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

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

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

Performance characteristics for influenza were established during a seasonal influenza viruses A/H1 and A/ H3 were the predominant influenza A viruses in circulation and during a season when the A/H1pdm09 influenza virus was the predominant influenza A virus in circulation. Performance characteristics may vary with other emerging influenza A viruses.

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 or from virus cultures requires additional laboratory testing, along with clinical and epidemiological assessment in consultation with national influenza surveillance experts.

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 a state or local health department for testing. Viral culture should not be attempted unless a BSL 3+ facility is available to receive and culture specimens.

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

Device Description

The CDC Human Influenza Real-Time RT-PCR Diagnostic Panel Influenza A/H5 Subtyping Kit is a real-time RT-PCR) assay that utilizes the Applied Biosystems® (ABI) 7500 Fast Dx Realtime PCR system. The Influenza A/H5 Subtyping Kit consists of oligonucleotide primers, fluorescently labeled hydrolysis probes, and controls which are used in rRT-PCR assays for the in vitro qualitative detection and characterization of influenza virus RNA in respiratory specimens from patients presenting with influenza-like illness (ILI). The oligonucleotide primers and probes for detection of influenza A viruses (InfA) were selected from highly conserved regions of the matrix (M) protein. Oligonucleotide primers and probes for characterization of avian influenza A/H5 (Asian lineage) viruses (H5a and H5b) were selected from highly conserved regions of their HA genes. The Influenza A/H5 Subtyping kit also contains primers and probes to detect the human RNase P gene (RP) in control samples and clinical specimens.

AI/ML Overview

Here's an analysis of the provided text regarding the acceptance criteria and the study that proves the device meets the acceptance criteria:

Device Name: CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel, Influenza A/H5 Subtyping Kit (VER 3)
K Number: K153148

1. Table of Acceptance Criteria and Reported Device Performance

The document describes the analytical and clinical performance studies, but directly stated "acceptance criteria" for each metric are not explicitly listed in a formatted table with pass/fail thresholds. Instead, the results are presented as the device's performance, implying these values met internal criteria for submission and approval. Inferring from the nature of diagnostic device validation, these would typically be high percentages for sensitivity and specificity.

Based on the provided data, here's a table summarizing the observed performance:

Performance Metric	Acceptance Criteria (Inferred)	Reported Device Performance
Analytical Sensitivity (LOD)	Specific LOD values for tested H5N1 and H5N8 strains. For example, the lowest concentration where ≥95% of replicates test positive.	- A/Vietnam/1203/2004 x A/Puerto Rico/8/34 reassortant (A/H5N1): - Invitrogen SuperScript™: 10^3.8 EID50/mL - Quanta qScript™: 10^2.4 EID50/mL - A/duck/Vietnam/NCVD-1544/2012 (A/H5N1): - Invitrogen SuperScript™: 10^3.1 EID50/mL - Quanta qScript™: 10^3.1 EID50/mL - A/gyrfalcon/Washington/41088-6/2014 (A/H5N8): - Invitrogen SuperScript™: 10^3.35 EID50/mL - Quanta qScript™: 10^3.35 EID50/mL Reported as the lowest concentration where InfA and both H5a/H5b showed uniform detection.
Analytical Inclusivity	Detection of all listed A/H5 (Asian lineage) isolates representative of different geographic locations and phylogenetic clades, at or near the established LOD.	- Reactive with all 15 tested H5 (Asian lineage) isolates (all 3/3 positive for InfA, H5a, and H5b) with both enzyme systems. - Predicted to be reactive with A/Bangladesh/3222/2011 (in silico analysis).
Analytical Specificity (Cross-Reactivity)	No detection of common human influenza A (non-H5), human influenza B, swine influenza, avian influenza (non-H5), canine influenza, or equine influenza viruses when tested at high titers.	- InfA assay detected all tested human influenza A and various animal influenza A strains (as expected for an InfA primer/probe set). - H5a and H5b assays showed no cross-reactivity with human influenza A (non-H5), human influenza B, most animal influenza A (non-H5), and specifically did not detect A/duck/Singapore-Q/F119-3/1997 (H5N3) when tested with Invitrogen SuperScript™. (Note: The H5a/H5b assays did detect A/duck/Singapore-Q/F119-3/1997 H5N3, which is an H5 subtype, albeit not specified as Asian lineage. This is an expected positive result for H5 detection.)
Analytical Specificity (Exclusivity)	No detection (no false positives) of common non-influenza human respiratory viruses, respiratory bacteria, and commensal organisms of the human respiratory tract when tested at high concentrations.	- No detection (all negative) for all 18 bacteria, 1 yeast, and 16 non-influenza human respiratory viruses tested with Invitrogen SuperScript™ for InfA, H5a, and H5b assays.
Clinical Performance (Positive Agreement)	High Positive Agreement (e.g., >90% with a reasonable lower bound for 95% CI).	- Quanta qScript™: 100% (95% CI: 92.9 – 100.00) (50/50) - Invitrogen SuperScript™: 88.0% (95% CI: 76.2 - 94.4) (44/50)
Clinical Performance (Negative Agreement)	High Negative Agreement (e.g., >90% with a reasonable lower bound for 95% CI).	- Quanta qScript™: 100% (95% CI: 94.4 – 100.00) (65/65) - Invitrogen SuperScript™: 100% (95% CI: 94.4 - 100.00) (65/65)

2. Sample Size Used for the Test Set and Data Provenance

Analytical Sensitivity (LOD):
- Test Set Sample Size: 20 replicates for each enzyme kit (Invitrogen SuperScript™ and Quanta qScript™) for the highest virus dilution for each of the three A/H5 strains to confirm LOD. Initial testing used 3 replicates.
- Data Provenance: Laboratory-generated (characterized viruses).
Analytical Inclusivity:
- Test Set Sample Size: 3 replicates per virus isolate for 15 A/H5 (Asian lineage) viruses for both enzyme systems. One virus was tested in silico. Total real wet-lab tests: 15 viruses * 3 replicates * 3 assays (InfA, H5a, H5b) * 2 enzyme systems = 270 individual reactions.
- Data Provenance: Laboratory-generated (characterized viruses representing different geographic locations and phylogenetic clades).
Analytical Specificity (Cross-Reactivity):
- Test Set Sample Size: 3 replicates per virus for 14 human and animal influenza A and influenza B viruses tested. Total: 14 viruses * 3 replicates * 3 assays (InfA, H5a, H5b) = 126 individual reactions.
- Data Provenance: Laboratory-generated (characterized viruses).
Analytical Specificity (Exclusivity):
- Test Set Sample Size: A single test for each of 35 non-influenza organisms (16 viruses, 18 bacteria, 1 yeast). Total: 35 organisms * 3 assays (InfA, H5a, H5b) = 105 individual reactions.
- Data Provenance: Laboratory-generated (propagated, titered, and characterized organisms).
Clinical Performance Evaluation:
- Test Set Sample Size:
  - Positive Contrived Samples: 50 samples.
  - Negative Specimens: 65 specimens.
- Data Provenance:
  - Positive Contrived Samples: Laboratory-generated (grown virus added to A549 cell suspension).
  - Negative Specimens: Obtained from a clinical study conducted during the 2011-2012 influenza season. This suggests the negative specimens were retrospective clinical samples (human respiratory specimens), originating likely from the US where CDC studies are typically conducted.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of Those Experts

The document does not explicitly state the number or qualifications of experts used to establish the ground truth for the test set.

For analytical studies (LOD, Inclusivity, Specificity), the ground truth is established by the known characteristics of the laboratory-controlled and characterized viral and bacterial stocks. This does not typically involve human expert consensus in the same way clinical diagnostics do.
For the clinical performance evaluation:
- The "positive contrived samples" have their ground truth by definition (known virus added).
- The "negative specimens" were previously tested as negative for influenza A by the CDC Human Influenza rRT-PCR Diagnostic Panel. The ground truth for these would have been established by the predicate device's results, which were likely interpreted by trained laboratory personnel, rather than experts in a concensus panel for the purpose of this study.

4. Adjudication Method for the Test Set

The document does not describe an adjudication method for the test set.

For analytical studies, the results are typically quantitative (e.g., Cq values) or qualitative (positive/negative call) based on predetermined assay thresholds. There's no indication of a need for expert adjudication of these raw results.
For clinical performance, the comparison is made against the known status of contrived samples or the prior result from a predicate device. An adjudication process (like 2+1, 3+1, etc.) is typically used when the ground truth itself is uncertain or to resolve discrepancies between readers/tests, which is not stated as part of this study design.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not done.

MRMC studies are typically used to compare the performance of human readers (e.g., radiologists, pathologists) with and without AI assistance or against another diagnostic method, often looking at reader agreement and diagnostic accuracy.
This submission describes a RT-PCR diagnostic panel, which is an in vitro diagnostic (IVD) device. The performance is determined by the assay's ability to detect viral RNA, not by human reader interpretation of complex images or clinical data in the same way an MRMC study would apply. Therefore, the concept of "how much human readers improve with AI vs without AI assistance" is not applicable here.

6. If a Standalone (Algorithm Only Without Human-in-the-Loop Performance) Was Done

Yes, the studies described are essentially standalone performance evaluations of the device (assay) itself.

The analytical studies (LOD, Inclusivity, Specificity, Exclusivity) inherently assess the algorithm/reagent's ability to detect targets or avoid off-target detection.
The 'clinical performance evaluation' also tests the device's ability to correctly identify positive and negative contrived/clinical samples.
While a human laboratory technologist performs the PCR steps and interprets the output (e.g., reads Cq values and applies the interpretation algorithm to call positive/negative), the study evaluates the device's performance characteristics given those outputs, not the technologist's diagnostic ability with or without the device. The data presented is the direct output of the device's reaction, not a human interpretation layer being separately assessed for its impact.

7. The Type of Ground Truth Used

Analytical Studies (LOD, Inclusivity, Cross-Reactivity, Exclusivity):
- Known Characteristics of Propagated and Characterized Viruses/Organisms: For these studies, the ground truth is established by the known identity, titer, and genetic makeup of the laboratory-prepared viral and bacterial stocks. For example, an A/H5N1 virus is known to be A/H5N1 because it was characterized as such.
Clinical Performance Evaluation:
- Contrived Samples: The ground truth for positive samples was established by the known addition of virus to a cell suspension.
- Negative Specimens: The ground truth for negative specimens was established by their prior categorization as "negative for influenza A" using the CDC Human Influenza rRT-PCR Diagnostic Panel (likely the predicate device or a similar established method) in a previous clinical study. This is a form of comparator method ground truth.

8. The Sample Size for the Training Set

The document does not explicitly mention a training set or its sample size.

For IVD assays like RT-PCR, the development process (which would involve "training" or optimization) typically focuses on primer/probe design, reaction conditions, and calibration using initial lab-generated samples. The 'training' in this context is implicit in the assay development and refinement, rather than a distinct, formal training set for a machine learning algorithm.
The studies described are for validation (testing), not for development or training.

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

As no explicit training set is mentioned (see point 8), the document does not describe how ground truth for a training set was established.

If an implicit 'training' phase (assay development) occurred, the ground truth would have been established through well-characterized laboratory reference materials and known viral isolates, similar to the ground truth for the analytical validation studies.

Summary

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Image /page/0/Picture/1 description: The image shows the logo for the U.S. Department of Health and Human Services. The logo consists of a circular seal with the text "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" around the perimeter. Inside the circle is a stylized image of three human profiles facing to the right, stacked on top of each other.

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

December 1, 2015

Centers For Disease Control And Prevention Yon Yu, Pharm. D. Associate Director For Regulatory Affairs 1600 Clifton Road, NE MS-C18 Atlanta, GA 30329-4027

Re: K153148

Trade/Device Name: CDC Human Influenza Virus Real- Time RT-PCR Diagnostic Panel. Influenza A/H5 Subtyping Kit (VER 3) Regulation Number: 21 CFR 866.3980 Regulation Name: Respiratory viral panel multiplex nucleic acid assay Regulatory Class: II Product Code: OZE, NSU, NXD Dated: October 29, 2015 Received: October 30, 2015

Dear Dr. Yu:

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

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

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

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

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

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

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

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.

Sincerely yours,

Tamara V. Feldblyum -S for

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

Enclosure

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Indications for Use

510(k) Number (if known) K153148

Device Name

CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel, Influenza A/H5 Subtyping Kit (VER 3)

Indications for Use (Describe)

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

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)

CONTINUE ON A SEPARATE PAGE IF NEEDED.

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

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9. 510(k) Summary

I. GENERAL INFORMATION

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

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

Date Prepared: October 22, 2015

II. DEVICE INFORMATION

Proprietary Name:	CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel,Influenza A/H5 Subtyping Kit (VER 3)
Common Name:	Influenza A/H5 Subtyping Kit
Regulation Section:	866.3980-Respiratory viral panel multiplex nucleic acid assay
Subsequent RegulationSections:	862.2570-Instrumentation for clinical multiplex test systems866.3332-Reagents for detection of specific novel influenza A viruses
Device Classification:	Class II
Product Code:	OZE
Subsequent Product Codes:	NSU, NXD
Panel:	Microbiology

III. PREDICATE DEVICE

CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel, Influenza A/H5 Subtyping Kit (K141859)

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IV. DEVICE DESCRIPTION

V. INTENDED USE

The Influenza A/H5 Subtyping Kit contains reagents and controls of the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel and is intended for use in real-time RT-PCR (rRT-PCR) assays on an Applied Biosystems (ABI) 7500 Fast Dx Real-Time PCR instrument in conjunction with clinical and epidemiological information:

. For the presumptive identification of virus in patients who may be infected with influenza A subtype A/H5 (Asian lineage) from viral RNA in human respiratory specimens and viral culture in conjunction with clinical and epidemiological risk factors;
. To provide epidemiologic information for surveillance of circulating influenza viruses.

Performance characteristics for influenza were established during a season when seasonal influenza viruses A/H1 and A/H3 were the predominant influenza A viruses in circulation and during a season when the A/H1pdm09 influenza virus was the predominant influenza A virus in circulation. Performance characteristics may vary with other emerging influenza A viruses.

Testing with the influenza H5a and H5b primer and probe sets should not be performed unless the patient meets 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.

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

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local health department for testing. Viral culture should not be attempted unless a BSL 3+ facility is available to receive and culture specimens.

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

VI. TECHNOLOGICAL CHARACTERISTICS

As circulating Asian lineage highly pathogenic avian influenza (HPAI) viruses continue to evolve, the HA gene target of certain phylogenetic groups has acquired changes in the oligonucleotide primer and probe regions that require modification to ensure comprehensive detection of circulating influenza A/H5 clades. The predicate Influenza A/H5 Subtyping Kit (K141859) includes reagents for two assays, referred to as H5a and H5b, specifically detecting influenza A H5 Asian lineage viruses. The predicate H5a assay consists of two primers and two probes. The predicate H5b assay consists of two primers and one probe. The oligonucleotide primers and probes used in the modified H5a and H5b assays target the HA gene at the same locations and include a total of five primers and three probes for H5a and three primers and one probe for H5b. In addition to evaluating the modified oligonucleotide primers and probes, CDC has evaluated the ZENTM Double-Quenched Probe technology (manufactured by Integrated DNA Technologies) as an alternate fluorescent hydrolysis probe quencher chemistry. The Influenza A/H5 Subtyping Kit assays contain ZENTM double quenched probes (ZEN probes) that include an internal ZEN 111 quencher located nine nucleotides away from the 5' FAM reporter dye in addition to an Iowa Black® FQ quencher (IABkFQ) at the 3' end of the probe.

SUBSTANTIAL EQUIVALENCE COMPARISON VII.

The CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel, Influenza A/H5 Subtyping Kit (K141859) will serve as the predicate for the intended change. See the table below for a detailed comparison.

Device Comparison
	CDC Human Influenza Virus Real-Time RT-PCR DiagnosticPanel Diagnostic Panel, Influenza A/H5 Subtyping Kit(K141859)	CDC Human Influenza VirusReal-Time RT-PCR DiagnosticPanel Diagnostic Panel,Influenza A/H5 Subtyping Kit
Intended Use	The Influenza A/H5 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) assayson an Applied Biosystems (ABI) 7500 Fast Dx Real-Time PCRinstrument in conjunction with clinical and epidemiologicalinformation: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;To provide epidemiologic information for surveillance ofcirculating influenza viruses.	Same
	Performance characteristics for influenza were established during aseason when seasonal influenza viruses A/H1 and A/H3 were thepredominant influenza A viruses in circulation and during a seasonwhen the A/H1pdm09 influenza virus was the predominant influenzaA virus in circulation. Performance characteristics may vary with
	other emerging influenza A viruses.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. Thedefinitive identification of influenza A/H5 (Asian lineage) eitherdirectly from patient specimens or from virus cultures requiresadditional laboratory testing, along with clinical and epidemiologicalassessment in consultation with national influenza surveillanceexperts.
	Negative results do not preclude influenza virus infection and shouldnot be used as the sole basis for treatment or other patientmanagement decisions. Conversely, positive results do not rule outbacterial infection or co-infection with other viruses. The agentdetected may not be the definite cause of disease.
	If infection with a novel influenza A virus is suspected based oncurrent clinical and epidemiological screening criteria recommendedby public health authorities, specimens should be collected withappropriate infection control precautions for novel virulent influenzaviruses and sent to state or local health department for testing. Viralculture should not be attempted unless a BSL 3+ facility is availableto receive and culture specimens.
	All users, and any person reporting results from use of this device should betrained to perform and interpret the results from this procedure by a competentinstructor prior to use. CDC Influenza Division will limit the distribution of this deviceto only those users who have successfully completed a training course provided byCDC instructors or designees.
OrganismDetected	Universal influenza A viruses (animal and human) and InfluenzaA/H5 Subtype (Asian lineage) viruses.	Same
Specimen Types	Human respiratory specimens and viral culture.	Same
Nucleic AcidExtraction	Yes	Same
ExtractionMethod	• QIAamp® DSP Viral RNA Mini Kit, Qiagen· MagNA Pure Compact -Nucleic Acid Isolation Kit I, Roche· MagNA Pure Compact - RNA Isolation Kit, Roche· MagNA Pure LC - Total Nucleic Acid Kit, Roche· Qiagen QIAcube - QIAamp® DSP Viral RNA Mini Kit, Qiagen· NucliSENS® easyMAG®, bioMerieux	Same
Enzyme MasterMix	Invitrogen SuperScript™ III Platinum® One-Step Quantitative RT-PCR Kit (with or without ROX)ORQuanta BioSciences qScript™ One-Step qRT-PCR Kit, Low ROX	Same
PCR Technology	Real-Time RT-PCR	Same
RequiredInstrumentation	Applied Biosystems 7500 Fast Dx Real-Time PCR Instrument withSDS software version 1.4	Same

Device Comparisor

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ProbeQuenchingMolecule	Black Hole Quencher Probe® (BHQ-1)	ZENTM Double-Quenched Probe(InfA, H5a, H5b, and RP assays)ORBlack Hole Quencher Probe®(InfA and RP assays)
Oligonucleotides	H5a assay-Targets a region of the HA geneH5b assay-Targets a region of the HA geneInfA assay-Targets a conserved region of the matrix gene inInfluenza A viruses	Gene targets of the oligonucleotideassays are the same as thepredicate; minor changes to theoligonucleotide sequences havebeen made

ANALYTICAL PERFORMANCE EVALUATION VIII.

Analytical Sensitivity - Limit of Detection Study (LOD)

Analytical sensitivity of the Influenza A/H5 Subtyping Kit was demonstrated by determining the LOD using Quanta qScript™ and Invitrogen SuperScript™ enzyme kits. Characterized viruses of known 50% infectious dose titers (EID50mL) were extracted, and the RNA was serially diluted and tested (n=3 replicates) in order to determine an apparent endpoint range. The LOD for each primer and probe set was confirmed by testing extraction replicates (n=20) of the highest virus dilution where ≥95% of all replicates tested positive. Virus dilutions were prepared in virus transport medium containing human A549 cells to emulate clinical specimen matrix. The lowest concentration where the InfA and both H5a and H5b primer and probe sets demonstrate uniform detection was reported as the LOD. The results are summarized in the table below.

LOD Summary
Influenza VirusTested	Influenza Strain Designation	LOD (EID50/mL)InvitrogenSuperScriptTM	LOD (EID50/mL)Quanta qScriptTM
A/H5N1	A/Vietnam/1203/2004×A/Puerto Rico/8/34reassortant(A/Vietnam/1203/2004 PR8- VNH5N1-PR8/CDC-RG)	10 3.8	10 2.4
A/H5N1	A/duck/Vietnam/NCVD-1544/2012	10 3.1	10 3.1
A/H5N8	A/gyrfalcon/Washington/41088-6/2014	10 3.35	10 3.35

Analytical Sensitivity - Inclusivity Testing

Inclusivity testing was conducted to demonstrate the capability of the oligonucleotide primers and probes in the Influenza A/H5 Subtyping Kit to detect strains of influenza A/H5 viruses (Asian lineage) representative of different geographic locations and phylogenetic clades at or near the established LOD. Inclusivity testing was performed with sixteen representative H5 viruses (Asian lineage). A virus of the phylogenetic clade 2.2.2.1 was unavailable for testing, therefore reactivity of the probes with A/Bangladesh/322/2011 was performed in silico. The remaining fifteen viruses were grown to high titer, harvested, and serially diluted to near the LOD of the assays. The diluted influenza A/H5 viruses were extracted and tested in triplicate with the InfA, and H5b assays to demonstrate

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reactivity. Inclusivity of the Influenza A/H5 Subtyping Kit was evaluated with both enzyme systems (i.e. Invitrogen SuperScript™ and Quanta qScript™) and one cleared extraction method.

The Influenza A/H5 Subtyping Kit was reactive with all H5 (Asian lineage) isolates that were tested and predicted to be reactive with the influenza A/Bangladesh/3222/2011 in silico. The inclusivity results are summarized in the table below.

Influenza VirusStrain Identification	Subtype	Clade	EID50/mL	InvitrogenSuperScript™			Quanta qScript™
				InfA	H5a	H5b	InfA	H5a	H5b
A/Northernpintail/Washington/40964/2014	H5N2	2.3.4.4	103.4	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)
A/common magpie/HongKong/645/2006	H5N1	2.3.4	103.2	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)
A/chicken/Bangladesh/11rs-1984-30/2011	H5N1	2.3.4.2	103.75	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)
A/chicken/Vietnam/NCVD-279/2009	H5N1	2.3.4.3	104.25	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)
A/Cambodia/W0526301/2012	H5N1	1.1	103.4	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)
A/Cambodia/R040505/2007	H5N1	1.1	103.5	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)
A/duck/Hunan/795/2002	H5N1	2.1.1	103.9	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)
A/Indonesia/NIHRD11771/2011	H5N1	2.1.3.2a	104.4	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)
A/turkey/Turkey/1/2005	H5N1	2.2.1	104.1	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)
A/Egypt/1050/NAMRU3/2013	H5N1	2.2.1	103.4	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)
A/Bangladesh/3233/2011 (insilico analysis)	H5N1	2.2.2.1	--	+	+	+	+	+	+
A/common magpie/HongKong/5052/2007	H5N1	2.3.2.1	103.75	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)
A/chicken/Bangladesh/42010/2012	H5N1	2.3.2.1a	103.2	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)
A/duck/Vietnam/NCVD-672/2011	H5N1	2.3.2.1b	103.8	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)
A/goose/Guiyang/337/2006	H5N1	4	104.1	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)
A/chicken/Vietnam/NCVD-1088/2013	H5N1	7.2	103.9	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)	3/3(+)

Inclusivity Results of the Influenza A/H5 Subtyping Kit

Analytical Specificity -Cross-Reactivity

Cross-reactivity of the Influenza A/H5 Subtyping Kit was evaluated by testing influenza A viruses of different types and subtypes that include viruses representing diverse geographic locations and different sources. Samples were tested in triplicate using RNA extracted from high titer preparations

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of viruses (≥ 10° EID50/mL). Cross-reactivity testing of the Influenza A/H5 Subtyping Kit was evaluated with the Invitrogen Superscript™ enzyme system and one cleared extraction method. The results are summarized in the tables below.

Virus Designation	Subtype	EID50/mL	Invitrogen SuperScript™
			InfA	H5a	H5b
A/Brisbane/59/07	A(H1N1)	108.4	(+)3/3	-	-
A/Fujian Gulou/1896/2009		109.1	(+)3/3	-	-
A/Perth/16/2009	A(H3N2)	108.9	(+)3/3	-	-
A/Texas/50/2012		109.2	(+)3/3	-	-
A/California/07/09	A(H1N1)pdm09	108.4	(+)3/3	-	-
A/Washington/24/2012		108.5	(+)3/3	-	-
B/Brisbane/60/2008	B(Victorialineage)	109.3	-	-	-
B/Montana/5/2012		108.4	-	-	-
B/Wisconsin/01/2010	B(Yamagatalineage)	109.2	-	-	-
B/Massachusetts/02/2012		109.2	-	-	-

Human Influenza Viruses for Cross-Reactivity Testing

Animal Influenza Viruses for Cross-Reactivity Testing

Species	Virus	Type	EID50/mL	Invitrogen SuperScript™
				InfA	H5a	H5b
Swine	A/swine/Wisconsin/125/1997	SW-H1N1	107.9	(+)3/3	-	-
Swine	A/Maryland/12/1991	SW-H1N1	109.0	(+)3/3	-	-
Swine	A/Iowa/1/2006	SW-H1N1	109.0	(+)3/3	-	-
Avian	A/chicken/Pennsylvania/298101-4/2004	H2N2	109.5	(+)3/3	-	-
Canine	A/canine/Florida/43/2004	H3N8	107.2	(+)3/3	-	-
Equine	A/equine/Ohio/01/2003	EQ-H3N8	107.5	(+)3/3	-	-
Avian	A/chicken/Alabama/1975	H4N8	1010.0	(+)3/3	-	-

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New Special 510(k)
CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel, Influenza A/H5 Subtyping Kit

Avian	A/duck/Singapore-Q/F119-3/1997	H5N3	$10^{8.5}$	(+)3/3	(+)3/3	(+)3/3
Avian	A/duck/Pennsylvania/1969	H6N1	$10^{9.2}$	(+)3/3	-	-
Avian	A/chicken/California/32213-1/2000	H6N2	$10^{9.1}$	(+)3/3	-	-
Avian	A/chicken/New York/13237-6/1998	H6N8	$10^{10.0}$	(+)3/3	-	-
Avian	A/chicken/New Jersey/15906-9/1996	H11N1	$10^{6.5}$	(+)3/3	-	-
Avian	A/duck/Memphis/546/1974	H11N9	$10^{9.8}$	(+)3/3	-	-
Avian	A/Taiwan/2/2013	H6N1	$10^{10.2}$	(+)3/3	-	-
Avian	A/Anhui/1/2013	H7N9	$10^{10.9}$	(+)3/3	-	-
Avian	A/mallard/Netherlands/12/2000	H7N3	$10^{9.5}$	(+)3/3	-	-
Avian	A/chicken/Arkansas/10/2008	H7N3	$10^{9.9}$	(+)3/3	-	-
Avian	A/Bangladesh/0994/2011	H9N2	$10^{10.5}$	(+)3/3	-	-
Swine	A/Indiana/21/2012	H3N2v	$10^{9.4}$	(+)3/3	-	-
Swine	A/Minnesota/11/2010	H3N2v	$10^{9.2}$	(+)3/3	-	-

Exclusivity Testing

An exclusivity study was performed to demonstrate the specificity of each primer and probe set of the Influenza A/H5 Subtyping Kit when tested with common non-influenza human respiratory viruses, respiratory bacteria, and commensal organisms of the human respiratory tract. All organisms used in the study were propagated, titered, and characterized to confirm identity prior to testing. Nucleic acids were purified from thirty-five (35) non-influenza organisms (16 viruses, 18 bacteria, and 1 yeast) representing common respiratory pathogens or flora commonly present in specimens collected from the human nasopharynx region. High titer preparations of bacteria and yeast, generally greater than or equal to106 cfu/mL, and non-influenza respiratory virus preparations at concentrations greater than 10° TCIDso/mL were tested (except in cases where production of high titer virus stock was not possible, e.g. parainfluenza virus type 2). The Influenza A/H5 Subtyping Kit was evaluated with the Invitrogen Superscript™ enzyme system and one cleared extraction method. The results are summarized in the table below.

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Organism Tested			Invitrogen SuperScript™
Bacteria and Yeast	Strain	cfu/mL	InfA	H5a	H5b
Bordetella pertussis	A639	108.3	-	-	-
Candida albicans (yeast)	2001-21-196	108.8	-	-	-
Chlamydia pneumoniae	TW183	40 IFU/mL1	-	-	-
Corynebacterium diphtheriae	-	1010	-	-	-
Escherichia coli	K12	109.6	-	-	-
Streptococcus pyogenes	7790-06	107.5	-	-	-
Haemophilus influenzae	M15709	106.4	-	-	-
Lactobacillus plantarum	-	108.8	-	-	-
Legionella pneumophila	-	107.1	-	-	-
Moraxella catarrhalis	M15757	109.5	-	-	-
Mycobacterium tuberculosis	H37Rv	100 ng/uL2	-	-	-
Mycoplasma pneumoniae	M129	107.7	-	-	-
Neisseria elongata	-	108.6	-	-	-
Neisseria meningitidis	M2578	107.9	-	-	-
Pseudomonas aeruginosa	-	1010.5	-	-	-
Staphylococcus aureus	-	1010.7	-	-	-
Staphylococcus epidermidis	-	1010.5	-	-	-
Streptococcus pneumoniae	249-06	106.6	-	-	-
Streptococcus salivarius	SS1672	108.4	-	-	-
Organism Tested			Invitrogen SuperScript™
Viruses	Strain	TCID50/mL	InfA	H5a	H5b
Human adenovirus type 1	Ad.71	109.2	-	-	-
Human adenovirus type 7a	S-1058	107.1	-	-	-
Human parainfluenza virus type 1	-	3.0 ng/µL2	-	-	-
Human parainfluenza virus type 2	Greer	103.1	-	-	-
Human parainfluenza virus type 3	C-243	107.9	-	-	-
Respiratory syncytial virus	CH93-18b	106.8	-	-	-
Human rhinovirus type A	1A	105.8	-	-	-
Enterovirus	Echo 6	106.9	-	-	-
Human coronavirus	299E	31.6ng/μL2	-	-	-
Human coronavirus	OC43	50.4ng/μL2	-	-	-
Herpes simplex virus	KOS	5 X 107.75	-	-	-
Varicella-zoster virus	AV92-3:H	5 X 103.75	-	-	-
Epstein-Barr virus	B95-8	1.7 ng/µL2	-	-	-
Measles virus (Paramyxoviridae)	Edmonston	5 X 104.5	-	-	-
Mumps virus	Enders	5 X 106.5	-	-	-
Human Cytomegalovirus	AD-169	5 X 106.25	-	-	-

Exclusivity results with Respiratory Pathogens and Flora

1Organism quantified by Infectious Forming Units (IFU)

²Organism nucleic acid quantified by spectrophotometry

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CLINICAL PERFORMANCE EVALUATION IX.

The clinical performance of oligonucleotide primer and probe sets of the Influenza A/H5 Subtyping Kit were evaluated using contrived samples of grown virus added to an A549 cell suspension to simulate positive clinical samples. A total of fifty positive contrived samples in high, moderate, and low concentrations were evaluated. In addition, sixty-five specimens that tested negative for influenza A with the CDC Human Influenza rRT-PCR Diagnostic Panel that were obtained from a clinical study conducted during the 2011-2012 influenza season were evaluated. Testing was performed with both enzymes cleared for use with the kit. The results are summarized in the table below.

EnzymeUtilized	# ofPositives1	% PositiveAgreement (95% CI)	# ofNegatives2	% NegativeAgreement (95% CI)
Quanta BioSciencesqScriptTM	50/50	100 (92.9 – 100.00)	65/65	100 (94.4 – 100.00)
InvitrogenSuperScriptTM	44/50	88.0 (76.2 - 94.4)	65/65	100 (94.4 - 100.00)

Clinical Performance Evaluation Results

1 Proportion of contrived samples correctly identified as positive by both influenza A H5a and H5b primer and probe sets. 2Proportion of negative samples correctly identified versus the comparator.

X. CONCLUSION

The modification of the CDC Human Influenza Virus rRT-PCR Diagnostic Panel, Influenza A/H5 Subtyping kit to ensure comprehensive detection of circulating influenza A/H5 clades does not substantially change the device. Analytical and clinical data demonstrate that the performance of the device to detect Asian lineage influenza A H5 viruses is accomplished with high positive and negative percent agreement in a manner substantially equivalent to the predicate.

XI. REFERENCES

Lednicky JA, Villanueva JM, Burke SA, Shively R, Shaw MW, Daniels DE, Hamilton SB, Donis RO. 2010. Validation of a method for preparing influenza H5N1 simulated samples. J Virol Methods. 2010 Aug;167(2):125-31.

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.