Predicate Devices

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The document describes a standard real-time RT-PCR assay kit and its performance characteristics. There is no mention of AI or ML in the device description, intended use, or performance studies. The analysis relies on the detection of specific genetic sequences using primers and probes, which is a traditional molecular diagnostic method.

Therapeutic

No
This device is an in vitro diagnostic (IVD) kit used for the detection and subtyping of influenza A viruses. It is intended for diagnostic purposes and does not provide any direct therapeutic benefit or treatment to patients.

Diagnostic

Yes

The "Intended Use / Indications for Use" section explicitly states that the kit is "intended for use in real-time RT-PCR (rRT-PCR) assays... For determination of the subtype of seasonal human influenza A viruses... from human patients with signs and symptoms of respiratory infection," which is a diagnostic purpose. The "Device Description" also refers to it as a "Diagnostic Panel."

SaMD (Software as a Medical Device)

No

The device is a kit containing reagents and controls for a real-time RT-PCR assay, which are physical components, not software.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

Here's why:

Intended Use: The "Intended Use / Indications for Use" section explicitly states that the kit is "intended for use in real-time RT-PCR (rRT-PCR) assays... For determination of the subtype of seasonal human influenza A viruses... from viral RNA in upper respiratory tract clinical specimens... and lower respiratory tract specimens... from human patients with signs and symptoms of respiratory infection and/or from viral culture." This clearly describes a test performed in vitro (outside the body) on biological specimens to diagnose or provide information about a disease state (influenza infection and subtyping).
Device Description: The "Device Description" further clarifies that it's an "in vitro qualitative detection and characterization of influenza virus RNA in respiratory specimens."
Specimen Types: The device is designed to analyze various clinical specimens (nasopharyngeal swabs, throat swabs, etc.), which are collected from the human body for diagnostic purposes.
Performance Evaluation: The document includes detailed information about analytical and clinical performance evaluations using clinical samples, which is standard for IVD devices.
Predicate Device: The mention of a "Predicate Device(s)" with a K number (K140851) indicates that this device is being compared to a previously cleared IVD device, further confirming its classification as an IVD.

All these points align with the definition and characteristics of an In Vitro Diagnostic device.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

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.

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.

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.

Product codes (comma separated list FDA assigned to the subject device)

OZE, NSU, NXD, OEP, OQW

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.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

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)

Indicated Patient Age Range

Not Found

Intended User / Care Setting

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.

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

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 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.

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.

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 with both enzyme systems (i.e., Invitrogen SuperScript™ and Quanta qScript™) and one cleared extraction method.

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.

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

Analytical Sensitivity - Limit of Detection Study (LOD):

Study Type: LOD study
Sample Size: n=3 replicates for range finding, n=20 replicates for LOD confirmation.
Key Results:
- A(H1)pdm09: LOD for A/West Virginia/01/2016 was 100.9 ID50/mL for both Invitrogen SuperScript™ and Quanta qScript™. LOD for A/California/07/2009 was 103.1 ID50/mL for Invitrogen SuperScript™ and 103.8 ID50/mL for Quanta qScript™.
- H3 assay: LOD equivalency between BHQ and ZEN probes demonstrated.

Analytical Sensitivity - Inclusivity Testing:

Study Type: Inclusivity testing
Sample Size: Ten representative H1pdm09 viruses, each tested in triplicate.
Key Results: The Influenza A Subtyping Kit was reactive with all H1pdm09 isolates that were tested (3/3 positives for InfA, pdmInfA, and pdmH1 assays with both enzyme systems for all 10 strains).

Analytical Specificity - Cross-Reactivity:

Study Type: Cross-reactivity
Sample Size: Various influenza A(H1) virus strains, tested in triplicate.
Key Results:
- A/Brisbane/59/07 (A(H1N1)) and A/Hawaii/15/2001 (A(H1N1)) showed positive results with InfA (3/3) but negative with pdmInfA and pdmH1.
- A/Iowa/1/2006 (A(H1N1v)), A/Texas/14/2008 (A(H1N1v)), and A/Ohio/09/2015 (A(H1N1v)) showed positive results with InfA, pdmInfA, and pdmH1 (3/3).
- A/Minnesota/19/2011 (A(H1N2v)) showed positive results with InfA and pdmInfA (3/3) but negative with pdmH1.

Clinical Performance Evaluation

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

Study Type: Retrospective Clinical Study
Sample Size: 42 samples previously positive with A(H1)pdm09.
Key Results:
- Invitrogen SuperScript™:
  - BW: 1/1 (100.0% Positive Agreement, 95% CI: 20.7-100.0)
  - NPS, NS: 34/35 (97.1% Positive Agreement, 95% CI: 85.5-99.5)
  - NW: 4/4 (100.0% Positive Agreement, 95% CI: 51.0-100.0)
  - TS: 2/2 (100.0% Positive Agreement, 95% CI: 34.2-100.0)
- Quanta qScript™:
  - BW: 1/1 (100.0% Positive Agreement, 95% CI: 20.7-100.0)
  - NPS, NS: 33/33 (100.0% Positive Agreement, 95% CI: 89.6-100.0)
  - NW: 4/4 (100.0% Positive Agreement, 95% CI: 51.0-100.0)
  - TS: 2/2 (100.0% Positive Agreement, 95% CI: 34.2-100.0)

Retrospective Positive Clinical Study Results-A(H3) Comparison:

Study Type: Retrospective Clinical Study
Sample Size: 32 samples previously positive with A(H3).
Key Results:
- Invitrogen SuperScript™:
  - NA: 1/1 (100.0% Positive Agreement, 95% CI: 20.7-100.0)
  - NPS, NS: 30/30 (100.0% Positive Agreement, 95% CI: 88.7-100.0)
  - NW: 1/1 (100.0% Positive Agreement, 95% CI: 20.7-100.0)
- Quanta qScript™:
  - NA: 1/1 (100.0% Positive Agreement, 95% CI: 20.7-100.0)
  - NPS, NS: 30/30 (100.00% Positive Agreement, 95% CI: 88.7-100.00)
  - NW: 1/1 (100.0% Positive Agreement, 95% CI: 20.7-100.0)

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

Study Type: Retrospective Clinical Study
Sample Size: 53 samples negative for influenza A.
Key Results:
- Invitrogen SuperScript™: NPS: 53/53 (100.00% Negative Agreement, 95% CI: 93.2-100.0)
- Quanta qScript™: NPS: 52/52 (100.00% Negative Agreement, 95% CI: 93.1-100.0)

Retrospective Negative Clinical Study Results-A(H3) Comparison:

Study Type: Retrospective Clinical Study
Sample Size: 30 samples negative for influenza A.
Key Results:
- Invitrogen SuperScript™: NPS: 29/29 (100.00% Negative Agreement, 95% CI: 88.3-100.0)
- Quanta qScript™: NPS: 28/28 (100.00% Negative Agreement, 95% CI: 87.9-100.0)

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

Positive Agreement, Negative Agreement.

Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.

K140851

Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).

Not Found

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.

Summary

0

Image /page/0/Picture/1 description: The image is a black and white logo for the U.S. Department of Health & Human Services. The logo features a stylized image of an eagle with three human profiles incorporated into its design. The text "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" is arranged in a circular pattern around the eagle.

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.

1

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:

· 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.

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.

3

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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 Regulation
Sections: | 866.3332-Reagents for detection of specific novel influenza A
viruses
862.2570-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.

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.

V. INTENDED USE

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.

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.

6

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, 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 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/H1,
A/H3, and/or A/H1pdm09 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. | Minor changes to the intended use were made to
reflect the current WHO nomenclature of 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; |
| Intended Use | Performance characteristics for influenza were
established during a season when 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.

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
3+ facility is available to receive and culture
specimens. | 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.

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
3+ facility is available to receive and culture
specimens. |
| | 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. | 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. |
| Organism
Detected | Universal influenza A viruses (animal and human),
Swine-origin influenza A viruses, Influenza A
subtypes: seasonal A(H1), A(H3), A(H1)pdm09 | Universal influenza A viruses (animal and human),
Swine-origin influenza A viruses, Influenza A
subtypes: seasonal A(H3) and A(H1)pdm09 |
| Specimen Types | Nasopharyngeal swabs, nasal swabs, throat swabs,
nasal aspirates, nasal washes and dual
nasopharyngeal/throat swabs, bronchoalveolar
lavages, bronchial aspirates, bronchial washes,
tracheal aspirates, sputum, and lung tissue. | Same |
| Nucleic Acid
Extraction | Yes | Same |
| Extraction
Method | • 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 Mini
Kit, Qiagen
• NucliSENS® easyMAG®, bioMerieux | Same |
| Enzyme Master
Mix | Invitrogen SuperScript™ III Platinum® One-Step
Quantitative RT-PCR Kit (with or without ROX)
OR
Quanta BioSciences qScript™ One-Step qRT-PCR
Kit, Low ROX | Same |
| PCR Technology | Real-Time RT-PCR | Same |
| Required
Instrumentation | Applied Biosystems 7500 Fast Dx Real-Time PCR
Instrument with SDS software version 1.4 | Same |
| Probe
Quenching
Molecule | Black Hole Quencher Probe® (BHQ-1) | ZEN™ Double-Quenched Probe (InfA, H3,
pdmInfA, pdmH1, and RP assays)
OR
Black Hole Quencher Probe® (InfA, H3, pdmInfA,
pdmH1, and RP assays) |
| Oligonucleotides | H1 Assay-Targets a region of the HA gene
H3 assay-Targets a region of the HA gene
pdmH1 assay-Targets a region of the HA gene
InfA assay-Targets a conserved region of the
matrix gene in Influenza A viruses
pdmInfA-Targets a conserved region of the
nucleoprotein gene in Influenza A(H1N1)pdm09
viruses | Gene targets of the oligonucleotide assays are the
same as the predicate; minor changes to the pdmH1
oligonucleotide sequences have been made; the H1
assay is not included in this version of the
Influenza A Subtyping Kit |

8

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

9

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 Virus

Tested	Influenza Strain Designation	LOD (ID50/mL)
		Invitrogen
SuperScriptTM	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

10

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 Virus
Strain Identification | Subtype | ID50/
mL | Invitrogen
SuperScript™ | | | Quanta qScript™ | | |
|------------------------------------------|--------------|-------------|----------------------------|-------------|------------|-----------------|-------------|------------|
| | | | InfA | pdm
InfA | pdm
H1 | InfA | pdm
InfA | pdm
H1 |
| 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.

11

| Strain Designation | Subtype | ID50/mL | Invitrogen
SuperScript™ | | | Quanta qScript™ | | |
|---------------------|----------|---------|----------------------------|-------------|------------|-----------------|-------------|------------|
| | | | InfA | pdm
InfA | pdm
H1 | InfA | pdm
InfA | pdm
H1 |
| 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	# of
Positives	% Positive
Agreement (95%
CI)	# of
Positives	% Positive
Agreement (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)

12

	Invitrogen SuperScript™		Quanta qScript™
Specimen Type	# of
Positives	% Positive
Agreement (95%
CI)	# of
Positives	% Positive
Agreement (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	# of
Negatives1	% Negative
Agreement (95%
CI)	# of
Negatives1	% Negative
Agreement (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.