantially change the device. Analytical and clinical data demonstrate that the performance of the device to detect influenza A(H1)pdm09 and influenza A(H3) viruses is accomplished with high positive and negative percent agreement in a manner substantially equivalent to the predicate.

Regulation Number and Section

§ 866.3980 Respiratory viral panel multiplex nucleic acid assay.

(a)
Identification. A respiratory viral panel multiplex nucleic acid assay is a qualitative in vitro diagnostic device intended to simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or viral culture. The detection and identification of a specific viral nucleic acid from individuals exhibiting signs and symptoms of respiratory infection aids in the diagnosis of respiratory viral infection when used in conjunction with other clinical and laboratory findings. The device is intended for detection and identification of a combination of the following viruses:(1) Influenza A and Influenza B;
(2) Influenza A subtype H1 and Influenza A subtype H3;
(3) Respiratory Syncytial Virus subtype A and Respiratory Syncytial Virus subtype B;
(4) Parainfluenza 1, Parainfluenza 2, and Parainfluenza 3 virus;
(5) Human Metapneumovirus;
(6) Rhinovirus; and
(7) Adenovirus.
(b)
Classification. Class II (special controls). The special controls are:(1) FDA's guidance document entitled “Class II Special Controls Guidance Document: Respiratory Viral Panel Multiplex Nucleic Acid Assay;”
(2) For a device that detects and identifies Human Metapneumovirus, FDA's guidance document entitled “Class II Special Controls Guidance Document: Testing for Human Metapneumovirus (hMPV) Using Nucleic Acid Assays;” and
(3) For a device that detects and differentiates Influenza A subtype H1 and subtype H3, FDA's guidance document entitled “Class II Special Controls Guidance Document: Testing for Detection and Differentiation of Influenza A Virus Subtypes Using Multiplex Nucleic Acid Assays.” See § 866.1(e) for the availability of these guidance documents.