CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel: Influenza B Lineage Genotyping Kit,

• K Number: K181736
• Device Name: CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel: Influenza B Lineage Genotyping Kit,
• Manufacturer: Centers for Disease Control and Prevention (CDC)
• Date Cleared: 2018-07-30 ( 28 days)
• Product Code: OZE NSU OOI
• Regulation Number: 866.3980

Intended Use

The Influenza B Lineage Genotyping Kit contains reagents and controls of the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel and is intended for use in real-time RT-PCR (rRT-PCR) assays on an Applied Biosystems (ABI) 7500 Fast Dx Real-Time PCR instrument in conjunction with clinical and epidemiological information: For the determination of the genetic lineage of human influenza B viruses as B/Victoria or B/Yamagata lineage from · viral RNA in upper respiratory tract clinical specimens (including nasopharyngeal swabs [NS], throat swabs [TS], nasal aspirates [NA], nasal washes [NW] and dual nasopharyngeal/throat swabs [NPS/TS]) from human patients with signs and symptoms of respiratory infection and/or from viral culture; · To provide epidemiologic information for surveillance of circulating influenza viruses. Performance characteristics for influenza B lineage genotyping were established during a season when influenza B/ Victoria and B/Yamagata lineages were in circulation. Negative results do not preclude influenza virus infection and should not be used as the sole basis for treatment or other patient management decisions. Conversely, positive results do not rule out bacterial infection with other viruses. The agent detected may not be the definite cause of disease. All users, and any person reporting results from use of this device should be trained to perform and interpret the results from this procedure by a competent instructor prior to use. CDC Influenza Division will limit the distribution of this device to only those users who have successfully completed a training course provided by CDC instructors or designees.

Device Description

The CDC Human Influenza Real-Time RT-PCR Diagnostic Panel is used in real-time RT-PCR (rRT-PCR) assays on the Applied Biosystems® (ABI) 7500 Fast Dx Real-time PCR system. The panel is configured in four separate kits. Each kit consists of oligonucleotide primers. fluorescently labeled hydrolysis probes, and controls which are used in rRT-PCR assays for the in vitro qualitative detection and characterization of influenza virus RNA in respiratory specimens from patients presenting with influenza-like illness (ILI). Oligonucleotide primers and probes included in the Influenza B Lineage Genotyping Kit for detection of influenza B and the two major genetic lineages of influenza B were selected from highly conserved regions of the non-structural (NS) and HA genes, respectively. Oligonucleotide primers and probes to detect the human RNase P gene (RP) in control samples and clinical specimens are also included in the kit.

Predicate Devices

K172091

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The summary describes a real-time RT-PCR assay kit for detecting and genotyping influenza B viruses. There is no mention of AI, ML, or any computational algorithms beyond standard data analysis for PCR results. The device relies on chemical reagents and a standard PCR instrument.

Therapeutic

No

This device is an in vitro diagnostic (IVD) kit used to determine the genetic lineage of influenza B viruses for diagnostic and surveillance purposes. It does not provide therapy or treatment.

Diagnostic

Yes

The "Intended Use / Indications for Use" states that the kit is for "determination of the genetic lineage of human influenza B viruses" and is part of the "CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel". The "Device Description" further clarifies its use for "in vitro qualitative detection and characterization of influenza virus RNA in respiratory specimens from patients presenting with influenza-like illness (ILI)". These phrases describe a device used to identify the presence or characteristics of a disease-causing agent, which is a diagnostic function.

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 the determination of the genetic lineage of human influenza B viruses... from viral RNA in upper respiratory tract clinical 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 provide diagnostic information about a patient's condition.
• Device Description: The "Device Description" further clarifies that the panel is used in "in vitro qualitative detection and characterization of influenza virus RNA in respiratory specimens from patients presenting with influenza-like illness (ILI)." This reinforces the in vitro nature of the test.
• Clinical Specimens: The device is designed to be used with "upper respiratory tract clinical specimens," which are biological samples taken from a patient.
• Diagnostic Information: The results are used to determine the genetic lineage of influenza B viruses, which provides information relevant to diagnosis and epidemiological surveillance.

Therefore, based on the provided information, the Influenza B Lineage Genotyping Kit meets the definition of an In Vitro Diagnostic device.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The Influenza B Lineage Genotyping Kit contains reagents and controls of the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel and is intended for use in real-time RT-PCR (rRT-PCR) assays on an Applied Biosystems (ABI) 7500 Fast Dx Real-Time PCR instrument in conjunction with clinical and epidemiological information:

For the determination of the genetic lineage of human influenza B viruses as B/Victoria or B/Yamagata lineage from · viral RNA in upper respiratory tract clinical specimens (including nasopharyngeal swabs [NS], throat swabs [TS], nasal aspirates [NA], nasal washes [NW] and dual nasopharyngeal/throat swabs [NPS/TS]) from human patients with signs and symptoms of respiratory infection and/or from viral culture;

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

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

Negative results do not preclude influenza virus infection and should not be used as the sole basis for treatment or other patient management decisions. Conversely, positive results do not rule out bacterial infection with other viruses. The agent detected may not be the definite cause of disease.

All users, and any person reporting results from use of this device should be trained to perform and interpret the results from this procedure by a competent instructor prior to use. CDC Influenza Division will limit the distribution of this device to only those users who have successfully completed a training course provided by CDC instructors or designees.

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

OZE, NSU, OOI

Device Description

The CDC Human Influenza Real-Time RT-PCR Diagnostic Panel is used in real-time RT-PCR (rRT-PCR) assays on the Applied Biosystems® (ABI) 7500 Fast Dx Real-time PCR system. The panel is configured in four separate kits. Each kit consists of oligonucleotide primers. fluorescently labeled hydrolysis probes, and controls which are used in rRT-PCR assays for the in vitro qualitative detection and characterization of influenza virus RNA in respiratory specimens from patients presenting with influenza-like illness (ILI). Oligonucleotide primers and probes included in the Influenza B Lineage Genotyping Kit for detection of influenza B and the two major genetic lineages of influenza B were selected from highly conserved regions of the non-structural (NS) and HA genes, respectively. Oligonucleotide primers and probes to detect the human RNase P gene (RP) in control samples and clinical specimens are also included in the kit.

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])

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 - Influenza B Assay:

• Sample size: Two characterized influenza vaccine reference viruses. Serially diluted and tested (n=3 replicates).
• Data source: Characterized influenza vaccine reference viruses.
• Annotation protocol: Apparent endpoint range determined. Acceptance criterion for LOD equivalency: 100% positivity (3 out of 3 replicates) at either the same endpoint LOD concentration or within a 5-fold dilution of each other.

Analytical Sensitivity - Influenza B Lineage Genotyping Assay Update:

• Sample size: One historical benchmark strain and one current strain of both B/Victoria and B/Yamagata lineages of influenza B virus. Serially diluted RNA extracted from characterized virus stocks. Three replicates per dilution were tested.
• Data source: Characterized virus stocks of known 50% egg infectious dose titer (EID50mL).
• Annotation protocol: Apparent endpoint range determined. Acceptance criterion for LOD equivalency: 100% positivity (3 out of 3 replicates) at either the same endpoint LOD concentration or within a 5-fold dilution of each other.

Analytical Sensitivity - Inclusivity:

• Sample size: Ten influenza B viruses of each lineage, representative of different geographic locations and phylogenetic clades. Samples were serially diluted to concentrations near the LOD and tested in triplicate.
• Data source: Influenza B viruses.
• Annotation protocol: Not explicitly stated, but implies detection at or near LOD.

Analytical Specificity – Cross-Reactivity:

• Sample size: High titer preparations of influenza B viruses of the opposite lineage and from diverse geographic locations.
• Data source: Influenza B viruses.
• Annotation protocol: Cross-reactivity evaluated with both enzyme systems, looking for no detection.

Analytical Specificity - Exclusivity (with Influenza A Viruses):

• Sample size: Influenza A viruses of various subtypes.
• Data source: Characterized, high titer stocks.
• Annotation protocol: Not explicitly stated, but looking for no detection.

Analytical Specificity - Exclusivity (with Non-Influenza Respiratory Pathogens):

• Sample size: 36 organisms (16 viruses, 19 bacteria, and 1 yeast).
• Data source: CDC repositories or American Type Culture Collection (ATCC). Tested at high titers.
• Annotation protocol: Not explicitly stated, but looking for no detection.

Influenza B Assay - Retrospective Study (Clinical Performance):

• Sample size: 30 positive and 50 negative upper respiratory tract samples.
• Data source: Retrospective clinical samples collected during the 2011-2012 influenza season.
• Annotation protocol: Compared to the cleared InfB assay using a BHQ quenched probe. Result interpretation followed the instructions of the Package Insert for the cleared Influenza B Lineage Genotyping Kit.

Influenza B Lineage Genotyping Kit (VER 2) - Prospective Study (Clinical Performance):

• Sample size: 592 upper respiratory tract specimens collected. 579 included in data analysis after exclusions.
• Data source: Clinical investigation at 3 U.S. public health laboratories using upper respiratory tract specimens collected during the 2016-2017 influenza season from individuals symptomatic for influenza-like illness.
• Annotation protocol: Tested with assays from the FDA-cleared CDC Human Influenza Real-Time RT-PCR Diagnostic Panel and the investigational Influenza B Lineage Genotyping Kit (VER 2).

Influenza B Lineage Genotyping Kit VER 1.1 and VER 2 - Retrospective Study Results (Clinical Performance):

• Sample size: 126 specimens positive for either influenza B/Victoria or influenza B/Yamagata viruses and 61 specimens negative for influenza B viruses. Four lung tissue specimens were included for informational purposes only.
• Data source: Upper respiratory tract clinical samples collected during the 2017 and 2017-2018 influenza seasons, previously determined positive or negative using the FDA-cleared Influenza B Lineage Genotyping Kit.
• Annotation protocol: Tested using the cleared InfB assay and the investigative VER 1.1 and VER 2 VIC and YAM assays. Result interpretation followed the instructions of the Package Insert for the cleared Influenza B Lineage Genotyping Kit.

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

Analytical Sensitivity - Influenza B Assay

• Study Type: Range finding study (LOD equivalency).
• Sample Size: Two influenza vaccine reference viruses, serially diluted and tested in triplicates.
• Key Results: All assays demonstrated similar reactivity, with LOD endpoints meeting predefined acceptance criteria (100% positivity at same concentration or within 5-fold dilution).

Analytical Sensitivity - Influenza B Lineage Genotyping Assay Update

• Study Type: Range finding study (LOD equivalency and improved reactivity).
• Sample Size: One historical benchmark strain and one current strain of both B/Victoria and B/Yamagata lineages, serially diluted RNA tested in triplicates.
• Key Results: All assays demonstrated similar reactivity with apparent LOD endpoints within one 5-fold dilution of each other.

Analytical Sensitivity - Inclusivity

• Study Type: Inclusivity testing.
• Sample Size: Ten influenza B viruses of each lineage, tested in triplicate.
• Key Results: The modified VER 1.1 and VER 2 VIC and YAM primer and probe sets successfully detected strains of the corresponding influenza B lineage at or near the established LOD. All were 3/3 positive.

Analytical Specificity – Cross-Reactivity

• Study Type: Cross-reactivity testing.
• Sample Size: High titer preparations of influenza B viruses of the opposite lineage.
• Key Results: No cross-reactivity was detected with either primer and probe set design.

Analytical Specificity - Exclusivity (with Influenza A Viruses)

• Study Type: Exclusivity testing.
• Sample Size: Influenza A viruses of various subtypes.
• Key Results: No cross-reactivity was detected for Influenza A viruses.

Analytical Specificity - Exclusivity (with Non-Influenza Respiratory Pathogens)

• Study Type: Exclusivity testing.
• Sample Size: 36 organisms (16 viruses, 19 bacteria, and 1 yeast).
• Key Results: No cross-reactivity was detected for non-influenza respiratory pathogens.

Clinical Performance Evaluation
Influenza B Assay - Retrospective Study

• Study Type: Retrospective clinical study.
• Sample Size: 30 positive and 50 negative upper respiratory tract samples.
• Key Metrics: Positive Agreement, Negative Agreement.
• Key Results:
  • Invitrogen SuperScript: 100.0% Positive Agreement (95% CI: 88.7-100.0), 100.0% Negative Agreement (95% CI: 92.9-100.0).
  • Quanta qScript: 100.0% Positive Agreement (95% CI: 88.7-100.0), 100.0% Negative Agreement (95% CI: 92.9-100.0).

Influenza B Lineage Genotyping Kit (VER 2) - Prospective Study

• Study Type: Prospective clinical investigation.
• Sample Size: 579 upper respiratory tract specimens.
• Key Metrics: Sensitivity, Specificity.
• Key Results (B/Victoria Assay - VER 2 Design):
  • Overall Sensitivity: 100.0% (95% CI: 56.6 – 100.0) for 5/5 positives.
  • Overall Specificity: 100.0% (95% CI: 99.3 – 100.0) for 574/574 negatives.
• Key Results (B/Yamagata Assay - VER 2 Design):
  • Overall Sensitivity: 100.0% (95% CI: 90.8 – 100.0) for 38/38 positives.
  • Overall Specificity: 99.8% (95% CI: 99.0 – 100.0) for 540/541 negatives.

Influenza B Lineage Genotyping Kit VER 1.1 and VER 2 - Retrospective Study Results

• Study Type: Retrospective clinical study.
• Sample Size: 126 positive (for B/Victoria or B/Yamagata) and 61 negative (for influenza B) specimens.
• Key Metrics: Positive Agreement, Negative Agreement.
• Key Results (VIC VER 1.1 Design):
  • Positive Agreement: 100.0% (95% CI: 88.7 - 100.0) for 30/30 positives.
  • Negative Agreement: 100.0% (95% CI: 97.6 - 100.0) for 157/157 negatives.
• Key Results (YAM VER 1.1 Design):
  • Positive Agreement: 100.0% (95% CI: 96.2 - 100.0) for 96/96 positives.
  • Negative Agreement: 100.0% (95% CI: 96.0 - 100.0) for 91/91 negatives.
• Key Results (VIC VER 2 Design):
  • Positive Agreement: 100.0% (95% CI: 88.7 – 100.0) for 30/30 positives.
  • Negative Agreement: 100.0% (95% CI: 97.6 – 100.0) for 157/157 negatives.
• Key Results (YAM VER 2 Design):
  • Positive Agreement: 100.0% (95% CI: 96.2 - 100.0) for 96/96 positives.
  • Negative Agreement: 100.0% (95% CI: 96.0 – 100.0) for 91/91 negatives.

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

• Analytical Sensitivity: determined by LOD (Limit of Detection) equivalency and inclusivity, showing 100% positivity at or near LOD for tested strains.
• Analytical Specificity: demonstrated by no cross-reactivity with opposite influenza B lineages, influenza A viruses, and non-influenza respiratory pathogens.
• Clinical Performance:
  • Retrospective Study for InfB Assay:
    • Positive Agreement: 100.0% (95% CI: 88.7-100.0) for both Invitrogen SuperScript and Quanta qScript.
    • Negative Agreement: 100.0% (95% CI: 92.9-100.0) for both Invitrogen SuperScript and Quanta qScript.
  • Prospective Study for B/Victoria Assay - VER 2 Design:
    • Sensitivity: 100.0% (95% CI: 56.6 – 100.0)
    • Specificity: 100.0% (95% CI: 99.3 - 100.0)
  • Prospective Study for B/Yamagata Assay - VER 2 Design:
    • Sensitivity: 100.0% (95% CI: 90.8 - 100.0)
    • Specificity: 99.8% (95% CI: 99.0 - 100.0)
  • Retrospective Study for VIC VER 1.1 Design:
    • Positive Agreement: 100.0% (95% CI: 88.3 - 100.0)
    • Negative Agreement: 100.0% (95% CI: 97.6 - 100.0)
  • Retrospective Study for YAM VER 1.1 Design:
    • Positive Agreement: 100.0% (95% CI: 96.1 - 100.0)
    • Negative Agreement: 100.0% (95% CI: 95.9 - 100.0)
  • Retrospective Study for VIC VER 2 Design:
    • Positive Agreement: 100.0% (95% CI: 88.3 - 100.0)
    • Negative Agreement: 100.0% (95% CI: 97.6 - 100.0)
  • Retrospective Study for YAM VER 2 Design:
    • Positive Agreement: 100.0% (95% CI: 96.1 - 100.0)
    • Negative Agreement: 100.0% (95% CI: 95.9 - 100.0)

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.

CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel: Influenza B Lineage Genotyping Kit (K172091)

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

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Image /page/0/Picture/0 description: The image shows the logo of the U.S. Food and Drug Administration (FDA). On the left is the Department of Health & Human Services logo. To the right of that is the FDA logo, with the letters "FDA" in a blue square. To the right of the blue square is the text "U.S. FOOD & DRUG ADMINISTRATION" in blue.

July 30, 2018

Centers for Disease Control and Prevention (CDC) Yon Yu, Pharm.D. Associate Director for Regulatory Affairs 1600 Clifton Road Ms E-51 Atlanta, GA 30329-4027

Re: K181736

Trade/Device Name: CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel: Influenza B Lineage Genotyping Kit Regulation Number: 21 CFR 866.3980 Regulation Name: Respiratory viral panel multiplex nucleic acid assay Regulatory Class: Class II Product Code: OZE, NSU, OOI Dated: June 29, 2018 Received: July 2, 2018

Dear Yon 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 Part 801 and Part 809); medical device 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

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Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

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 comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/) and CDRH Learn (http://www.fda.gov/Training/CDRHLearn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (http://www.fda.gov/DICE) for more information or contact DICE by email ([e-mail]) or phone [phone] or 301-796-7100).

Sincerely,

Steven R. Gitterman -S
for

Uwe Scherf, 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) K181736

Device Name

CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel: Influenza B Lineage Genotyping Kit

Indications for Use (Describe)

The Influenza B Lineage Genotyping Kit contains reagents and controls of the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel and is intended for use in real-time RT-PCR (rRT-PCR) assays on an Applied Biosystems (ABI) 7500 Fast Dx Real-Time PCR instrument in conjunction with clinical and epidemiological information:

For the determination of the genetic lineage of human influenza B viruses as B/Victoria or B/Yamagata lineage from · viral RNA in upper respiratory tract clinical specimens (including nasopharyngeal swabs [NS], throat swabs [TS], nasal aspirates [NA], nasal washes [NW] and dual nasopharyngeal/throat swabs [NPS/TS]) from human patients with signs and symptoms of respiratory infection and/or from viral culture;

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

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

Negative results do not preclude influenza virus infection and should not be used as the sole basis for treatment or other patient management decisions. Conversely, positive results do not rule out bacterial infection with other viruses. The agent detected may not be the definite cause of disease.

All users, and any person reporting results from use of this device should be trained to perform and interpret the results from this procedure by a competent instructor prior to use. CDC Influenza Division will limit the distribution of this device to only those users who have successfully completed a training course provided by CDC instructors or designees.

Type of Use (Select one or both, as applicable)
Prescription Use (Part 21 CFR 801 Subpart D)	Over-The-Counter Use (21 CFR 801 Subpart C)

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8. 510(k) Summary

I. GENERAL INFORMATION

Submitter: Centers for Disease Control and Prevention 1600 Clifton Road, NE Atlanta, GA 30329

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 RD, MS E-51; Atlanta, GA 30329-4027 Phone: [phone] Fax: [phone] Email: [e-mail]

Date Prepared: June 28, 2018

DEVICE INFORMATION II.

| Proprietary Name: | CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel
Influenza B Lineage Genotyping Kit (VER 1.1) and Influenza B
Lineage Genotyping Kit (VER 2) |
|------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Common Name: | Influenza B Lineage Genotyping Kit |
| Regulation Section: | 866.3980-Respiratory viral panel multiplex nucleic acid assay |
| Subsequent Regulation
Sections: | 862.2570-Instrumentation for clinical multiplex systems |
| Device Classification: | Class II |
| Product Code: | OZE |
| Subsequent Product Codes: | NSU, OOI |
| Panel: | Microbiology |

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III. PREDICATE DEVICE

CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel: Influenza B Lineage Genotyping Kit (K172091)

IV. DEVICE DESCRIPTION

The CDC Human Influenza Real-Time RT-PCR Diagnostic Panel is used in real-time RT-PCR (rRT-PCR) assays on the Applied Biosystems® (ABI) 7500 Fast Dx Real-time PCR system. The panel is configured in four separate kits. Each kit consists of oligonucleotide primers. fluorescently labeled hydrolysis probes, and controls which are used in rRT-PCR assays for the in vitro qualitative detection and characterization of influenza virus RNA in respiratory specimens from patients presenting with influenza-like illness (ILI). Oligonucleotide primers and probes included in the Influenza B Lineage Genotyping Kit for detection of influenza B and the two major genetic lineages of influenza B were selected from highly conserved regions of the non-structural (NS) and HA genes, respectively. Oligonucleotide primers and probes to detect the human RNase P gene (RP) in control samples and clinical specimens are also included in the kit.

INTENDED USE V.

The Influenza B Lineage Genotyping Kit contains reagents and controls of the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel and is intended for use in real-time RT-PCR (rRT-PCR) assays on an Applied Biosystems (ABI) 7500 Fast Dx Real-Time PCR instrument in conjunction with clinical and epidemiological information:

. For the determination of the genetic lineage of human influenza B viruses as B/Victoria or B/Yamagata lineage from viral RNA in upper respiratory tract clinical specimens (including nasopharyngeal swabs [NPS], nasal swabs [NS], throat swabs [TS], nasal aspirates [NA], nasal washes [NW] and dual nasopharyngeal/throat swabs [NPS/TS]) from human patients with signs and symptoms of respiratory infection and/or from viral culture;

• To provide epidemiologic information for surveillance of circulating influenza viruses. .
  Performance characteristics for influenza B lineage genotyping were established during a season when influenza B/Victoria and B/Yamagata lineages were in circulation.

Negative results do not preclude influenza virus infection and should not be used as the sole basis for treatment or other patient management decisions. Conversely, positive results do not rule out bacterial infection or co-infection with other viruses. The agent detected may not be the definite cause of disease.

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.

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VI. TECHNOLOGICAL CHARACTERISTICS

The technological characteristics of the modified CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel- Influenza B Lineage Genotyping Kit (VER 1.1. and VER 2) remain the same as the predicate device. Modifications were made primarily to address recent evolutionary changes in circulating influenza B viruses that may impact the reactivity of the current Influenza B Lineage Genotyping Kit. Two design approaches were evaluated that address specific genetic mutations in the targeted hemagglutinin (HA) gene of influenza B viruses.

CDC also evaluated the ZENTM Double-Quenched Probe technology (manufactured by Integrated DNA Technologies) as an alternate fluorescent hydrolysis probe quencher chemistry for the InfB assay of the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel.

SUBSTANTIAL EQUIVALENCE COMPARISON VII.

The CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel, Influenza B Lineage Genotyping Kit (K172091), will serve as the predicate for the proposed change. See table 8-1 below for a detailed comparison of the modified device to the predicate.

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Table 8-1: Device Comparison
Item	Predicate Device	Proposed Device
	CDC Human Influenza Virus Real-Time RT-PCR
Diagnostic Panel, Influenza B Lineage Genotyping
Kit [K172091]	CDC Human Influenza Virus Real-Time RT-
PCR Diagnostic Panel, Influenza B Lineage
Genotyping Kit (VER 1.1 and VER 2)
	The Influenza B Lineage Genotyping Kit contains
reagents and controls of the CDC Human Influenza
Virus Real-Time RT-PCR Diagnostic Panel and is
intended for use in real-time RT-PCR (rRT-PCR)
assays on an Applied Biosystems (ABI) 7500 Fast Dx
Real-Time PCR instrument in conjunction with
clinical and epidemiological information:	Same
	For the determination of the genetic lineage of
human influenza B viruses as B/Victoria or
B/Yamagata lineage from viral RNA in upper
respiratory tract clinical specimens (including
nasopharyngeal swabs [NPS], nasal swabs [NS],
throat swabs [TS], nasal aspirates [NA], nasal
washes [NW] and dual nasopharyngeal/throat
swabs [NPS/TS]) from human patients with
signs and symptoms of respiratory infection
and/or from viral culture; To provide epidemiologic information for
surveillance of circulating influenza viruses.
Intended Use	Performance characteristics for influenza B lineage
genotyping were established during a season when
influenza B/Victoria and B/Yamagata lineages were
found in approximately equal proportion.

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. | |
| | 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 | Influenza B virus, lineages B/Victoria and
B/Yamagata | Same |
| Specimen Types | Nasopharyngeal swabs, nasal swabs, throat swabs,
nasal aspirates, nasal washes and dual
nasopharyngeal/throat swabs from human patients
with signs and symptoms of respiratory infection
and/or from viral culture | Same |
| Technological
Characteristics | Real-time RT-PCR based assay | Same |
| Nucleic Acid
Extraction | 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 QIAcube - QIAamp® DSP Viral RNA Mini Kit, QIAGEN NucliSENS® easyMAG®, bioMérieux EZ1 Advanced XL – EZ1 DSP Virus Kit and EZ1 RNA
Tissue Mini Kit, QIAGEN MagNA Pure 96 - DNA and Viral NA Small Volume Kit,
Roche | Same |
| Enzyme Master
Mix | Invitrogen SuperScript™ III Platinum® One-Step
| Same |
| | Quanta BioSciences qScript™ One-Step qRT-PCR
Kit, Low ROX | |
| 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) [InfB assay]
Black Hole Quencher Plus (BHQ Plus ) [VIC, YAM assays] | ZEN™ or BHQ-1 (InfB assay)
BHQ Plus or BHQ-1 (VIC, YAM assays) |

Table 8-1: Device Comparison

7

VIII. ANALYTICAL PERFORMANCE EVALUATION

Analytical Sensitivity - Influenza B Assay

A range finding study was performed to demonstrate LOD equivalency between the currently cleared InfB assay probe quenched with BHQ and the same probe quenched with ZEN. Two characterized influenza vaccine reference viruses of a known 50% egg infectious dose titer (EID50/mL) were extracted using the Roche MagNA Pure Compact RNA Isolation Kit. The RNA was serially diluted and tested (n=3 replicates) in order to determine the apparent endpoint range using both enzymes cleared for use with the CDC Human Influenza Real-Time RT-PCR Diagnostic Panel. The acceptance criterion for LOD equivalency was defined as demonstrating 100% positivity (3 out of 3 replicates) at either the same endpoint LOD concentration or within a 5-fold dilution of each other. All assays demonstrated similar reactivity (Tables 8-2 and 8-3).

| Influenza
B/Nevada/03/2011
Titer (EID50/mL) | Invitrogen
Superscript™ | | Quanta qScript™ | |
|---------------------------------------------------|----------------------------|------------|-------------------|------------|
| | InfB (BHQ)
IVD | InfB (ZEN) | InfB (BHQ)
IVD | InfB (ZEN) |
| 104.2 | 3/3*
(+) | 3/3
(+) | 3/3
(+) | 3/3
(+) |
| 103.5 | 3/3
(+) | 3/3
(+) | 3/3
(+) | 3/3
(+) |
| 102.8 | 3/3
(+) | 3/3
(+) | 3/3
(+) | 3/3
(+) |
| 102.1 | 1/3
(+) | 1/3
(+) | 0/3
(-) | 0/3
(-) |
| 101.4 | 2/3
(+) | 0/3
(-) | 0/3
(-) | 0/3
(-) |
| 100.7 | 0/3
(-) | 0/3
(-) | 0/3
(-) | 0/3
(-) |
| 100.0 | 0/3
(-) | 0/3
(-) | 0/3
(-) | 0/3
(-) |

Table 8-2: Influenza B (InfB) Assay LOD Equivalencv - B/Nevada/03/2011

Indicates number of positive replicates out of three total.

8

| Influenza
B/Wisconsin/1/2010

Titer (EID50/mL)	Invitrogen Superscript™		Quanta qScript™
	InfB (BHQ)
IVD	InfB (ZEN)	InfB (BHQ)
IVD	InfB (ZEN)
104.2	3/3*
(+)	3/3
(+)	3/3
(+)	3/3
(+)
103.5	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)
102.8	1/3
(+)	0/3
(-)	0/3
(-)	0/3
(-)
102.1	0/3
(-)	0/3
(-)	0/3
(-)	0/3
(-)
101.4	0/3
(-)	0/3
(-)	0/3
(-)	0/3
(-)
100.7	0/3
(-)	0/3
(-)	0/3
(-)	0/3
(-)
100.0	0/3
(-)	0/3
(-)	0/3
(-)	0/3
(-)

Table 8-3: Influenza B (InfB) Assay LOD Equivalency - B/Wisconsin/1/2010

Indicates number of positive replicates out of three total.

Analytical Sensitivity- Influenza B Lineage Genotyping Assay Update

A range finding study was performed with the currently cleared assays of the Influenza B Lineage Genotyping Kit and with modified VIC and YAM assays (VER 1.1) as well as newly designed VIC and Y AM assays (VER 2). These studies were performed to demonstrate the LOD equivalency and improved reactivity of the VER 1.1 and VER 2 VIC and YAM assays with one historical benchmark strain and one current strain of both the B/Victoria and B/Yamagata lineages of influenza B virus. The test samples consisted of serially diluted RNA extracted with the Roche MagNA Pure Compact RNA Isolation Kit from characterized virus stocks of known 50% egg infectious dose titer (EID50mL). Three replicates per dilution were tested to determine the apparent endpoint range (Tables 8-4 to 8-7) using both enzymes cleared for use with the CDC Human Influenza Real-Time RT-PCR Diagnostic Panel. The acceptance criterion for LOD equivalency was defined as demonstrating 100% positivity (3 out of 3 replicates) at either the same endpoint LOD concentration or within a 5-fold dilution of each other. All assays demonstrated similar reactivity with apparent LOD endpoints within one 5-fold dilution of each other.

9

| Influenza
B/Nevada/03/2011

Titer (EID50/mL)	Invitrogen Superscript™				Quanta qScript™
	InfB
IVD	VIC
IVD	VIC
VER 1.1	VIC
VER 2	InfB
IVD	VIC
IVD	VIC
VER 1.1	VIC
VER 2
104.2	3/3*	3/3	3/3	3/3	3/3	3/3	3/3	3/3
	(+)	(+)	(+)	(+)	(+)	(+)	(+)	(+)
103.5	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
	(+)	(+)	(+)	(+)	(+)	(+)	(+)	(+)
102.8	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
	(+)	(+)	(+)	(+)	(+)	(+)	(+)	(+)
102.1	3/3	3/3	3/3	3/3	3/3	3/3	3/3	3/3
	(+)	(+)	(+)	(+)	(+)	(+)	(+)	(+)
101.4	3/3	3/3	3/3	3/3	3/3	0/3	3/3	3/3
	(+)	(+)	(+)	(+)	(+)	(+)	(+)	(+)
100.7	1/3	0/3	2/3	3/3	0/3	0/3	1/3	0/3
	(+)	(-)	(+)	(+)	(-)	(+)	(+)	(-)
100.0	0/3	0/3	0/3	0/3	0/3	0/3	0/3	0/3
	(-)	(-)	(-)	(-)	(-)	(-)	(-)	(-)
10-0.7	0/3	0/3	0/3	0/3	0/3	0/3	0/3	0/3
	(-)	(-)	(-)	(-)	(-)	(-)	(-)	(-)

Table 8-4: B/Victoria Assay LOD Equivalency- VER 1.1 and VER 2 Designs - B/Nevada/03/2011

• Indicates number of positive replicates out of three total.

Table 8-5: B/Victoria Assay LOD Equivalency- VER 1.1 and VER 2 Designs - B/Marryand/15/2016

| Influenza
B/Maryland/15/2016

Titer (EID50/mL)	Invitrogen Superscript™				Quanta qScript™
	InfB
IVD	VIC
IVD	VIC
VER 1.1	VIC
VER 2	InfB
IVD	VIC
IVD	VIC
VER 1.1	VIC
VER 2
104.5	3/3*
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)
103.8	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)
103.1	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)
102.4	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)
101.7	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	2/3
(+)	3/3
(+)	3/3
(+)
101.0	3/3
(+)	0/3
(-)	3/3
(+)	3/3
(+)	0/3
(-)	0/3
(-)	0/3
(-)	0/3
(-)
100.3	1/3
(+)	0/3
(-)	0/3
(-)	0/3
(-)	0/3
(-)	0/3
(-)	0/3
(-)	0/3
(-)
10-0.4	0/3
(-)	0/3
(-)	0/3
(-)	0/3
(-)	0/3
(-)	0/3
(-)	0/3
(-)	0/3
(-)

• Indicates number of positive replicates out of three total.

10

| Influenza
B/Texas/06/2011

Titer (EID50/mL)	Invitrogen Superscript™				Quanta qScript™
	InfB
IVD	YAM
IVD	YAM
VER 1.1	YAM
VER 2	InfB
IVD	YAM
IVD	YAM
VER 1.1	YAM
VER 2
104.9	3/3*
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)
104.2	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)
103.5	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	2/3
(+)	3/3
(+)
102.8	3/3
(+)	3/3
(+)	0/3
(-)	3/3
(+)	3/3
(+)	2/3
(+)	0/3
(-)	3/3
(+)
102.1	2/3
(+)	1/3
(+)	1/3
(+)	1/3
(+)	0/3
(-)	2/3
(+)	0/3
(-)	2/3
(+)
101.4	2/3
(+)	0/3
(-)	0/3
(-)	1/3
(+)	0/3
(-)	1/3
(+)	0/3
(-)	2/3
(+)
100.7	0/3
(-)	0/3
(-)	0/3
(-)	0/3
(-)	0/3
(-)	0/3
(-)	0/3
(-)	0/3
(-)
100.0	0/3
(-)	0/3
(-)	0/3
(-)	0/3
(-)	0/3
(-)	0/3
(-)	0/3
(-)	0/3
(-)

Table 8-6: B/Yamagata Assay LOD Equivalency- VER 1.1 and VER 2 Designs - B/Texas/06/2011

• Indicates number of positive replicates out of three total.

Table 8-7: B/Yamagata Assay LOD Equivalency- VER 1.1 and VER 2 Designs - B/Texas/81/2016

| Influenza
B/Texas/81/2016

Titer (EID50/mL)	Invitrogen SuperscriptTM				Quanta qScriptTM
	InfB
IVD	YAM
IVD	YAM
VER 1.1	YAM
VER 2	InfB
IVD	YAM
IVD	YAM
VER 1.1	YAM
VER 2
104.3	3/3*
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)
103.6	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)
102.9	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)
102.2	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)
101.5	3/3
(+)	2/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	3/3
(+)	1/3
(+)	2/3
(+)
100.8	0/3
(-)	0/3
(-)	0/3
(-)	0/3
(-)	0/3
(-)	1/3
(+)	0/3
(-)	0/3
(-)
100.1	1/3
(+)	0/3
(-)	0/3
(-)	0/3
(-)	0/3
(-)	2/3
(+)	0/3
(-)	0/3
(-)
10-0.6	0/3
(-)	0/3
(-)	0/3
(-)	0/3
(-)	0/3
(-)	0/3
(-)	0/3
(-)	0/3
(-)

• Indicates number of positive replicates out of three total.

11

A confirmation of the LOD for each modified or new assay was performed using a current strain of the corresponding influenza B/Victoria or B/Yamagata lineage. The VER 1.1 and VER 2 VIC and YAM primer and probe sets were tested alongside the currently cleared InfB assay as required for interpretation by the routine testing algorithm. The confirmatory LOD testing for each primer and probe set was performed on extraction replicates (n=20) of each dilution. The lowest virus concentration where InfB and VIC or InfB and YAM primer and probes sets demonstrated ≥ 95% detection is reported as the LOD for each virus (Table 8-8).

Influenza B Lineage	Influenza Strain Designation	Assay Design	LOD (EID50/mL)
			Invitrogen SuperScriptTM	Quanta qScriptTM
Victoria	B/Maryland/15/2016	VER 1.1	101.7	101.7
		VER 2	101.7	101.7
Yamagata	B/Texas/81/2016	VER 1.1	102.2	101.5
		VER 2	102.2	101.5

Table 8-8: LOD Confirmation Summary

Analytical Sensitivity - Inclusivity

Inclusivity testing was performed to demonstrate the capability of the modified VER 1.1 and VER 2 VIC and YAM primer and probe sets to detect strains of the corresponding influenza B lineage at or near the established LOD. Ten influenza B viruses of each lineage and representative of different geographic locations and phylogenetic clades were selected. Characterized stocks were serially diluted to concentrations near the LOD of the assays and extracted using the Roche MagNA Pure Compact RNA Isolation Kit. Samples were tested in triplicate with the VER 2 VIC and YAM assays with both enzyme systems cleared for use with the CDC Human Influenza Real-Time RT-PCR Diagnostic Panel. The inclusivity results are summarized in Tables 8-9 and 8-10.

12

| Influenza Virus
Strain Designation | EID50/mL or
TCID50/mL | Invitrogen
SuperScript™ | | Quanta qScript™ | |
|---------------------------------------|--------------------------|----------------------------|--------------|-----------------|--------------|
| | | VIC
VER 1.1 | VIC
VER 2 | VIC
VER 1.1 | VIC
VER 2 |
| B/Hong Kong/259/2010 | 10 1.2 | 3/3*
(+) | 3/3
(+) | 3/3
(+) | 3/3
(+) |
| B/Bolivia/1526/2010 | 10 2.4 | 3/3
(+) | 3/3
(+) | 3/3
(+) | 3/3
(+) |
| B/Laos/89/2011 | 10 1.2 | 3/3
(+) | 3/3
(+) | 3/3
(+) | 3/3
(+) |
| B/Michigan/09/2011 | 10 1.5 | 3/3
(+) | 3/3
(+) | 3/3
(+) | 3/3
(+) |
| B/New Jersey/01/2012 | 10 1.9 | 3/3
(+) | 3/3
(+) | 3/3
(+) | 3/3
(+) |
| B/Montana/05/2012 | 10 2.4 | 3/3
(+) | 3/3
(+) | 3/3
(+) | 3/3
(+) |
| B/Texas/02/2013 | 10 2.1 | 3/3
(+) | 3/3
(+) | 3/3
(+) | 3/3
(+) |
| B/Florida/103/2016 | 10 0.3 | 3/3
(+) | 3/3
(+) | 3/3
(+) | 3/3
(+) |
| B/Florida/76/2016 | 10 1.5 | 3/3
(+) | 3/3
(+) | 3/3
(+) | 3/3
(+) |
| B/Hong Kong/269/2017 | 10 -0.8 | 3/3
(+) | 3/3
(+) | 3/3
(+) | 3/3
(+) |

Table 8-9: B/Victoria Assay Inclusivity VER 1.1 and VER 2 Designs

• Indicates number of positive replicates out of three total.

13

| Influenza Virus
Strain Designation | EID50 /mL
or
TCID50/mL | Invitrogen
SuperScript™ | | Quanta qScript™ | |
|---------------------------------------|------------------------------|----------------------------|--------------|-----------------|--------------|
| | | YAM
VER 1.1 | YAM
VER 2 | YAM
VER 1.1 | YAM
VER 2 |
| B/Brisbane/03/2007 | 10 2.4 | 3/3*
(+) | 3/3
(+) | 3/3
(+) | 3/3
(+) |
| B/Pennsylvania/07/2007 | 10 2.2 | 3/3
(+) | 3/3
(+) | 3/3
(+) | 3/3
(+) |
| B/Hubei-
Wujiagang/158/2009 | 10 2.2 | 3/3
(+) | 3/3
(+) | 3/3
(+) | 3/3
(+) |
| B/Wisconsin/01/2010 | 10 2.9 | 3/3
(+) | 3/3
(+) | 3/3
(+) | 3/3
(+) |
| B/Finland/39/2010 | 10 2.1 | 3/3
(+) | 3/3
(+) | 3/3
(+) | 3/3
(+) |
| B/Estonia/55669/2011 | 10 2.8 | 3/3
(+) | 3/3
(+) | 3/3
(+) | 3/3
(+) |
| B/Taiwan/1242/2011 | 10 3.9 | 3/3
(+) | 3/3
(+) | 3/3
(+) | 3/3
(+) |
| B/Massachusetts/02/2012 | 10 2.2 | 3/3
(+) | 3/3
(+) | 3/3
(+) | 3/3
(+) |
| B/Phuket/3073/2013 | 10 2.9 | 3/3
(+) | 3/3
(+) | 3/3
(+) | 3/3
(+) |
| B/Guangdong-
Liwan/1133/2014 | 10 3.9 | 3/3
(+) | 3/3
(+) | 3/3
(+) | 3/3
(+) |

Table 8-10: B/Yamagata Assay Inclusivity VER 1.1 and VER 2 Designs

Indicates number of positive replicates out of three total.

Analytical Specificity – Cross-Reactivity

Cross-reactivity of the modified VER 1.1 and VER 2 VIC and YAM primer and probe sets was evaluated by testing each set with influenza B viruses of the opposite lineage and from diverse geographic locations. Samples were extracted from high titer preparations of viruses (≥ 106 EID50 or TCID50/mL) using the Roche MagNA Pure Compact RNA Isolation Kit. Cross-reactivity was evaluated with both enzyme systems cleared for use with the CDC Human Influenza Real-Time RT-PCR Diagnostic Panel. No cross-reactivity was detected with either primer and probe set design (Tables 8-11 and 8-12).

|--|

| Influenza Virus
Strain Designation
B/Yamagata Lineage | EID50/mL
or
TCID50/mL | Invitrogen
SuperScript™ | | Quanta qScript™ | |
|-------------------------------------------------------------|-----------------------------|----------------------------|--------------|-----------------|--------------|
| | | VIC
VER 1.1 | VIC
VER 2 | VIC
VER 1.1 | VIC
VER 2 |
| B/Brisbane/03/2007 | 10 8.2 | - | - | - | - |
| B/Pennsylvania/07/2007 | 10 8.4 | - | - | - | - |
| B/Hubei-Wujiagang/158/2009 | 10 6.2 | - | - | - | - |
| B/Wisconsin/01/2010 | 10 8.5 | - | - | - | - |
| B/Finland/39/2010 | 10 8.9 | - | - | - | - |
| B/Estonia/55669/2011 | 10 8.4 | - | - | - | - |
| B/Taiwan/1242/2011 | 10 9.1 | - | - | - | - |
| B/Massachusetts/02/2012 | 10 6.3 | - | - | - | - |
| B/Phuket/3073/2013 | 10 6.5 | - | - | - | - |
| B/Guangdong-Liwan/1133/2014 | 10 4.2 | - | - | - | - |

14

| Influenza Virus
Strain Designation
B/Victoria Lineage | EID50/mL
or
TCID50/mL | Invitrogen
SuperScript™ | | Quanta
qScript™ | |
|-------------------------------------------------------------|-----------------------------|----------------------------|--------------|--------------------|--------------|
| | | YAM
VER 1.1 | YAM
VER 2 | YAM
VER 1.1 | YAM
VER 2 |
| B/Hong Kong/259/2010 | 10 $8.4$ | - | - | - | - |
| B/Bolivia/1526/2010 | 10 $8.2$ | - | - | - | - |
| B/Laos/89/2011 | 10 $8.2$ | - | - | - | - |
| B/Michigan/09/2011 | 10 $8.9$ | - | - | - | - |
| B/New Jersey/01/2012 | 10 $8.1$ | - | - | - | - |
| B/Montana/05/2012 | 10 $7.8$ | - | - | - | - |
| B/Texas/02/2013 | 10 $9.9$ | - | - | - | - |
| B/Florida/103/2016 | 10 $9.2$ | - | - | - | - |
| B/Florida/76/2016 | 10 $9.9$ | - | - | - | - |
| B/Hong Kong/269/2017 | 10 $9.9$ | - | - | - | - |

Table 8-12: B/Yamagata Assay Cross-Reactivity VER 1.1 and VER 2 Designs

Analytical Specificity - Exclusivity

Exclusivity with Influenza A Viruses

Exclusivity of the VER 1.1 and VER 2 VIC and YAM assays was examined with influenza A viruses of various subtypes that circulate in humans and from animal origin that infect humans (Table 8-13 and 8-14). Samples were prepared from characterized, high titer stocks (≥106 TCID50 or EID50/mL) by extracting RNA using the Roche MagNA Pure Compact RNA Isolation Kit. Testing was performed using both of the currently cleared enzyme systems.

	Table 8-13: Exclusivity with Influenza A Viruses- VER 1.1 and VER 2 -Invitrogen SuperScript™

| Influenza Virus
Strain Designation | Origin | Subtype | EID50/mL
or
TCID50/mL | Invitrogen SuperScript™ | | | |
|----------------------------------------------|--------|------------------|-----------------------------|-------------------------|--------------|----------------|--------------|
| | | | | VIC
VER 1.1 | VIC
VER 2 | YAM
VER 1.1 | YAM
VER 2 |
| A/Michigan/45/2015 | Human | A(H1N1)
pdm09 | $10^{8.3}$ | - | - | - | - |
| A/Hong Kong/4801/2014 | Human | A(H3N2) | $10^{7.9}$ | - | - | - | - |
| A/Ohio/35/2017 | Swine | A(H1N2)v | $10^{6.9}$ | - | - | - | - |
| A/Ohio/13/2017 | Swine | A(H3N2)v | $10^{6.9}$ | - | - | - | - |
| A/gyrfalcon/
Washington/
41088-6/2014 | Avian | A(H5N8) | $10^{9.75}$ | - | - | - | - |
| A/Northern pintail/Washington/
40964/2014 | Avian | A(H5N2) | $10^{9.4}$ | - | - | - | - |
| A/Bangladesh/
0994/2011 | Avian | A(H9N2) | $10^{10.5}$ | - | - | - | - |
| A/Anhui/01/2013 | Avian | A(H7N9) | $10^{10.9}$ | - | - | - | - |

15

| Influenza Virus
Strain Designation | Origin | Subtype | EID50/mL
or
TCID50/mL | Quanta qScript™ | | | |
|----------------------------------------------|--------|------------------|-----------------------------|-----------------|--------------|----------------|--------------|
| | | | | VIC
VER 1.1 | VIC
VER 2 | YAM
VER 1.1 | YAM
VER 2 |
| A/Michigan/45/
2015 | Human | A(H1N1)
pdm09 | 108.3 | - | - | - | - |
| A/Hong Kong/4801/2014 | Human | A(H3N2) | 107.9 | - | - | - | - |
| A/Ohio/35/2017 | Swine | A(H1N2)v | 106.9 | - | - | - | - |
| A/Ohio/13/2017 | Swine | A(H3N2)v | 106.9 | - | - | - | - |
| A/gyrfalcon/
Washington/
41088-6/2014 | Avian | A(H5N8) | 109.75 | - | - | - | - |
| A/Northern pintail/Washington/
40964/2014 | Avian | A(H5N2) | 109.4 | - | - | - | - |
| A/Bangladesh/
0994/2011 | Avian | A(H9N2) | 1010.5 | - | - | - | - |
| A/Anhui/01/2013 | Avian | A(H7N9) | 1010.9 | - | - | - | - |

Table 8-14: Exclusivity with Influenza A Viruses- VER 1.1 and VER 2 -Quanta qScript™

Exclusivity with Non-Influenza Respiratory Pathogens

Exclusivity of the VER 2 VIC and YAM assays was examined with non-influenza human respiratory viruses, bacteria, and yeast (Table 8-15). Nucleic acids were extracted using the Roche MagNA Pure Compact RNA Isolation Kit from 36 organisms (16 viruses, 19 bacteria, and 1 yeast) representing common respiratory pathogens or flora commonly present in specimens collected from the nasopharynx region. All bacteria, yeast, and non-influenza viruses were from CDC repositories or acquired from American Type Culture Collection (ATCC, Manassas, VA) and tested at high titers, typically ≥ 100 TCID50 or EID50/mL, ≥ 10° CFU/mL, or as high as culture allowed. Testing was performed using both of the currently cleared enzyme systems.

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Organism Tested			SuperScript		qScript
Bacteria and Yeast	Strain	cfu / mL	VIC
VER2	YAM
VER2	VIC
VER2	YAM
VER2
Bordetella pertussis	A639	10 8.3	-	-	-	-
Candida albicans	2001-21-196	10 8.8	-	-	-	-
Chlamydia pneumoniae1	TW183	40 IFU/mL	-	-	-	-
Corynebacterium diphtheriae	NA2	10 10	-	-	-	-
Escherichia coli	K12	10 9.6	-	-	-	-
Haemophilus influenzae	M15709	10 6.4	-	-	-	-
Lactobacillus plantarum	NA	10 8.8	-	-	-	-
Legionella pneumophila	NA	10 10.3	-	-	-	-
Moraxella catarrhalis	M15757	10 9.5	-	-	-	-
Mycobacterium tuberculosis3	H37Rv	95 ng/ µL	-	-	-	-
Mycoplasma pneumoniae	MI-29	10 7.7	-	-	-	-
Neisseria elongata	NA	10 8.6	-	-	-	-
Neisseria meningitidis	M2578	10 7.9	-	-	-	-
Pseudomonas aeruginosa	NA	10 10.5	-	-	-	-
Staphylococcus epidermidis	NA	10 10.5	-	-	-	-
Staphylococcus aureus	NA	10 10.7	-	-	-	-
Streptococcus pneumoniae	249-06
(Thailand)	10 6.6	-	-	-	-
Streptococcus pyogenes	7790-06	10 7.5	-	-	-	-
Streptococcus salivarius	SS1672	10 8.4	-	-	-	-
Viruses	Strain	EID50/mL
or
TCID50/mL	VIC
VER2	YAM
VER2	VIC
VER2	YAM
VER2
Enterovirus	Echo 6	10 6.9	-	-	-	-
Human Adenovirus, type 1	Ad.71	10 9.2	-	-	-	-
Human Adenovirus, type 7a	S-1058	10 7.1	-	-	-	-
Human Coronavirus virus3	OC43	50.4 ng/µL	-	-	-	-
Human Coronavirus virus3	299E	31.6 ng/µL	-	-	-	-
Human Rhinovirus A	1A	10 5.8	-	-	-	-
Human Parainfluenza 1 virus3	NA	3.0 ng/ µL	-	-	-	-
Human Parainfluenza 2 virus	Greer	10 3.1	-	-	-	-
Human Parainfluenza 3 virus	C-243	10 7.9	-	-	-	-
Respiratory Syncytial virus	CH93-18b	10 6.8	-	-	-	-
Herpes Simplex Virus	KOS	10 8.4	-	-	-	-
Varicella-zoster Virus	AV92-3	10 4.4	-	-	-	-
Epstein Barr Virus3	B95-8	1.7 ng/µL	-	-	-	-
Measles Virus	Edmonston	10 5.2	-	-	-	-
Mumps Virus	Enders	10 7.2	-	-	-	-
Cytomegalovirus	AD-169	10 6.9	-	-	-	-

Table 8-15: Assay Exclusivity with Respiratory Viruses, Bacteria, and Yeast- VER 2 Design

1 Organism quantified by Infectious Forming Units (IFU)

² NA = not applicable

3 Organism quantified by spectrophotometry (ng/μL)

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CLINICAL PERFORMANCE EVALUATION IX.

Influenza B Assay -Retrospective Study

The clinical performance of the InfB oligonucleotide probe quenched with ZEN was evaluated using retrospective clinical samples collected during the 2011-2012 influenza season that were previously determined to be positive or negative for influenza B virus. A total of 30 positive and 50 negative upper respiratory tract samples were evaluated with the cleared InfB assay using either the existing oligonucleotide probe quenched with BHQ or the investigational probe quenched with ZEN. Testing was performed with both enzymes cleared for use with the CDC Human Influenza Real-Time RT-PCR Diagnostic Panel and one of the currently cleared extraction methods. Result interpretation followed the instructions of the Package Insert for the cleared Influenza B Lineage Genotyping Kit. The InfB assay containing the investigational probe quenched with ZEN demonstrated 100% positive and negative agreement with the cleared oligonucleotide probe quenched with BHQ (Tables 8-16 and 8-17).

		Invitrogen SuperScript™	Quanta qScript™
Specimen Type	# of
Positives1	% Positive Agreement
(95% CI)	# of
Positives1	% Positive Agreement
(95% CI)
NPS, NS	18/18	100.0 (82.4-100.0)	18/18	100.0 (82.4-100.0)
NPS/TS	11/11	100.0 (74.1-100.0)	11/11	100.0 (74.1-100.0)
NW	1/1	100.0 (20.7-100.0)	1/1	100.0 (20.7-100.0)
Total	30/30	100.0 (88.7-100.0)	30/30	100.0 (88.7-100.0)

Table 8-16: InfB Assay-Retrospective Positive Clinical Study Results

1 Proportion of positive samples correctly identified versus the comparator.

Table 8-17: InfB Assay-Retrospective Negative Clinical Study Results

	Invitrogen SuperScript™		Quanta qScript™
Specimen Type	# of
Negatives1	% Negative Agreement
(95% CI)	# of
Negatives1	% Negative Agreement
(95% CI)
NPS, NS	48/48	100.0 (92.6-100.0)	48/48	100.0 (92.6-100.0)
TS	2/2	100.0 (34.2-100.0)	2/2	100.0 (34.2-100.0)
Total	50/50	100.0 (92.9-100.0)	50/50	100.0 (92.9-100.0)

1 Proportion of negative samples correctly identified versus the comparator.

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Influenza B Lineage Genotyping Kit (VER 2) - Prospective Study

A prospective clinical investigation was conducted at 3 U.S. public health laboratories using upper respiratory tract specimens collected during the 2016-2017 influenza season. Samples were taken from specimens collected for routine influenza testing at each site from individuals symptomatic for influenza-like illness. The range of patient ages and specimen types for the total of 592 samples collected are represented in Table 8-18. A total of 13 were excluded for reasons of unspecified specimen type, inconclusive result of the comparator, or technician testing error. A total of 579 upper respiratory tract specimens were included in the data analysis.

Patient Age	Totals
0-16	149
17-54	192
≥55	249
Not Reported	2
Specimen Type1	Totals
NPS	374
NPS/TS	44
NA	12
NW	34
TS	13
NS	114
Not Reported	1

Table 8-18: Specimen Information

1 NPS=nasopharyngeal swab, NPS/TS=dual nasopharyngeal and throat swab, NA=nasal aspirate, NW=nasal wash, TS=throat swab, NS=nasal swab

Specimens were tested with assays from the FDA-cleared CDC Human Influenza Real-Time RT-PCR Diagnostic Panel and the investigational Influenza B Lineage Genotyping Kit (VER 2). The performance is summarized in Tables 8-19 and 8-20. Due to the low prevalence of influenza B/Victoria lineage viruses in specimens collected during the prospective study, the performance of the Influenza B Lineage Genotyping Kit (VER 2) was also evaluated in a retrospective study.

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| Specimen Type1 | # of
Positives2 | % Sensitivity (95% CI) | # of Negatives3 | % Specificity (95%
CI) |
|----------------|--------------------|------------------------|-----------------|---------------------------|
| NPS, NS | 5/5 | 100.0 (56.6 – 100.0) | 472/472 | 100.0 (99.2 - 100.0) |
| NPS/TS | 0/0 | NA4 | 44/44 | 100.0 (92.0 - 100.0) |
| TS | 0/0 | NA | 13/13 | 100.0 (77.2 – 100.0) |
| NA, NW | 0/0 | NA | 45/45 | 100.0 (92.1 – 100.0) |
| Total5 | 5/5 | 100.0 (56.6 – 100.0) | 574/574 | 100.0 (99.3 – 100.0) |

Table 8-19: B/Victoria Assay - VER 2 Design- Prospective Study Results

1 NPS=nasopharyngeal swab. NPS/TS=dual nasopharyngeal and throat swab. NA=nasal wash. TS=throat swab, NS=nasal swab

2Proportion of positive samples correctly identified versus the comparator.

3 Proportion of negative samples correctly identified versus the comparator.

4NA=not applicable

| Specimen Type1 | # of
Positives2 | % Sensitivity (95% CI) | # of Negatives3 | % Specificity (95%
CI) |
|----------------|--------------------|------------------------|-----------------|---------------------------|
| NPS, NS | 31/31 | 100.0 (89.0 – 100.0) | 446/446 | 100.0 (99.1 - 100.0) |
| NPS/TS | 2/2 | 100.0 (34.2 – 100.0) | 42/42 | 100.0 (91.6 - 100.0) |
| TS | 0/0 | NA4 | 13/13 | 100.0 (77.2 - 100.0) |
| NA, NW | 5/5 | 100.0 (56.6 - 100.0) | 39/40 | 97.5 (87.1 - 99.6) |
| Total5 | 38/38 | 100.0 (90.8 - 100.0) | 540/541 | 99.8 (99.0 - 100.0) |

Table 8-20: B/Yamagata Assay - VER 2 Design - Prospective Study Results

1 NPS=nasopharyngeal swab, NPS/TS=dual nasopharyngeal and throat swab, NA=nasal wash, TS=throat swab, NS=nasal swab

2Proportion of positive samples correctly identified versus the comparator.

3 Proportion of negative samples correctly identified versus the comparator.

4NA=not applicable

Influenza B Lineage Genotyping Kit VER 1.1 and VER 2 - Retrospective Study Results

A retrospective study was performed using the VER 1.1 and YER 2 VIC and Y AM assays to evaluate their clinical performance. Upper respiratory tract clinical samples were collected during the 2017 and 2017-2018 influenza seasons and determined to be positive for influenza B/Victoria or B/Yamagata viruses using the FDA-cleared Influenza B Lineage Genotyping Kit. In the current study, specimens were tested using the cleared InfB assay and the investigative VER 1.1 and VER 2 VIC and Y AM assays. A total of 126 specimens positive for either influenza B/Victoria or influenza B/Yamagata viruses and 61 specimens negative for influenza B viruses were tested. Four lung tissue specimens were included in the testing, but were used for informational purposes only and not included in calculations of assay performance (Tables 8-21 to 8-24). Result interpretation followed the instructions of the Package Insert for the cleared Influenza B Lineage Genotyping Kit.

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| Specimen Type | # of
Positives1 | % Positive Agreement
(95% CI) | # of
Negatives2 | % Negative Agreement
(95% CI) |
|---------------|--------------------|----------------------------------|--------------------|----------------------------------|
| NPS, NS | 29/29 | 100.0 (88.3 - 100.0) | 156/156 | 100.0 (97.6 - 100.0) |
| TS | 1/1 | 100.0 (20.7 - 100.0) | 1/1 | 100.0 (20.7 - 100.0) |
| Total | 30/30 | 100.0 (88.7 - 100.0) | 157/157 | 100.0 (97.6 - 100.0) |

Table 8-21: VIC VER 1.1 Design-Retrospective Study Results

1Proportion of positive samples correctly identified versus the comparator.

2Proportion of negative samples correctly identified versus the comparator.

| Specimen Type | # of
Positives1 | % Positive Agreement
(95% CI) | # of
Negatives2 | % Negative Agreement
(95% CI) |
|---------------|--------------------|----------------------------------|--------------------|----------------------------------|
| NPS, NS | 95/95 | 100.0 (96.1 – 100.0) | 90/90 | 100.0 (95.9 – 100.0) |
| TS | 1/1 | 100.0 (20.7 - 100.0) | 1/1 | 100.0 (20.7 - 100.0) |
| Total | 96/96 | 100.0 (96.2 - 100.0) | 91/91 | 100.0 (96.0 – 100.0) |

Table 8-22: YAM VER 1.1 Design-Retrospective Study Results

1Proportion of positive samples correctly identified versus the comparator.

2Proportion of negative samples correctly identified versus the comparator.

| Specimen Type | # of
Positives1 | % Positive Agreement
(95% CI) | # of
Negatives2 | % Negative Agreement
(95% CI) |
|---------------|--------------------|----------------------------------|--------------------|----------------------------------|
| NPS, NS | 29/29 | 100.0 (88.3 – 100.0) | 156/156 | 100.0 (97.6 – 100.0) |
| TS | 1/1 | 100.0 (20.7 - 100.0) | 1/1 | 100.0 (20.7 - 100.0) |
| Total | 30/30 | 100.0 (88.7 – 100.0) | 157/157 | 100.0 (97.6 – 100.0) |

Table 8-23: VIC VER 2 Design-Retrospective Study Results

1Proportion of positive samples correctly identified versus the comparator.

2Proportion of negative samples correctly identified versus the comparator.

| Specimen Type | # of
Positives1 | % Positive Agreement
(95% CI) | # of
Negatives2 | % Negative Agreement
(95% CI) |
|---------------|--------------------|----------------------------------|--------------------|----------------------------------|
| NPS, NS | 95/95 | 100.0 (96.1 – 100.0) | 90/90 | 100.0 (95.9 – 100.0) |
| TS | 1/1 | 100.0 (20.7 - 100.0) | 1/1 | 100.0 (20.7 - 100.0) |
| Total | 96/96 | 100.0 (96.2 - 100.0) | 91/91 | 100.0 (96.0 – 100.0) |

Table 8-24: YAM VER 2 Design-Retrospective Study Results

1Proportion of positive samples correctly identified versus the comparator.

2Proportion of negative samples correctly identified versus the comparator.

X. CONCLUSION

The modification of the Influenza B Lineage Genotyping Kit of the CDC Human Influenza Virus rRT-PCR Diagnostic Panel to ensure comprehensive detection of influenza B viruses does not

21

change the fundamental scientific technology of the device. Analytical and clinical data demonstrate that the performance of the modified device to detect and characterize influenza B viruses is accomplished with high positive and negative percent agreement in a manner substantially equivalent to the predicate. The indications for use remain the same.