(32 days)
Not Found
No
The device description and performance studies focus on traditional RT-PCR methods and do not mention any AI or ML components for analysis or interpretation.
No.
This device is an in vitro diagnostic (IVD) test kit used to detect influenza A/H5 virus RNA for diagnostic and surveillance purposes; it does not directly treat or alleviate a disease or condition.
Yes
This device is clearly identified as a "Diagnostic Panel" and its intended use is for the "presumptive identification of virus in patients who may be infected with influenza A subtype A/HS". It involves detecting viral RNA in human respiratory specimens, which is a diagnostic process.
No
The device is a kit containing reagents, controls, oligonucleotide primers, and probes for a real-time RT-PCR assay. These are physical components, not software. While it utilizes a PCR instrument, the core of the device as described is a collection of chemical and biological materials.
Yes, this device is an IVD (In Vitro Diagnostic).
Here's why:
- Intended Use: The intended use explicitly states it's for the "presumptive identification of virus in patients... from viral RNA in human respiratory specimens and viral culture". This is a diagnostic purpose performed in vitro (outside the body) on biological samples.
- Device Description: The description details a "real-time RT-PCR assay" that utilizes primers, probes, and controls for the "in vitro qualitative detection and characterization of influenza virus RNA in respiratory specimens". This directly aligns with the definition of an in vitro diagnostic test.
- Performance Studies: The document describes various performance studies (Analytical Sensitivity, Analytical Specificity, Clinical Performance Evaluation) conducted to validate the device's ability to detect and characterize the target virus in biological samples. This is a standard requirement for IVDs.
- Key Metrics: The inclusion of metrics like % Positive Agreement and % Negative Agreement from the Clinical Performance Evaluation further confirms its role as a diagnostic test.
- Predicate Device: The mention of a predicate device (K141859; CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel, Influenza A/H5 Subtyping Kit) indicates that this device is being compared to a previously cleared IVD.
The entire document describes a test kit designed to be used in a laboratory setting to analyze human biological samples for the presence of a specific pathogen, which is the core function of an In Vitro Diagnostic device.
N/A
Intended Use / Indications for 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.
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, analysts, and any person reporting results from use of this device should be trained to perform and interpret the results from this procedure by a competent instructor prior to use. CDC Influenza Division will limit the distribution of this device to only those users who have successfully completed a training course provided by CDC instructors or designees.
Product codes
OZE, NSU, NXD
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.
Mentions image processing
Not Found
Mentions AI, DNN, or ML
Not Found
Input Imaging Modality
Not Found
Anatomical Site
Human respiratory specimens and viral culture.
Indicated Patient Age Range
Not Found
Intended User / Care Setting
All users, analysts, and any person reporting results from use of this device should be trained to perform and interpret the results from this procedure by a competent instructor prior to use. CDC Influenza Division will limit the distribution of this device to only those users who have successfully completed a training course provided by CDC instructors or designees.
Description of the training set, sample size, data source, and annotation protocol
Not Found
Description of the test set, sample size, data source, and annotation protocol
Not Found
Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)
Analytical Performance Evaluation
-
Analytical Sensitivity - Limit of Detection Study (LOD):
- Study Type: Analytical sensitivity study.
- Sample Size: Viruses of known EID50/mL titers were extracted, serially diluted, and tested in n=3 replicates 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.
- Key Results: The lowest concentration where the InfA and both H5a and H5b primer and probe sets demonstrate uniform detection was reported as the LOD.
- A/Vietnam/1203/2004 x A/Puerto Rico/8/34 reassortant: LOD (Invitrogen SuperScriptTM) = 10^3.8 EID50/mL, LOD (Quanta qScriptTM) = 10^2.4 EID50/mL.
- A/duck/Vietnam/NCVD-1544/2012: LOD (Invitrogen SuperScriptTM) = 10^3.1 EID50/mL, LOD (Quanta qScriptTM) = 10^3.1 EID50/mL.
- A/gyrfalcon/Washington/41088-6/2014: LOD (Invitrogen SuperScriptTM) = 10^3.35 EID50/mL, LOD (Quanta qScriptTM) = 10^3.35 EID50/mL.
-
Analytical Sensitivity - Inclusivity Testing:
- Study Type: Inclusivity testing.
- Sample Size: Sixteen representative H5 viruses (Asian lineage) to demonstrate the capability of the oligonucleotide primers and probes to detect different geographic locations and phylogenetic clades. A virus of phylogenetic clade 2.2.2.1 was unavailable, so reactivity was performed in silico with A/Bangladesh/322/2011. The remaining fifteen viruses were grown to high titer, harvested, and serially diluted to near the LOD, extracted, and tested in triplicate with the InfA, and H5b assays.
- Key Results: 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. All tested viruses showed 3/3 positive results for InfA, H5a, and H5b using both Invitrogen SuperScript™ and Quanta qScript™ enzyme systems.
-
Analytical Specificity - Cross-Reactivity:
- Study Type: Cross-reactivity testing.
- Sample Size: Influenza A viruses of different types and subtypes, including human and animal influenza viruses (e.g., A/Brisbane/59/07, A/swine/Wisconsin/125/1997, A/duck/Singapore-Q/F119-3/1997). Samples were tested in triplicate using RNA extracted from high titer preparations (>= 10^6 EID50/mL).
- Key Results: The Influenza A/H5 Subtyping Kit showed reactivity with InfA for all influenza A viruses tested but generally no reactivity for H5a and H5b with human and animal influenza A viruses other than Asian lineage H5. A/duck/Singapore-Q/F119-3/1997 (H5N3) showed positive results for InfA, H5a, and H5b (3/3). This is an avian H5 influenza.
-
Exclusivity Testing:
- Study Type: Exclusivity study.
- Sample Size: Nucleic acids purified from thirty-five (35) non-influenza organisms (16 viruses, 18 bacteria, and 1 yeast) representing common respiratory pathogens or flora. High titer preparations (generally >= 10^6 cfu/mL for bacteria and yeast, and > 10^3 TCID50/mL for non-influenza respiratory viruses) were tested.
- Key Results: The Influenza A/H5 Subtyping Kit showed no reactivity (all negative results) for InfA, H5a, and H5b with all tested non-influenza human respiratory viruses, respiratory bacteria, and commensal organisms.
Clinical Performance Evaluation
- Study Type: Clinical performance evaluation.
- Sample Size: Fifty positive contrived samples (grown virus added to A549 cell suspension) in high, moderate, and low concentrations. Sixty-five specimens that tested negative for influenza A with the CDC Human Influenza rRT-PCR Diagnostic Panel, obtained from a clinical study during the 2011-2012 influenza season.
- Key Results:
- Quanta BioSciences qScriptTM:
-
of Positives: 50/50
- % Positive Agreement (95% CI): 100 (92.9 – 100.00)
-
of Negatives: 65/65
- % Negative Agreement (95% CI): 100 (94.4 – 100.00)
-
- Invitrogen SuperScriptTM:
-
of Positives: 44/50
- % Positive Agreement (95% CI): 88.0 (76.2 - 94.4)
-
of Negatives: 65/65
- % Negative Agreement (95% CI): 100 (94.4 - 100.00)
-
- Quanta BioSciences qScriptTM:
Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)
Not directly reported as sensitivity/specificity in the clinical study table, but rather "Positive Agreement" and "Negative Agreement".
- Quanta BioSciences qScriptTM:
- % Positive Agreement: 100% (50/50)
- % Negative Agreement: 100% (65/65)
- Invitrogen SuperScriptTM:
- % Positive Agreement: 88.0% (44/50)
- % Negative Agreement: 100% (65/65)
Predicate Device(s)
Reference Device(s)
Not Found
Predetermined Change Control Plan (PCCP) - All Relevant Information
Not Found
§ 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.
0
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.
1
If you desire specific advice for your device on our labeling regulations (21 CFR Parts 801 and 809), please contact the Division of Industry and Consumer Education at its toll-free number (800) 638 2041 or (301) 796-7100 or at its Internet address
http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to
http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.
You may obtain other general information on your responsibilities under the Act from the Division of Industry and Consumer Education at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address
http://www.fda.gov/MedicalDevices/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
2
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)
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.
| 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.
3
This section applies only to requirements of the Paperwork Reduction Act of 1995.
DO NOT SEND YOUR COMPLETED FORM TO THE PRA STAFF EMAIL ADDRESS BELOW.
The burden time for this collection of information is estimated to average 79 hours per response, including the time to review instructions, search existing data sources, gather and maintain the data needed and complete and review the collection of information. Send comments regarding this burden estimate or any other aspect of this information collection, including suggestions for reducing this burden, to:
Department of Health and Human Services Food and Drug Administration Office of Chief Information Officer Paperwork Reduction Act (PRA) Staff PRAStaff@fda.hhs.gov
"An agency may not conduct or sponsor, and a person is not required to respond to, a collection of information unless it displays a currently valid OMB number."
4
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 Regulation
Sections: | 862.2570-Instrumentation for clinical multiplex test systems
866.3332-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)
5
IV. 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.
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.
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
6
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 Diagnostic | ||
| Panel Diagnostic Panel, Influenza A/H5 Subtyping Kit | ||
| (K141859) | CDC Human Influenza Virus | |
| Real-Time RT-PCR Diagnostic | ||
| Panel Diagnostic Panel, | ||
| Influenza A/H5 Subtyping Kit | ||
| 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. | Same |
| | 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. | |
| | If infection with a novel influenza A virus is suspected based on
current clinical and epidemiological screening criteria recommended
by public health authorities, specimens should be collected with
appropriate infection control precautions for novel virulent influenza
viruses and sent to state or local health department for testing. Viral
culture should not be attempted unless a BSL 3+ facility is available
to receive and culture specimens. | |
| | All users, and any person reporting results from use of this device should be
trained to perform and interpret the results from this procedure by a competent
instructor prior to use. CDC Influenza Division will limit the distribution of this device
to only those users who have successfully completed a training course provided by
CDC instructors or designees. | |
| Organism
Detected | Universal influenza A viruses (animal and human) and Influenza
A/H5 Subtype (Asian lineage) viruses. | Same |
| Specimen Types | Human respiratory specimens and viral culture. | Same |
| Nucleic Acid
Extraction | Yes | Same |
| Extraction
Method | • QIAamp® DSP Viral RNA Mini Kit, Qiagen
· MagNA Pure Compact -Nucleic Acid Isolation Kit I, Roche
· MagNA Pure Compact - RNA Isolation Kit, Roche
· MagNA Pure LC - Total Nucleic Acid Kit, Roche
· Qiagen QIAcube - QIAamp® DSP Viral RNA Mini Kit, Qiagen
· NucliSENS® easyMAG®, bioMerieux | Same |
| Enzyme Master
Mix | Invitrogen SuperScript™ III Platinum® One-Step Quantitative RT-
PCR Kit (with or without ROX)
OR
Quanta BioSciences qScript™ One-Step qRT-PCR Kit, Low ROX | Same |
| PCR Technology | Real-Time RT-PCR | Same |
| Required
Instrumentation | Applied Biosystems 7500 Fast Dx Real-Time PCR Instrument with
SDS software version 1.4 | Same |
Device Comparisor
7
8
| Probe
Quenching
Molecule | Black Hole Quencher Probe® (BHQ-1) | ZENTM Double-Quenched Probe
(InfA, H5a, H5b, and RP assays)
OR
Black Hole Quencher Probe®
(InfA and RP assays) |
|--------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------|
| Oligonucleotides | H5a assay-Targets a region of the HA gene
H5b assay-Targets a region of the HA gene
InfA assay-Targets a conserved region of the matrix gene in
Influenza A viruses | Gene targets of the oligonucleotide
assays are the same as the
predicate; minor changes to the
oligonucleotide sequences have
been 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 Virus | |||
| Tested | Influenza Strain Designation | LOD (EID50/mL) | |
| Invitrogen | |||
| SuperScriptTM | LOD (EID50/mL) | ||
| Quanta qScriptTM | |||
| A/H5N1 | A/Vietnam/1203/2004×A/Puerto Rico/8/34 | ||
| reassortant | |||
| (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
9
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 Virus
Strain Identification | Subtype | Clade | EID50/
mL | Invitrogen
SuperScript™ | | | Quanta qScript™ | | |
|------------------------------------------------|---------|----------|--------------|----------------------------|------------|------------|-----------------|------------|------------|
| | | | | InfA | H5a | H5b | InfA | H5a | H5b |
| A/Northern
pintail/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/Hong
Kong/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 (in
silico analysis) | H5N1 | 2.2.2.1 | -- | + | + | + | + | + | + |
| A/common magpie/Hong
Kong/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
10
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(Victoria | ||||
| lineage) | 109.3 | - | - | - | |
| B/Montana/5/2012 | 108.4 | - | - | - | |
| B/Wisconsin/01/2010 | B(Yamagata | ||||
| lineage) | 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 | - | - |
11
| 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.
12
| 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
13
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.
| Enzyme
Utilized | # of
Positives1 | % Positive
Agreement (95% CI) | # of
Negatives2 | % Negative
Agreement (95% CI) |
|---------------------------------|--------------------|----------------------------------|--------------------|----------------------------------|
| Quanta BioSciences
qScriptTM | 50/50 | 100 (92.9 – 100.00) | 65/65 | 100 (94.4 – 100.00) |
| Invitrogen
SuperScriptTM | 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.