The Influenza A/B Typing Kit contains reagents and controls of the CDC Human Influenza Virus RT-PCR Diagnostic Panel and is intended for use in real-time RT-PCR (rRT-PCR) assays on an in vitro diagnostic real-time PCR instrument that has been FDA-cleared for use with this kit in conjunction with clinical and evidemiological information:

· For qualitative detection of influenza virus type A or B viral RNA in upper respiratory tract clinical specimens (including nasopharyngeal swabs [NPS], throat swabs [TS], nasal aspirates [NA], nasal washes [NW and dual nasopharyngeal/throat swabs [NPS/TS]) and lower respiratory tract specimens (including bronchoalveolar lavage [BAL], bronchial wash [BW], tracheal aspirate [TA], sputum, and lung tissue) from human patients with signs and symptoms of respiratory infection and/or from viral culture.

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

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

The Influenza A Subtyping Kit contains reagents and controls of the CDC Human Influenza Virus RT-PCR Diagnostic Panel and is intended for use in real-time RT-PCR (rRT-PCR) assays on an in vitro diagnostic real-time PCR instrument that has been FDA-cleared for use with this kit in conjunction with clinical and epidemiological information:

· For determination of the subtype of seasonal human influenza A viruses as seasonal A(H3) and/or A(H1)pdm09 from viral RNA in upper respiratory tract clinical specimens (including nasopharyngeal swabs [NS], throat swabs [TS], nasal aspirates [NA], nasal washes [NW] and dual nasopharyngeal/throat swabs [NPS/TS]) and lower respiratory tract specimens (including bronchoalveolar lavage [BAL], bronchial wash [BW], tracheal aspirate [TA], sputum, and lung tissue) from human patients with signs and symptoms of respiratory infection and/or from viral culture:

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

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

The Influenza B Lineage Genotyping Kit contains reagents and controls of the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel and is intended for use in real-time RT-PCR (rRT-PCR) assays on an in vitro diagnostic realtime PCR instrument that has been FDA-cleared for use with this kit in conjunction with clinical and epidemiological information:

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

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

CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel: Influenza A/H5 Subtyping Kit (VER 4)

The Influenza A/H5 Subtyping Kit contains reagents and controls of the CDC Human Influenza Virus RT-PCR Diagnostic Panel and is intended for use in real-time RT-PCR (rRT-PCR) assays on an in vitro diagnostic real-time PCR instrument that has been FDA-cleared for use with this kit in conjunction with clinical and epidemiological information:

· For the presumptive identification of virus in patients who may be infected with influenza A subtype A(H5) (Asian lineage) from viral RNA in human respiratory specimens, conjunctival swabs, and viral culture in conjunction with clinical and epidemiological risk factors;

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

Device Description

The CDC Human Influenza Real-Time RT-PCR Diagnostic Panel consists of four real-time RT-PCR (rRT-PCR) assays used on IVD-labeled real-time PCR instruments that has been FDA-cleared for use with this device. The panel is configured in four separate kits: Influenza A/B Typing kit, Influenza A/H5 Subtyping kit, and Influenza B Genotyping kit. Each kit consists of oligonucleotide primers, fluorescently labeled hydrolysis probes, and controls which are used in rRT-PCR assays for the in vitro qualitative detection of influenza virus RNA in respiratory and conjunctival specimens from patients presenting with influenza-like illness (IL). Oligonucleotide primers and probes for detection of influenza A, influenza B, and 2009 influenza A (swine origin) were selected from highly conserved regions of the matrix (M), non-structural (NS), and nucleoprotein (NP) genes, respectively. Oligonucleotide primers and probes for characterization and differentiation of influenza A(H3) and A(H1)pdm09 viruses, genetic lineages of influenza A(H5) viruses were selected from highly conserved regions of their HA genes. Oligonucleotide primers and probes to detect the human RNase P gene (RP) in control samples and clinical specimens are also included in the panel.

AI/ML Overview

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

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

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

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

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

Performance Measure	Value/Description	Context/Details
Limit of Detection (LoD)	10^-5 EID50/ml	Confirmed for conjunctival swab specimens.
Interfering Substances	No interference observed	Tested with two common over-the-counter eyedrops (containing olopatadine hydrochloride and naphazoline hydrochloride respectively). Tested at 2x LoD in contrived specimens (conjunctival matrix and simulated respiratory matrix) and by adding directly to RT-PCR reaction.
Clinical Performance (on Confirmed Cases with Conjunctivitis Symptoms)	26/33 (78.8%) positive, 3/33 (9.1%) inconclusive	For A/H5 on Conjunctival Swab (CS) specimens.
Clinical Performance (on Confirmed Cases with Conjunctivities but URT Positive Only)	4/33 (15.2%) positive	For A/H5 on Upper Respiratory Tract (URT) specimens only.
Clinical Performance (on Confirmed Cases without Conjunctivitis Symptoms)	1/2 (50%) positive on CS, 1/2 (50%) positive on URT only	One case had CS sample, other had URT only.
Clinical Performance (on Probable Cases with Conjunctivitis Symptoms)	2/2 (100%) negative on CS
Subject Matched Paired URT and CS Specimens (Confirmed Cases)	10/32 (31.3%) positive on at least one URT and CS specimens
	15/32 (46.9%) positive in CS specimens only
	4/32 (12.5%) positive in at least one URT specimen only
	3/32 (9.4%) CS inconclusive, URT negative
Subject Matched Paired URT and CS Specimens (Probable Cases)	All URT and CS specimens tested negative	From 1 probable human case.

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

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

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

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

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

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

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

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

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

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

7. The type of ground truth used:

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

8. The sample size for the training set:

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

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

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

Summary

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Image /page/0/Picture/0 description: The image contains the logos of the U.S. Department of Health & Human Services and the U.S. Food & Drug Administration (FDA). The Department of Health & Human Services logo is on the left, and the FDA logo is on the right. The FDA logo is a blue square with the letters "FDA" in white, followed by the words "U.S. Food & Drug Administration" in blue.

March 14, 2025

Centers for Disease Control and Prevention Melissa Ivev Regulatory Affairs Specialist 1600 Clifton Rd Atlanta, Georgia 30329

Re: K243931

Trade/Device Name: CDC Human Influenza Virus Real-time RT-PCR Diagnostic Panel: Influenza A/B Typing Kit (VER 2); Influenza A Subtyping Kit (VER 4); Influenza B Lineage Genotyping Kit (VER 1.1 and 2); and Influenza A/H5 Subtyping Kit (VER 4) Regulation Number: 21 CFR 866.3980 Regulation Name: Respiratory Viral Panel Multiplex Nucleic Acid Assay Regulatory Class: Class II Product Code: OZE Dated: December 16, 2024 Received: December 20, 2024

Dear Melissa Ivey:

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 (the 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. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database available at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. 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.

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Additional information about changes that may require a new premarket notification are provided in the FDA guidance documents entitled "Deciding When to Submit a 510(k) for a Change to an Existing Device" (https://www.fda.gov/media/99812/download) and "Deciding When to Submit a 510(k) for a Software Change to an Existing Device" (https://www.fda.gov/media/99785/download).

Your device is also subject to, among other requirements, the Quality System (OS) regulation (21 CFR Part 820), which includes, but is not limited to, 21 CFR 820.30, Design controls; 21 CFR 820.90, Nonconforming product; and 21 CFR 820.100, Corrective and preventive action. Please note that regardless of whether a change requires premarket review, the QS regulation requires device manufacturers to review and approve changes to device design and production (21 CFR 820.30 and 21 CFR 820.70) and document changes and approvals in the device master record (21 CFR 820.181).

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 Part 803) for devices or postmarketing safety reporting (21 CFR Part 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safetyreporting-combination-products); good manufacturing practice requirements as set forth in the quality systems (OS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR Part 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR Parts 1000-1050.

All medical devices, including Class I and unclassified devices and combination product device constituent parts are required to be in compliance with the final Unique Device Identification System rule ("UDI Rule"). The UDI Rule requires, among other things, that a device bear a unique device identifier (UDI) on its label and package (21 CFR 801.20(a)) unless an exception or alternative applies (21 CFR 801.20(b)) and that the dates on the device label be formatted in accordance with 21 CFR 801.18. The UDI Rule (21 CFR 830.300(a) and 830.320(b)) also requires that certain information be submitted to the Global Unique Device Identification Database (GUDID) (21 CFR Part 830 Subpart E). For additional information on these requirements, please see the UDI System webpage at https://www.fda.gov/medical-device-advicecomprehensive-regulatory-assistance/unique-device-identification-system-udi-system.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about medical devices and radiation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). 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 (https://www.fda.gov/medical-device-advice-comprehensive-regulatory

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assistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely,

Anna M. Mielech -S

Anna Mielech, PhD. Deputy Branch Chief (Acting) Viral Respiratory and HPV Branch Division of Microbiology Devices OHT7: Office of In Vitro Diagnostics Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K243931

Device Name

CDC Human Influenza Real-Time RT-PCR Diagnostic Panel:Influenza A/B Typing Kit (VER 2), Influenza A Subtyping Kit (VER 4), Influenza B Lineage Genotyping Kit (VER 1.1 and 2), and Influenza A/H5 Subtyping Kit (VER 4)

Indications for Use (Describe)

CDC Human Influenza Real-Time RT-PCR Diagnostic Panel: Influenza A/B Typing Kit (VER 2)

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

Performance characteristics for influenza were established during a seasonal influenza viruses A(H1N1) 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.

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

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respiratory tract specimens (including bronchoalveolar lavage [BAL], bronchial wash [BW], tracheal aspirate [TA], sputum, and lung tissue) from human patients with signs and symptoms of respiratory infection and/or from viral culture:

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

Performance characteristics for influenza were established during a 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 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.

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

· 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.

CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel: Influenza A/H5 Subtyping Kit (VER 4)

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

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

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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510(k) Summary

I. GENERAL INFORMATION

Submitter

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

Contact Person

Marie Kirby Lead, Genomics and Diagnostics Team Virology, Surveillance and Diagnostic Branch, Influenza Division National Center for Immunization and Respiratory Diseases Centers for Disease Control and Prevention mkirby@cdc.gov (404)718-7689

Date Prepared: December 16, 2024

II. DEVICE INFORMATION

Proprietary Name:	CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel: Influenza A/B Typing Kit(VER 2), Influenza A Subtyping Kit (VER 4), Influenza B Lineage Genotyping Kit (VER 1.1 and2), and Influenza A/H5 Subtyping Kit (VER 4)
Common Name:	CDC Flu rRT-PCR Dx Panel: Influenza A/B Typing Kit, Influenza A Subtyping Kit, Influenza BLineage Genotyping Kit, and Influenza A/H5 Subtyping Kit
Regulation Section:	866.3980-Respiratory viral panel multiplex nucleic acid assay

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Device Classification: Class II

Product Code: OZE

Panel: Microbiology

PREDICATE DEVICES III.

K 190302 - CDC Human Influenza Virus Real-time RT-PCR Diagnostic Panel: Influenza A Subtyping Kit (VER 2), Influenza B Lineage Genotyping Kit (VER 1.1 and 2), and Influenza A/H5 Subtyping Kit (VER 3)

DEVICE DESCRIPTION IV.

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V. INTENDED USE

Influenza A/B Typing Kit (VER 2)

The Influenza A/B Typing Kit contains reagents and controls of the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel and is intended for use in real-time RT-PCR) assays on an in vitro diagnostic real-ime PCR instrument that has been FDA-cleared for use with this kit in conjunction with clinical and epidemiological information:

· For qualitative detection of influenza virus type A or B viral RNA in upper respiratory tract clinical specimens (including nasopharyngeal swabs [NPS], nasal swabs [TS], throat swabs [TS], nasal washes [NW] and dual nasopharyneal/throat swabs [NPSTS]) and lower respiratory tract specimens (including bronchoalveolar lavage [BAL], bronchial wash [BW], tracheal aspirate [TA], sputum, and lung tissue) from human patients with signs and symptoms of respiratory infection and/or from viral culture.

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

Performance characteristics for influenza were established during a season when seasonal influenza viruses A(HIN) and A(H3N2) were the predominant influenza A viruses in circulation and during a season when the A(H1N) 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 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 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.

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Influenza A Subtvping Kit (VER 4)

The Influenza A Subtying Kit contains reagents and controls of the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel and is intended for use in real-time RT-PCR) assays on an in vitro diagnostic real-time PCR instrument that has been FDAcleared for use with this kit in conjunction with clinical and epidemiological information:

• For determination of the subtype of seasonal human influenza A viruses as seasonal A(H3) and/or A(H1) pdm09 from viral RNA in upper respiratory tract clinical specimens (including nasopharyngeal swabs [NS], throat swabs [TS], nasal aspirates [NA], nasal washes [NW] and dual nasopharyngeal/throat swabs [NPS/TS]) and lower respiratory tract specimens (including bronchoalveolar lavage [BAL], bronchial wash [BW], tracheal aspirate [TA], sputum, and lung tissue) from human patients with signs and symptoms of respiratory infection and/or from viral culture;

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

Performance characteristics for influenza were established during a season influenza viruses A(HIN) and A(H3N2) were the predominant influenza A viruses in circulation and during a season when the A(H1N) 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 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 epideria recommended by public health authorities, specimens should be collected with appropriate infection 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.

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Influenza B Genotyping Kit (VER 1.1 and 2)

The Influenza B Lineage Genotyping Kit contains reagents and controls of the CDC Human Influenza Virus RT-PCR Diagnostic Panel and is intended for use in real-time RT-PCR) assays on an in vitro diagnostic real-time PCR instrument that has been FDA-cleared for use with this kit in conjunction with clinical and epidemiological information:

• For the determination of the genetic lineage of human influenza B viruses as B/Victoria or B/Y amagata 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 respuntory 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.

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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Influenza A/H5 Subtyping Kit (VER 4)

The Influenza A/H5 Subtyping Kit contains reagents and controls of the Centers for Disease Control and Prevention (CDC) Human Influenza Virus Real-Time RT-PCR Diagnostic Panel and is intended for use in real-time RT-PCR (rRT-PCR) assays on an in vitro diagnostic real-time PCR instrument that has been FDA-cleared for use with this kit in conjunction with clinical and epidemiological information:

· For the presumptive identification of virus in patients who mfluenza A subtype A(HS) (Asian lineage) from viral RNA in human respiratory specimens, conjunctival swabs and viral culture in conjunction with clinical and epidemiological risk factors;

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

Testing with the influenza H5a and H56 primer and probe sets should not be patient meets the most current U.S. Department of Health and Human Services (DHHS) clinical and epidemiologic criteria for testing suspect A(HS) specimens. The definitive identification of influenza A(H5) (Asian linectly from patient specimens or from virus cultures requires additional laboratory testing, along with clinical assessment in consultation with national influenza survellance experts.

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 infections 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 facilable to receive and culture specimens.

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

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VI. TECHNOLOGICAL CHARACTERISTICS

The technological characteristics of the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel remains the same as the predicate device.

VII. SUBSTANTIAL EQUIVALENCE COMPARISON

	CDC Human Influenza Virus Real-Time RT-PCRDiagnostic Panel: Influenza A/B Typing Kit(K190302)	CDC Human Influenza Virus Real-Time RT-PCRDiagnostic Panel: Influenza A/B Typing Kit (VER 2)(K243931)
General DeviceCharacteristicSimilarities
Intended Use	The Influenza A/B Typing Kit contains reagents andcontrols of the CDC Human Influenza Virus Real-TimeRT-PCR Diagnostic Panel and is intended for use in real-time RT-PCR (rRT-PCR) assays on an in vitro diagnosticreal-time PCR instrument that has been FDA-cleared foruse with this kit in conjunction with clinical andepidemiological information:For qualitative detection of influenza virus type A or Bviral RNA in upper respiratory tract clinical specimens(including nasopharyngeal swabs [NPS], nasal swabs[NS], throat swabs [TS], nasal aspirates [NA], nasalwashes [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 ofrespiratory infection and/or from viral culture.To provide epidemiological information for surveillance	Same

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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. [f 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. [black box: 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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Specimen Types	Upper respiratory tract clinical specimens (includingnasopharyngeal swabs [NPS], nasal swabs [NS], throatswabs [TS], nasal aspirates [NA], nasal washes [NW] anddual nasopharyngeal/throat swabs [NPS/TS]) and lowerrespiratory tract specimens (including bronchoalveolarlavage [BAL], bronchial wash [BW], tracheal aspirate[TA], sputum, and lung tissue), and/or viral culture	Same
OrganismsDetected	Influenza A and Influenza B	Same
Analytes	RNA- InfA, InfB, RP	Same
TechnologicalPrinciples	Real-time RT-PCR	Same
Instrumentation	Applied Biosystems 7500 Fast Dx Real-time PCR systemwith SDS software version 1.4Applied Biosystems QuantStudio Dx with version 1.0.3softwareQIAGEN Rotor-Gene Q MDx with AssayManager1.0.4.1 and Epsilon version 1.0.1 software	Same
Enzyme MasterMix	Invitrogen SuperScript III Platinum One-StepQuantitative RT-PCR Kit (with or without ROX)Quanta BioSciences qScript One-Step qRT PCR Kit, LowROX	Same
Nucleic AcidExtraction	QIAamp DSP Viral RNA Mini Kit, QIAGENMagNA Pure Compact - Nucleic Acid Isolation Kit I,RocheMagNA Pure Compact – RNA Isolation Kit, Roche	Same

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	MagNA Pure LC – Total Nucleic Acid Kit, RocheMagNA Pure 96 - DNA and Viral NA Small Volume Kit,Roche
	QIAcube - QIAamp DSP Viral RNA Mini Kit, QIAGEN
	NucliSENS easyMAG, bioMérieuxEMAG, bioMérieux*
	EZ1 Advanced XL - EZ1 DSP Virus Kit and EZ1 RNATissue Mini Kit, QIAGEN
	CDC Human Influenza Virus Real-Time RT-PCRDiagnostic Panel: Influenza A Subtyping Kit (VER 2)(K190302)	CDC Human Influenza Virus Real-Time RT-PCRDiagnostic Panel: Influenza A Subtyping Kit (VER 4)(K243931)
General DeviceCharacteristicSimilarities
	The Influenza A Subtyping Kit contains reagents and controls ofthe CDC Human Influenza Virus Real-Time RT-PCR DiagnosticPanel and is intended for use in real-time RT-PCR (rRT-PCR)assays on an in vitro diagnostic real-time PCR instrument that hasbeen FDA-cleared for use with this kit in conjunction withclinical and epidemiological information:
Intended Use	· For determination of the subtype of seasonal human influenzaA viruses as seasonal A(H3), and/or A(H1)pdm09 from viralRNA in upper respiratory tract clinical specimens (includingnasopharyngeal swabs [NPS], nasal swabs [NS], throat swabs[TS], nasal aspirates [NA], nasal washes [NW] and dualnasopharyngeal/throat swabs [NPS/TS]) and lower respiratorytract specimens (including bronchoalveolar lavage [BAL]	Same

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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.
[black box: 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

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	instructors or designees.]
Specimen Types	Upper respiratory tract clinical specimens (includingnasopharyngeal swabs [NPS], nasal swabs [NS], throat swabs[TS], nasal aspirates [NA], nasal washes [NW] and dualnasopharyngeal/throat swabs [NPS/TS]) and lower respiratorytract specimens (including bronchoalveolar lavage [BAL],bronchial wash [BW], tracheal aspirate [TA], sputum, and lungtissue) from human patients with signs and symptoms ofrespiratory infection and/or from viral culture	Same
OrganismsDetected	Influenza A viruses (animal and human), swine-origin influenzaA viruses, influenza A subtypes: seasonal A(H3) andA(H1)pdm09	Same
Analytes	RNA- InfA, H3, pdmInfA, pdmH1 and RP	Same
TechnologicalPrinciples	Real-time RT-PCR	Same
Instrumentation	Applied Biosystems 7500 Fast Dx Real-time PCR system withSDS software version 1.4Applied Biosystems QuantStudio Dx with version 1.0.3 softwareQIAGEN Rotor-Gene Q MDx with AssayManager 1.0.4.1 andEpsilon version 1.0.1 software	Same
Enzyme MasterMix	Invitrogen SuperScript III Platinum One-Step Quantitative RT-PCR Kit (with or without ROX)Quanta BioSciences qScript One-Step qRT PCR Kit, Low ROX	Same
Nucleic AcidExtraction	QuIAamp DSP Viral RNA Mini Kit, QIAGENMagNA Pure Compact – Nucleic Acid Isolation Kit I,Roche	QIAamp DSP Viral RNA Mini Kit, QIAGENMagNA Pure LC – Total Nucleic Acid Kit, Roche
MagNA Pure Compact - RNA Isolation Kit, Roche	MagNA Pure 96 - DNA and Viral NA Small Volume Kit, Roche
MagNA Pure LC – Total Nucleic Acid Kit, Roche	QIAcube – QIAamp DSP Viral RNA Mini Kit, QIAGEN
MagNA Pure 96 - DNA and Viral NA Small Volume Kit, Roche	NucliSENS easyMAG, bioMérieux
QIAcube - QIAamp DSP Viral RNA Mini Kit, QIAGEN	EMAG, bioMérieux*EZ1 Advanced XL – EZ1 DSP Virus Kit and EZ1 RNA
NucliSENS easyMAG, bioMérieux	Tissue Mini Kit, QIAGEN
EMAG, bioMérieux*
EZ1 Advanced XL - EZ1 DSP Virus Kit and EZ1 RNATissue Mini Kit, QIAGEN
CDC Human Influenza Virus Real-Time RT-PCRDiagnostic Panel: Influenza B Genotyping Kit (VER 1.1 and 2)(K190302)	CDC Human Influenza Virus Real-Time RT-PCRDiagnostic Panel: Influenza B Genotyping Kit (VER 1.1 and 2)(K243931)
General DeviceCharacteristicSimilarities
	nasopharyngeal/throat swabs [NPS/TS]) from human patientswith signs and symptoms of respiratory infection and/or fromviral culture;
	• To provide epidemiologic information for surveillance ofcirculating influenza viruses.
	Performance characteristics for influenza B lineage genotypingwere established during a season when influenza B/Victoria andB/Yamagata lineages were in circulation.
	Negative results do not preclude influenza virus infection andshould not be used as the sole basis for treatment or other patientmanagement decisions. Conversely, positive results do not ruleout bacterial infection or co-infection with other viruses. Theagent detected may not be the definite cause of disease.
	[black box: All users, analysts, and any person reportingresults from use of this device should be trained to performand interpret the results from this procedure by a competentinstructor prior to use. CDC Influenza Division will limit thedistribution of this device to only those users who havesuccessfully completed a training course provided by CDCinstructors or designees.]
Specimen Types	Upper respiratory tract clinical specimens (includingnasopharyngeal swabs [NPS], nasal swabs [NS], throat swabs[TS], nasal aspirates [NA], nasal washes [NW] and dualnasopharyngeal/throat swabs [NPS/TS]) and/or viral culture	Same
OrganismDetected	Influenza B	Same
Analytes	RNA- InfB, VIC, YAM, RP	Same
TechnologicalPrinciples	Real-time RT-PCR	Same
	Applied Biosystems 7500 Fast Dx Real-time PCR system withSDS software version 1.4	Same
Instrumentation	Applied Biosystems QuantStudio Dx with version 1.0.3 software	Same
	QIAGEN Rotor-Gene Q MDx with AssayManager 1.0.4.1 andEpsilon version 1.0.1 software
Enzyme MasterMix	Invitrogen SuperScript III Platinum One-StepQuantitative RT-PCR Kit (with or without ROX)	Same
	Quanta BioSciences qScript One-Step qRT PCR Kit, Low ROX
Nucleic AcidExtraction	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
	MagNA Pure 96 - DNA and Viral NA Small Volume Kit,Roche	Same
	QIAcube - QIAamp DSP Viral RNA Mini Kit, QIAGEN
	NucliSENS easyMAG, bioMérieux
	EMAG, bioMérieux
	EZ1 Advanced XL - EZ1 DSP Virus Kit and EZ1 RNATissue Mini Kit, QIAGEN
	CDC Human Influenza Virus Real-Time RT-PCRDiagnostic Panel: Influenza A/H5 Subtyping Kit (VER 3)(K190302)	CDC Human Influenza Virus Real-Time RT-PCRDiagnostic Panel: Influenza A/H5 Subtyping Kit (VER 4)(K243931)
General DeviceCharacteristicSimilarities
OrganismsDetected	Influenza A viruses (animal and human), Influenza A subtypeA(H5) (Asian lineage)	Same
Analyte	RNA- InfA, H5a, H5b, RP	Same
TechnologicalPrinciples	Real-time RT-PCR	Same
Instrumentation	Applied Biosystem 7500 Fast Dx Real-time PCR system withSDS software version 1.4	Same
	Applied Biosystems QuantStudio Dx with version 1.0.3software
	QIAGEN Rotor-Gene Q MDx with AssayManager 1.0.4.1and Epsilon version 1.0.1 software
Enzyme MasterMix	Invitrogen SuperScript III Platinum One-Step Quantiative RT-PCR Kit (with or without ROX)	Same
	Quanta BioSciences qScript One-Step qRT-PCR Kit, LowROX
Nucleic AcidExtraction	QIAamp DSP Viral RNA Mini Kit, QIAGEN	Same
	MagNA Pure Compact – Nucleic Acid Isolation Kit I,Roche
	MagNA Pure Compact – RNA Isolation Kit, Roche
	MagNA Pure LC – Total Nucleic Acid Kit, Roche
MagNA Pure 96 - DNA and Viral NA Small Volume Kit,RocheQIAcube - QIAamp DSP Viral RNA Mini Kit, QIAGENNucliSENS easyMAG, bioMérieuxEMAG, bioMérieux*EZ1 Advanced XL - EZ1 DSP Virus Kit and EZ1 RNATissue Mini Kit, QIAGEN
GeneralDeviceCharacteristicDifferences
The Influenza A/H5 Subtyping Kit contains reagents andcontrols of the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel and is intended for use in real-time RT-PCR (rRT-PCR) assays on an in vitro diagnostic real-timePCR instrument that has been FDA-cleared for use with theCDC device in conjunction with clinical and epidemiologicalinformation:	The Influenza A/H5 Subtyping Kit contains reagents and controlsof the Centers for Disease Control and Prevention (CDC) HumanInfluenza Virus Real-Time RT-PCR Diagnostic Panel and isintended for use in real-time RT-PCR (rRT-PCR) assays on an invitro diagnostic real-time PCR instrument that has been FDA-cleared for use with this kit in conjunction with clinical andepidemiological information:
Intended Use	• For the presumptive identification of virus in patients whomay be infected with influenza A subtype A(H5) (Asianlineage) from viral RNA in human respiratory specimens andviral culture in conjunction with clinical and epidemiologicalrisk factors;	• For the presumptive identification of virus in patients who maybe infected with influenza A subtype A(H5) (Asian lineage) fromviral RNA in human respiratory specimens, conjunctival swabsand viral culture in conjunction with clinical andepidemiological risk factors;
	• To provide epidemiologic information for surveillance ofcirculating influenza viruses.	• To provide epidemiologic information for surveillance ofcirculating influenza viruses.
	Performance characteristics for influenza were establishedduring a season when seasonal influenza viruses A(H1N1)	Performance characteristics for influenza were established duringa season when seasonal influenza viruses A(H1N1) and A(H3N2)
and A(H3N2) were the predominant influenza A viruses incirculation and during a season when the A(H1N1)pdm09influenza virus was the predominant influenza A virus incirculation. Performance characteristics may vary with otheremerging influenza A viruses.	were the predominant influenza A viruses in circulation andduring a season when the A(H1N1)pdm09 influenza virus was thepredominant influenza A virus in circulation. Performancecharacteristics may vary with other emerging influenza A viruses.
Testing with the influenza H5a and H5b primer and probesets should not be performed unless the patient meets themost current U.S. Department of Health and Human Services(DHHS) clinical and epidemiologic criteria for testingsuspect A(H5) specimens. The definitive identification ofinfluenza A(H5) (Asian lineage) either directly from patientspecimens or from virus cultures requires additionallaboratory testing, along with clinical and epidemiologicalassessment in consultation with national influenzasurveillance experts.	Testing with the influenza H5a and H5b primer and probe setsshould not be performed unless the patient meets the most currentU.S. Department of Health and Human Services (DHHS) clinicaland epidemiologic criteria for testing suspect A(H5) specimens.The definitive identification of influenza A(H5) (Asian lineage)either directly from patient specimens or from virus culturesrequires additional laboratory testing, along with clinical andepidemiological assessment in consultation with nationalinfluenza surveillance experts.
Negative results do not preclude influenza virus infection andshould not be used as the sole basis for treatment or otherpatient management decisions. Conversely, positive resultsdo not rule out bacterial infection or co-infection with otherviruses. The agent detected may not be the definite cause ofdisease.	Negative results do not preclude influenza virus infection andshould not be used as the sole basis for treatment or other patientmanagement decisions. Conversely, positive results do not ruleout bacterial infection or co-infection with other viruses. Theagent detected may not be the definite cause of disease.
If infection with a novel influenza A virus is suspected basedon current clinical and epidemiological screening criteriarecommended by public health authorities, specimens shouldbe collected with appropriate infection control precautions fornovel virulent influenza viruses and sent to state or localhealth department for testing. Viral culture should not beattempted unless a BSL 3E facility is available to receive andculture specimens.	If infection with a novel influenza A virus is suspected based oncurrent clinical and epidemiological screening criteriarecommended by public health authorities, specimens should becollected with appropriate infection control precautions for novelvirulent influenza viruses and sent to state or local healthdepartment for testing. Viral culture should not be attemptedunless a BSL 3E facility is available to receive and culturespecimens.
[black box: All users, analysts, and any person reportingresults from use of this device should be trained to perform	[black box: All users, analysts, and any person reporting resultsfrom use of this device should be trained to perform and interpretthe results from this procedure by a competent instructor prior touse. CDC Influenza Division will limit the distribution of thisdevice to only those users who have successfully completed a
	and interpret the results from this procedure by a competentinstructor prior to use. CDC Influenza Division will limit thedistribution of this device to only those users who havesuccessfully completed a training course provided by CDCinstructors or designees.]	training course provided by CDC instructors or designees.]
Specimen Types	Human respiratory specimens and viral culture	Human respiratory specimens, conjunctival swabs, and viral culture

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Analytical Performance Evaluation VIII.

Confirmation of Limit of Detection

To assess the Limit of Detection (LoD) of the Influenza A/H5 Subtyping Kit testing conjunctival swab collected in VTM/UTM specimens, an initial range-finding experiment using BPL inactivated A/American Wigeon/South Carolina/22-000345-001/2021 H5N1 was performed in a background of VTM + A549 human lung epithelial cells (a simulated respiratory VTM matrix). A 5-fold dilution series of the virus was prevared using this simulated matrix as the dilution step was tested in triplicate using the CDC Flu rRT-PCR Dx Page Influeriza A/H5 Subtyping assay. The range finding imit of detection was defined as the viral titer of the last dilution step demonstrating 100% positive results (3/3) in this experiment. Results demonstrated a preliminary LoD with an approximate titer of 1055 EIDsg/ml.

Due to Iimited availability of negative conjunctiv material, the estimated LoD was confirmed by testing replicates at concentrations of 2x and 5x preliminary LoD using contrived specimens in either influenza A/H5 negative conjunctival swab matrix or simulated respiratory swabs collected in VTM matrix. Contrived specimens were spiked with BPL inactivated A/American Wigeon/South Carolina/22-000345-001/2021 H5N1 virus. Five replicates of each contrived specimen type at either 2x LoD or 5x preliminary LoD were tested along with 10 negatives for each matrix. Each specimen was extracted on the Qiagen EZ1 Advanced XL with the EZ1 DSP Virus kit, amplified by the Invirogen SuperScript™ III Platinum® One-Step Quantitative RT-PCR System (without Rox) and run on the Applied Biosystems 7500 Fast Dx PCR platform. Detection levels and Ct values of the conjunctival swab matrix were similar to that observed in the simulated respiratory matrix in VTM at both the 2x and 5x preliminary LoD concentrations. Based on this data, the LoD for the conjunctival swab specimens was determined to be 10-5 EIDsolml.

Interfering Substances

Two studies were performed to evaluate the potential impact of exogenous interfering substances in conjunctival swab specimen matrix. Studies were conducted using two common over the counter eye drops containing olopatadine hydrocholoride.

For the first study, to evaluate the effect of eyedrops on the assay, a set of contrived specimens (N=5) was constructed with a measured volune of each eyedrop plus A/American Wigeon South Carolina/22-000345-001/2021 H5N1 at 2x LoD in conjunctival swab matrix. An additional set of contrived specimens (N=5) was constructed with a measured volume of each eyedrop plus A/American Wigeon/South Carolina/22-000345-001/2021 H5N1 at 2x LoD in VTM + A549 cells (a simulated respiratory matrix). Each contrived specimen contained 2.31% Clearlye eyedrop (naphazoline hydrochloride as the main ingredient) and 2.08% Patadine hydrochloride as the main ingredient), and was extracted on the Qagen EZ1 Advanced XL with the EZ1 DSP Virus kit, amplified by the Invitrogen SuperScrip™ III Platinum® One-Step Quantitative RT-PCR System (without Rox) and run on the Applied Biosystems 7500 Fast Dx PCR platform. Due to a lack of sufficient volume of natural conjunctival swabs, the study was conducted by mixing both the olopatadine hydrochloride into a single reaction. Cycle threshold (Ct) results were compared to contrived specimens tested at 2x LoD without the addition of eyedrops as the control condition.

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No interference was seen with the addition of olopatadine hydrochloride in either the contrived specimens in conjunctival matrix or the contrived specimens in VTM + A549 cells matrix in this study.

The second study was performed by adding a measured amount of each eyedrop (2.46% Clear Eyes, respectively) directly into the

RT-PCR enzyme master mix and tested in 20 replicates to evaluate the effect of the A/H5 assay and determine if autofluorescence is produced due to the eyedrop additions that could generate a false possive A/H5 assay result was seen when the eyedrops were added directly to the RT-PCR reaction in this study.

IX. Clinical Performance Evaluation

Human upper respiratory tract (URT) swab specimens, including nasopharyngeal swab (NPS) specimens, combined nasal swab (NS) and oropharyngeal swab (OS) specimens, NS and/or OS specimens, as well as human conjunctival swab (CS) specimens were collected by health care professionals (HCPs) as part of public health investigations into outbreaks of influenza A/H5 in dairy cows and poultry in the US from March to November 2024. All available URT specimens from individuals with either a presumptive influenza A/H5 positive laboratory result (in any specimen) at the state laboratories, or a strong epidemiological link and clinical suspicion that indicate suspected influenza A/H5 infection, were sent to the CDC laboratory in Atlanta for confirmatory testing and evaluation.

URT specimens and CS specimens collected from a total of 44 confirmed and 2 probable human cases of A/H5N1 were received and tested using the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel Influenza A/H5 Subtyping Kit (VER 4) (i.e., the CDC A/H5 assay). The influenza A/H5N1 confirmed or probable case status for each case on the criteria described in the current Council of State and Territory Epidemiologists (CSTE) document 24-ID-09.

Of the 44 confirmed human cases and 2 probable cases, 42 (95.5%) and 2 (100%), respectively, reported conjunctivitis symptoms. While 28 confirmed cases and 1 probable case reported both respiratory and conunctivits symptoms, 16 confirmed cases and 1 probable case reported conjunctivitis symptoms only. The 46 confirmed and probable cases was 36.5 years (range: 18-57 years); 87.0% were male and 13.0% were female.

Due to a field correction regarding the H5b component assay reagents required for testing and result interpretation using the CDC AH5 assay, clinical specimens from 9 of the 44 confirmed cases were tested without the H5b component assay reagents. As a result, clinical specimens from these 9 confirmed cases were excluded from the performance assessment (i.e., 35 confirmed cases were assessed).

Of the 35 confirmed cases assessed. 33/35 (94.3%) reported conjunctivitis symptoms. Of those 33 cases, 26/33 (78.8%) tested positive and 3/33 (9.1%) tested inconclusive for A/H5 on CS specimens using the CDC A/H5 assay. Four (4) of the 33 confirmed cases with conjunctivits symptoms (15.2%) tested positive for A/H5 on URT specimens only using the CDC A/H5 assay.

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Of the 2 confirmed cases without conimons reported, 1/2 (50%) tested positive for A/H5 on a CS specimen, and 1/2 (50%) tested positive for A/H5 on a URT specimen only using the CDC A/H5 assay.

Of the 2 probable cases with conjunctivits symptoms reported, 2/2 (100%) tested negative on CS specimens using the CDC AHS assay.

Subject matched paired URT swab specimens were available for testing in 32 out of 35 confirmed cases and 1 of the probable cases. For 3 of the 35 confirmed cases and 1 of the 2 probable cases, only CS specimens were available for testing.

Of the subject matched paired URT and CS specimens from 32 confirmed human cases, 10/32 (31.3%) were positive on at least one URT and the CS specimens; 15/32 (46.9%) were positive in CS specimens only and 4/32 (12.5%) were positive in at least one URT specimen only. For 3/32 (9.4%) confirmed human cases the CS specimens tested inconclusive and URT specimens tested negative.

Of the subject matched paired URT and CS specimens from 1 probable human case, all URT and the CS specimen tested negative.

For 3 of the 35 confirmed cases where only CS specimens were available for testing, the CS specimens tested positive in 2 cases, and inconclusive in 1 case.

For 1 of the 2 probable cases where only a CS specimen was available for testing, the result was negative.

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X. Conclusion

Validation data demonstrates that the modification of the CDC Human Influenza Virus RT-PCR Diagnostic Panel to add conjunctival swabs for use with the Influenza A/H5 Subtyping Kit, which is one of the four component assay kits of the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel, is substantially equivalent to the predicate device.

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.