(28 days)
The Influenza B Lineage Genotyping Kit contains reagents and controls of the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel and is intended for use in real-time RT-PCR (rRT-PCR) assays on an Applied Biosystems (ABI) 7500 Fast Dx Real-Time PCR instrument in conjunction with clinical and epidemiological information:
For the determination of the genetic lineage of human influenza B viruses as B/Victoria or B/Yamagata lineage from · viral RNA in upper respiratory tract clinical specimens (including nasopharyngeal swabs [NS], throat swabs [TS], nasal aspirates [NA], nasal washes [NW] and dual nasopharyngeal/throat swabs [NPS/TS]) from human patients with signs and symptoms of respiratory infection and/or from viral culture;
· To provide epidemiologic information for surveillance of circulating influenza viruses.
Performance characteristics for influenza B lineage genotyping were established during a season when influenza B/ Victoria and B/Yamagata lineages were in circulation.
Negative results do not preclude influenza virus infection and should not be used as the sole basis for treatment or other patient management decisions. Conversely, positive results do not rule out bacterial infection with other viruses. The agent detected may not be the definite cause of disease.
All users, and any person reporting results from use of this device should be trained to perform and interpret the results from this procedure by a competent instructor prior to use. CDC Influenza Division will limit the distribution of this device to only those users who have successfully completed a training course provided by CDC instructors or designees.
The CDC Human Influenza Real-Time RT-PCR Diagnostic Panel is used in real-time RT-PCR (rRT-PCR) assays on the Applied Biosystems® (ABI) 7500 Fast Dx Real-time PCR system. The panel is configured in four separate kits. Each kit consists of oligonucleotide primers. fluorescently labeled hydrolysis probes, and controls which are used in rRT-PCR assays for the in vitro qualitative detection and characterization of influenza virus RNA in respiratory specimens from patients presenting with influenza-like illness (ILI). Oligonucleotide primers and probes included in the Influenza B Lineage Genotyping Kit for detection of influenza B and the two major genetic lineages of influenza B were selected from highly conserved regions of the non-structural (NS) and HA genes, respectively. Oligonucleotide primers and probes to detect the human RNase P gene (RP) in control samples and clinical specimens are also included in the kit.
Here's a summary of the acceptance criteria and study details for the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel: Influenza B Lineage Genotyping Kit, based on the provided document:
This document describes a 510(k) premarket notification for modifications to an existing influenza B lineage genotyping kit (K172091). The changes primarily address recent evolutionary changes in circulating influenza B viruses that could impact the reactivity of the current kit, and the evaluation of an alternative fluorescent hydrolysis probe quencher chemistry (ZEN™). The study aims to demonstrate that the modified device remains substantially equivalent to the predicate device.
1. Table of Acceptance Criteria and Reported Device Performance
The acceptance criteria are generally demonstrated through 100% agreement or high percentage sensitivity/specificity with a clear lower bound for the 95% Confidence Interval (CI).
| Test Type | Acceptance Criteria | Reported Device Performance |
|---|---|---|
| Analytical Sensitivity (LOD Equivalency) | Demonstrating 100% positivity (3 out of 3 replicates) at either the same endpoint LOD concentration or within a 5-fold dilution of each other. | Influenza B Assay (BHQ vs. ZEN quencher): Both InfB (BHQ) and InfB (ZEN) showed similar reactivity and endpoint range with Influenza B/Nevada/03/2011 and B/Wisconsin/1/2010. For B/Nevada/03/2011, LOD for both was generally around 10^2.8 EID50/mL with Invitrogen Superscript™ and 10^3.5 EID50/mL with Quanta qScript™. For B/Wisconsin/1/2010, LOD for both was generally around 10^3.5 EID50/mL with Invitrogen Superscript™ and 10^4.2 EID50/mL with Quanta qScript™. Influenza B Lineage Genotyping Assay (VER 1.1 and VER 2 VIC & YAM assays): All assays demonstrated similar reactivity with apparent LOD endpoints within one 5-fold dilution of each other for various B/Victoria and B/Yamagata strains across both enzyme systems (Invitrogen Superscript™ and Quanta qScript™). For example, LOD for B/Maryland/15/2016 (VER 1.1 & VER 2) was 10^1.7 EID50/mL for both enzymes. |
| Analytical Sensitivity (LOD Confirmation) | Lowest virus concentration where InfB and VIC or InfB and YAM primer and probe sets demonstrated ≥ 95% detection. | Demonstrated LODs for B/Maryland/15/2016 (Victoria) were 10^1.7 EID50/mL for both VER 1.1 and VER 2 with both enzyme systems. Demonstrated LODs for B/Texas/81/2016 (Yamagata) were 10^2.2 EID50/mL (VER 1.1 & VER 2 with Invitrogen) and 10^1.5 EID50/mL (VER 1.1 & VER 2 with Quanta). |
| Analytical Sensitivity (Inclusivity) | 100% positivity (3 out of 3 replicates) for the tested strains at or near the established LOD. | B/Victoria (VER 1.1 & VER 2): All 10 tested B/Victoria strains showed 3/3 positive replicates (100% inclusivity) at or near their LOD for both VER 1.1 and VER 2, across both enzyme systems. B/Yamagata (VER 1.1 & VER 2): All 10 tested B/Yamagata strains showed 3/3 positive replicates (100% inclusivity) at or near their LOD for both VER 1.1 and VER 2, across both enzyme systems. |
| Analytical Specificity (Cross-Reactivity) | No cross-reactivity detected with the opposite influenza B lineage. | No cross-reactivity was detected with either VER 1.1 or VER 2 VIC and YAM primer/probe sets when tested against 10 high-titer B/Yamagata lineage viruses (for VIC assays) and 10 high-titer B/Victoria lineage viruses (for YAM assays), across both enzyme systems. (Results consistently showed '-') |
| Analytical Specificity (Exclusivity with Influenza A Viruses) | No cross-reactivity detected with various influenza A viruses. | No cross-reactivity was detected with either VER 1.1 or VER 2 VIC and YAM assays when tested against 8 high-titer Influenza A viruses of various human and animal subtypes, across both enzyme systems. (Results consistently showed '-') |
| Analytical Specificity (Exclusivity with Non-Influenza Respiratory Pathogens) | No cross-reactivity detected with common non-influenza respiratory pathogens or flora. | No cross-reactivity was detected with VER 2 VIC and YAM assays when tested against 36 organisms (16 viruses, 19 bacteria, and 1 yeast) at high titers, across both enzyme systems. (Results consistently showed '-') |
| Clinical Performance (Retrospective InfB Assay) | 100% positive and negative agreement with the cleared oligonucleotide probe (BHQ quencher). | 100.0% Positive Agreement (95% CI: 88.7-100.0) for 30/30 positive samples. 100.0% Negative Agreement (95% CI: 92.9-100.0) for 50/50 negative samples. (Both enzyme systems showed identical results). |
| Clinical Performance (VER 2 B/Victoria Prospective Study) | High sensitivity and specificity, typically aiming for >90% sensitivity and >95% specificity with a tight 95% CI. | Sensitivity: 100.0% (5/5 positives) (95% CI: 56.6 – 100.0). Specificity: 100.0% (574/574 negatives) (95% CI: 99.3 – 100.0). (Note: Low number of positives impacts CI width for sensitivity). |
| Clinical Performance (VER 2 B/Yamagata Prospective Study) | High sensitivity and specificity, typically aiming for >90% sensitivity and >95% specificity with a tight 95% CI. | Sensitivity: 100.0% (38/38 positives) (95% CI: 90.8 – 100.0). Specificity: 99.8% (540/541 negatives) (95% CI: 99.0 – 100.0). |
| Clinical Performance (Retrospective VER 1.1 & VER 2 VIC & YAM assays) | 100% positive and negative agreement with the FDA-cleared Influenza B Lineage Genotyping Kit. | VIC VER 1.1: 100.0% Positive Agreement (30/30) (95% CI: 88.7 - 100.0); 100.0% Negative Agreement (157/157) (95% CI: 97.6 - 100.0). YAM VER 1.1: 100.0% Positive Agreement (96/96) (95% CI: 96.2 - 100.0); 100.0% Negative Agreement (91/91) (95% CI: 96.0 - 100.0). VIC VER 2: 100.0% Positive Agreement (30/30) (95% CI: 88.7 - 100.0); 100.0% Negative Agreement (157/157) (95% CI: 97.6 - 100.0). YAM VER 2: 100.0% Positive Agreement (96/96) (95% CI: 96.2 - 100.0); 100.0% Negative Agreement (91/91) (95% CI: 96.0 - 100.0). |
2. Sample Size for the Test Set and Data Provenance
Test Set Sample Sizes:
- Analytical Sensitivity (LOD Equivalency & Inclusivity): Varied. Typically, 3 replicates per dilution/strain. For LOD confirmation, 20 extraction replicates per dilution.
- Analytical Specificity (Cross-Reactivity & Exclusivity): For cross-reactivity, 10 B/Yamagata strains for VIC assays and 10 B/Victoria strains for YAM assays. For exclusivity with Influenza A, 8 Influenza A strains. For exclusivity with non-influenza pathogens, 36 organisms (16 viruses, 19 bacteria, 1 yeast). All tested with both enzyme systems.
- Clinical Performance (InfB Assay Retrospective): 30 positive and 50 negative upper respiratory tract clinical samples.
- Clinical Performance (VER 2 Prospective Study):
- Total collected: 592 samples.
- Analyzed: 579 upper respiratory tract clinical specimens.
- B/Victoria: 5 positive, 574 negative.
- B/Yamagata: 38 positive, 541 negative.
- Clinical Performance (VER 1.1 & VER 2 Retrospective Study):
- 126 specimens positive for either influenza B/Victoria or B/Yamagata viruses.
- 61 specimens negative for influenza B viruses.
- (An additional 4 lung tissue specimens were tested for informational purposes but not included in performance calculations).
Data Provenance:
- Analytical studies: Laboratory-characterized virus stocks and reference strains (e.g., CDC repositories, ATCC).
- Clinical Performance (InfB Assay Retrospective): Retrospective clinical samples collected during the 2011-2012 influenza season.
- Clinical Performance (VER 2 Prospective Study): Prospective clinical investigation conducted at 3 U.S. public health laboratories using upper respiratory tract specimens collected during the 2016-2017 influenza season.
- Clinical Performance (VER 1.1 & VER 2 Retrospective Study): Retrospective upper respiratory tract clinical samples collected during the 2017 and 2017-2018 influenza seasons.
Country of Origin: Primarily the United States (CDC, U.S. public health laboratories) with some virus strains noted as originating from various international locations (e.g., Hong Kong, Bolivia, Laos, Michigan, etc.) for inclusivity and exclusivity testing, reflecting global diversity.
3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications
The ground truth for the test sets (both analytical and clinical) appears to be established by molecular diagnostic methods using an FDA-cleared predicate device, the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel: Influenza B Lineage Genotyping Kit (K172091), and/or by using well-characterized virus stocks with known titers (EID50/mL or TCID50/mL).
For clinical samples, they were "previously determined to be positive or negative for influenza B virus" or "determined to be positive for influenza B/Victoria or B/Yamagata viruses using the FDA-cleared Influenza B Lineage Genotyping Kit."
The document does not explicitly mention the use of a specific "number of experts" (e.g., clinicians, virologists, or laboratory professionals) for establishing the individual patient-level clinical ground truth for the test sets in the same way one might describe image interpretation by radiologists. Instead, the ground truth is primarily based on the results of the already FDA-cleared predicate diagnostic test or characterized reference materials. The "competent instructor" trained individuals (users, analysts, and those reporting results) on how to perform and interpret the device results.
4. Adjudication Method for the Test Set
The document does not describe an adjudication method for conflicting results in the test set, such as a 2+1 or 3+1 system. This is typical for molecular diagnostic studies where the comparison is generally made against a well-established reference method (the predicate device or characterized stocks). Any discrepancies would likely be investigated to determine the cause (e.g., retesting, review of sample integrity, or confirmation by alternative gold standard) rather than being resolved by expert consensus in a traditional adjudication panel format. For example, in the LOD studies, "3 out of 3 replicates" or "≥ 95% detection" criteria are used, indicating a reliance on consistent assay performance rather than expert adjudication.
5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study
No, a multi-reader multi-case (MRMC) comparative effectiveness study was not explicitly described. MRMC studies are typically performed for imaging devices or other diagnostics where human interpretation is a key component and the effect of AI assistance on human performance is being evaluated. This submission is for a molecular diagnostic (RT-PCR kit) where the output is a quantitative measurement (Ct values) and qualitative result (positive/negative, lineage determination) interpreted directly by the instrument software or a trained individual following predefined rules, not a subjective interpretation by multiple human readers.
Therefore, there is no mention of an effect size of how much human readers improve with AI vs. without AI assistance.
6. Standalone (Algorithm Only) Performance
Yes, the studies presented primarily represent standalone performance of the device (or its modified components). The analytical performance evaluations (sensitivity, inclusivity, specificity, exclusivity) directly assess the algorithm's (reagent kit's) ability to detect and differentiate influenza B lineages from viral RNA. The clinical performance studies compare the results of the investigational device directly against the FDA-cleared predicate device on clinical samples. While human technicians perform the laboratory procedures, the "algorithm" here refers to the specific RT-PCR assay chemistry, probes, and primer sets that yield a result, which is then interpreted according to established cut-offs/rules, not an AI algorithm that assists human interpretation.
7. Type of Ground Truth Used
The ground truth used for the studies is a combination of:
- Reference standard/Predicate device: For clinical samples, the ground truth was largely established by the "FDA-cleared CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel: Influenza B Lineage Genotyping Kit."
- Characterized virus stocks: For analytical studies (LOD, inclusivity, cross-reactivity, exclusivity), the ground truth was established using well-characterized influenza virus stocks and other respiratory pathogens with known strain designations, subtypes, and titers (EID50/mL, TCID50/mL, CFU/mL, or ng/µL). This implies a high level of confidence in the identity and concentration of the biological material.
8. Sample Size for the Training Set
The document does not explicitly state a sample size for a "training set" in the context of an algorithm's development. This is because the device described is a molecular diagnostic kit (RT-PCR) with specific primers and probes designed based on biological knowledge of viral sequences, rather than a machine learning or artificial intelligence algorithm that requires a distinct training phase with labeled data.
The "modifications" were "primarily to address recent evolutionary changes in circulating influenza B viruses that may impact the reactivity of the current Influenza B Lineage Genotyping Kit. Two design approaches were evaluated that address specific genetic mutations in the targeted hemagglutinin (HA) gene..." This indicates a design process driven by biological knowledge and engineering based on viral genetics, not data-driven machine learning training.
9. How the Ground Truth for the Training Set was Established
Since there's no explicitly defined "training set" for an AI algorithm in the provided text, the concept of establishing ground truth for it doesn't directly apply. The design of the new VER 1.1 and VER 2 assays was based on scientific understanding of circulating influenza B virus evolutionary changes and specific genetic mutations in the HA gene. This informed the selection of the oligonucleotide primers and probes. The efficacy of these new designs was then evaluated using the analytical and clinical studies described in the rest of the document, where the ground truth was established as detailed in point 7.
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Image /page/0/Picture/0 description: The image shows the logo of the U.S. Food and Drug Administration (FDA). On the left is the Department of Health & Human Services logo. To the right of that is the FDA logo, with the letters "FDA" in a blue square. To the right of the blue square is the text "U.S. FOOD & DRUG ADMINISTRATION" in blue.
July 30, 2018
Centers for Disease Control and Prevention (CDC) Yon Yu, Pharm.D. Associate Director for Regulatory Affairs 1600 Clifton Road Ms E-51 Atlanta, GA 30329-4027
Re: K181736
Trade/Device Name: CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel: Influenza B Lineage Genotyping Kit Regulation Number: 21 CFR 866.3980 Regulation Name: Respiratory viral panel multiplex nucleic acid assay Regulatory Class: Class II Product Code: OZE, NSU, OOI Dated: June 29, 2018 Received: July 2, 2018
Dear Yon Yu:
We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.
If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.
Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR
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Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.
Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm.
For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/) and CDRH Learn (http://www.fda.gov/Training/CDRHLearn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (http://www.fda.gov/DICE) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).
Sincerely,
Steven R. Gitterman -S
for
Uwe Scherf, Ph.D. Director Division of Microbiology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health
Enclosure
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Indications for Use
510(k) Number (if known) K181736
Device Name
CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel: Influenza B Lineage Genotyping Kit
Indications for Use (Describe)
The Influenza B Lineage Genotyping Kit contains reagents and controls of the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel and is intended for use in real-time RT-PCR (rRT-PCR) assays on an Applied Biosystems (ABI) 7500 Fast Dx Real-Time PCR instrument in conjunction with clinical and epidemiological information:
For the determination of the genetic lineage of human influenza B viruses as B/Victoria or B/Yamagata lineage from · viral RNA in upper respiratory tract clinical specimens (including nasopharyngeal swabs [NS], throat swabs [TS], nasal aspirates [NA], nasal washes [NW] and dual nasopharyngeal/throat swabs [NPS/TS]) from human patients with signs and symptoms of respiratory infection and/or from viral culture;
· To provide epidemiologic information for surveillance of circulating influenza viruses.
Performance characteristics for influenza B lineage genotyping were established during a season when influenza B/ Victoria and B/Yamagata lineages were in circulation.
Negative results do not preclude influenza virus infection and should not be used as the sole basis for treatment or other patient management decisions. Conversely, positive results do not rule out bacterial infection with other viruses. The agent detected may not be the definite cause of disease.
All users, and any person reporting results from use of this device should be trained to perform and interpret the results from this procedure by a competent instructor prior to use. CDC Influenza Division will limit the distribution of this device to only those users who have successfully completed a training course provided by CDC instructors or designees.
| Type of Use (Select one or both, as applicable) | |
|---|---|
| Prescription Use (Part 21 CFR 801 Subpart D) | Over-The-Counter Use (21 CFR 801 Subpart C) |
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8. 510(k) Summary
I. GENERAL INFORMATION
Submitter: Centers for Disease Control and Prevention 1600 Clifton Road, NE Atlanta, GA 30329
Contact Person: CDR Yon Yu, Pharm. D. Associate Director for Regulatory Affairs Office of the Director National Center for Emerging and Zoonotic Infectious Diseases Centers for Disease Control and Prevention 1600 Clifton RD, MS E-51; Atlanta, GA 30329-4027 Phone: 404-639-3046 Fax: 404-498-1106 Email: fkb8@cdc.gov
Date Prepared: June 28, 2018
DEVICE INFORMATION II.
| Proprietary Name: | CDC Human Influenza Virus Real-Time RT-PCR Diagnostic PanelInfluenza B Lineage Genotyping Kit (VER 1.1) and Influenza BLineage Genotyping Kit (VER 2) |
|---|---|
| Common Name: | Influenza B Lineage Genotyping Kit |
| Regulation Section: | 866.3980-Respiratory viral panel multiplex nucleic acid assay |
| Subsequent RegulationSections: | 862.2570-Instrumentation for clinical multiplex systems |
| Device Classification: | Class II |
| Product Code: | OZE |
| Subsequent Product Codes: | NSU, OOI |
| Panel: | Microbiology |
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III. PREDICATE DEVICE
CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel: Influenza B Lineage Genotyping Kit (K172091)
IV. DEVICE DESCRIPTION
The CDC Human Influenza Real-Time RT-PCR Diagnostic Panel is used in real-time RT-PCR (rRT-PCR) assays on the Applied Biosystems® (ABI) 7500 Fast Dx Real-time PCR system. The panel is configured in four separate kits. Each kit consists of oligonucleotide primers. fluorescently labeled hydrolysis probes, and controls which are used in rRT-PCR assays for the in vitro qualitative detection and characterization of influenza virus RNA in respiratory specimens from patients presenting with influenza-like illness (ILI). Oligonucleotide primers and probes included in the Influenza B Lineage Genotyping Kit for detection of influenza B and the two major genetic lineages of influenza B were selected from highly conserved regions of the non-structural (NS) and HA genes, respectively. Oligonucleotide primers and probes to detect the human RNase P gene (RP) in control samples and clinical specimens are also included in the kit.
INTENDED USE V.
The Influenza B Lineage Genotyping Kit contains reagents and controls of the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel and is intended for use in real-time RT-PCR (rRT-PCR) assays on an Applied Biosystems (ABI) 7500 Fast Dx Real-Time PCR instrument in conjunction with clinical and epidemiological information:
. For the determination of the genetic lineage of human influenza B viruses as B/Victoria or B/Yamagata lineage from viral RNA in upper respiratory tract clinical specimens (including nasopharyngeal swabs [NPS], nasal swabs [NS], throat swabs [TS], nasal aspirates [NA], nasal washes [NW] and dual nasopharyngeal/throat swabs [NPS/TS]) from human patients with signs and symptoms of respiratory infection and/or from viral culture;
- To provide epidemiologic information for surveillance of circulating influenza viruses. .
Performance characteristics for influenza B lineage genotyping were established during a season when influenza B/Victoria and B/Yamagata lineages were in circulation.
Negative results do not preclude influenza virus infection and should not be used as the sole basis for treatment or other patient management decisions. Conversely, positive results do not rule out bacterial infection or co-infection with other viruses. The agent detected may not be the definite cause of disease.
All users, analysts, and any person reporting results from use of this device should be trained to perform and interpret the results from this procedure by a competent instructor prior to use. CDC Influenza Division will limit the distribution of this device to only those users who have successfully completed a training course provided by CDC instructors or designees.
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VI. TECHNOLOGICAL CHARACTERISTICS
The technological characteristics of the modified CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel- Influenza B Lineage Genotyping Kit (VER 1.1. and VER 2) remain the same as the predicate device. Modifications were made primarily to address recent evolutionary changes in circulating influenza B viruses that may impact the reactivity of the current Influenza B Lineage Genotyping Kit. Two design approaches were evaluated that address specific genetic mutations in the targeted hemagglutinin (HA) gene of influenza B viruses.
CDC also evaluated the ZENTM Double-Quenched Probe technology (manufactured by Integrated DNA Technologies) as an alternate fluorescent hydrolysis probe quencher chemistry for the InfB assay of the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel.
SUBSTANTIAL EQUIVALENCE COMPARISON VII.
The CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel, Influenza B Lineage Genotyping Kit (K172091), will serve as the predicate for the proposed change. See table 8-1 below for a detailed comparison of the modified device to the predicate.
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| Table 8-1: Device Comparison | ||
|---|---|---|
| Item | Predicate Device | Proposed Device |
| CDC Human Influenza Virus Real-Time RT-PCRDiagnostic Panel, Influenza B Lineage GenotypingKit [K172091] | CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel, Influenza B LineageGenotyping Kit (VER 1.1 and VER 2) | |
| The Influenza B Lineage Genotyping Kit containsreagents and controls of the CDC Human InfluenzaVirus Real-Time RT-PCR Diagnostic Panel and isintended for use in real-time RT-PCR (rRT-PCR)assays on an Applied Biosystems (ABI) 7500 Fast DxReal-Time PCR instrument in conjunction withclinical and epidemiological information: | Same | |
| For the determination of the genetic lineage ofhuman influenza B viruses as B/Victoria orB/Yamagata lineage from viral RNA in upperrespiratory tract clinical specimens (includingnasopharyngeal swabs [NPS], nasal swabs [NS],throat swabs [TS], nasal aspirates [NA], nasalwashes [NW] and dual nasopharyngeal/throatswabs [NPS/TS]) from human patients withsigns and symptoms of respiratory infectionand/or from viral culture; To provide epidemiologic information forsurveillance of circulating influenza viruses. | ||
| Intended Use | Performance characteristics for influenza B lineagegenotyping were established during a season wheninfluenza B/Victoria and B/Yamagata lineages werefound in approximately equal proportion.Negative results do not preclude influenza virusinfection and should not be used as the sole basis fortreatment or other patient management decisions.Conversely, positive results do not rule out bacterialinfection or co-infection with other viruses. The agentdetected may not be the definite cause of disease. | |
| All users, analysts, 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 | Influenza B virus, lineages B/Victoria andB/Yamagata | Same |
| Specimen Types | Nasopharyngeal swabs, nasal swabs, throat swabs,nasal aspirates, nasal washes and dualnasopharyngeal/throat swabs from human patientswith signs and symptoms of respiratory infectionand/or from viral culture | Same |
| TechnologicalCharacteristics | Real-time RT-PCR based assay | Same |
| 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 QIAcube - QIAamp® DSP Viral RNA Mini Kit, QIAGEN NucliSENS® easyMAG®, bioMérieux EZ1 Advanced XL – EZ1 DSP Virus Kit and EZ1 RNATissue Mini Kit, QIAGEN MagNA Pure 96 - DNA and Viral NA Small Volume Kit,Roche | Same |
| Enzyme MasterMix | Invitrogen SuperScript™ III Platinum® One-Step | Same |
| Quanta BioSciences qScript™ One-Step qRT-PCRKit, Low ROX | ||
| RequiredInstrumentation | Applied Biosystems 7500 Fast Dx Real-Time PCRInstrument with SDS software version 1.4 | Same |
| Probe QuenchingMolecule | Black Hole Quencher Probe® (BHQ-1) [InfB assay]Black Hole Quencher Plus (BHQ Plus ) [VIC, YAM assays] | ZEN™ or BHQ-1 (InfB assay)BHQ Plus or BHQ-1 (VIC, YAM assays) |
Table 8-1: Device Comparison
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VIII. ANALYTICAL PERFORMANCE EVALUATION
Analytical Sensitivity - Influenza B Assay
A range finding study was performed to demonstrate LOD equivalency between the currently cleared InfB assay probe quenched with BHQ and the same probe quenched with ZEN. Two characterized influenza vaccine reference viruses of a known 50% egg infectious dose titer (EID50/mL) were extracted using the Roche MagNA Pure Compact RNA Isolation Kit. The RNA was serially diluted and tested (n=3 replicates) in order to determine the apparent endpoint range using both enzymes cleared for use with the CDC Human Influenza Real-Time RT-PCR Diagnostic Panel. The acceptance criterion for LOD equivalency was defined as demonstrating 100% positivity (3 out of 3 replicates) at either the same endpoint LOD concentration or within a 5-fold dilution of each other. All assays demonstrated similar reactivity (Tables 8-2 and 8-3).
| InfluenzaB/Nevada/03/2011Titer (EID50/mL) | InvitrogenSuperscript™ | Quanta qScript™ | ||
|---|---|---|---|---|
| InfB (BHQ)IVD | InfB (ZEN) | InfB (BHQ)IVD | InfB (ZEN) | |
| 104.2 | 3/3*(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| 103.5 | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| 102.8 | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| 102.1 | 1/3(+) | 1/3(+) | 0/3(-) | 0/3(-) |
| 101.4 | 2/3(+) | 0/3(-) | 0/3(-) | 0/3(-) |
| 100.7 | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) |
| 100.0 | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) |
Table 8-2: Influenza B (InfB) Assay LOD Equivalencv - B/Nevada/03/2011
Indicates number of positive replicates out of three total.
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| InfluenzaB/Wisconsin/1/2010Titer (EID50/mL) | Invitrogen Superscript™ | Quanta qScript™ | ||
|---|---|---|---|---|
| InfB (BHQ)IVD | InfB (ZEN) | InfB (BHQ)IVD | InfB (ZEN) | |
| 104.2 | 3/3*(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| 103.5 | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| 102.8 | 1/3(+) | 0/3(-) | 0/3(-) | 0/3(-) |
| 102.1 | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) |
| 101.4 | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) |
| 100.7 | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) |
| 100.0 | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) |
Table 8-3: Influenza B (InfB) Assay LOD Equivalency - B/Wisconsin/1/2010
Indicates number of positive replicates out of three total.
Analytical Sensitivity- Influenza B Lineage Genotyping Assay Update
A range finding study was performed with the currently cleared assays of the Influenza B Lineage Genotyping Kit and with modified VIC and YAM assays (VER 1.1) as well as newly designed VIC and Y AM assays (VER 2). These studies were performed to demonstrate the LOD equivalency and improved reactivity of the VER 1.1 and VER 2 VIC and YAM assays with one historical benchmark strain and one current strain of both the B/Victoria and B/Yamagata lineages of influenza B virus. The test samples consisted of serially diluted RNA extracted with the Roche MagNA Pure Compact RNA Isolation Kit from characterized virus stocks of known 50% egg infectious dose titer (EID50mL). Three replicates per dilution were tested to determine the apparent endpoint range (Tables 8-4 to 8-7) using both enzymes cleared for use with the CDC Human Influenza Real-Time RT-PCR Diagnostic Panel. The acceptance criterion for LOD equivalency was defined as demonstrating 100% positivity (3 out of 3 replicates) at either the same endpoint LOD concentration or within a 5-fold dilution of each other. All assays demonstrated similar reactivity with apparent LOD endpoints within one 5-fold dilution of each other.
{9}------------------------------------------------
| InfluenzaB/Nevada/03/2011Titer (EID50/mL) | Invitrogen Superscript™ | Quanta qScript™ | ||||||
|---|---|---|---|---|---|---|---|---|
| InfBIVD | VICIVD | VICVER 1.1 | VICVER 2 | InfBIVD | VICIVD | VICVER 1.1 | VICVER 2 | |
| 104.2 | 3/3* | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| (+) | (+) | (+) | (+) | (+) | (+) | (+) | (+) | |
| 103.5 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| (+) | (+) | (+) | (+) | (+) | (+) | (+) | (+) | |
| 102.8 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| (+) | (+) | (+) | (+) | (+) | (+) | (+) | (+) | |
| 102.1 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| (+) | (+) | (+) | (+) | (+) | (+) | (+) | (+) | |
| 101.4 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 0/3 | 3/3 | 3/3 |
| (+) | (+) | (+) | (+) | (+) | (+) | (+) | (+) | |
| 100.7 | 1/3 | 0/3 | 2/3 | 3/3 | 0/3 | 0/3 | 1/3 | 0/3 |
| (+) | (-) | (+) | (+) | (-) | (+) | (+) | (-) | |
| 100.0 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| (-) | (-) | (-) | (-) | (-) | (-) | (-) | (-) | |
| 10-0.7 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| (-) | (-) | (-) | (-) | (-) | (-) | (-) | (-) |
Table 8-4: B/Victoria Assay LOD Equivalency- VER 1.1 and VER 2 Designs - B/Nevada/03/2011
- Indicates number of positive replicates out of three total.
| Table 8-5: B/Victoria Assay LOD Equivalency- VER 1.1 and VER 2 Designs - B/Marryand/15/2016 |
|---|
| InfluenzaB/Maryland/15/2016Titer (EID50/mL) | Invitrogen Superscript™ | Quanta qScript™ | ||||||
|---|---|---|---|---|---|---|---|---|
| InfBIVD | VICIVD | VICVER 1.1 | VICVER 2 | InfBIVD | VICIVD | VICVER 1.1 | VICVER 2 | |
| 104.5 | 3/3*(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| 103.8 | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| 103.1 | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| 102.4 | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| 101.7 | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 2/3(+) | 3/3(+) | 3/3(+) |
| 101.0 | 3/3(+) | 0/3(-) | 3/3(+) | 3/3(+) | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) |
| 100.3 | 1/3(+) | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) |
| 10-0.4 | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) |
- Indicates number of positive replicates out of three total.
{10}------------------------------------------------
| InfluenzaB/Texas/06/2011Titer (EID50/mL) | Invitrogen Superscript™ | Quanta qScript™ | ||||||
|---|---|---|---|---|---|---|---|---|
| InfBIVD | YAMIVD | YAMVER 1.1 | YAMVER 2 | InfBIVD | YAMIVD | YAMVER 1.1 | YAMVER 2 | |
| 104.9 | 3/3*(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| 104.2 | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| 103.5 | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 2/3(+) | 3/3(+) |
| 102.8 | 3/3(+) | 3/3(+) | 0/3(-) | 3/3(+) | 3/3(+) | 2/3(+) | 0/3(-) | 3/3(+) |
| 102.1 | 2/3(+) | 1/3(+) | 1/3(+) | 1/3(+) | 0/3(-) | 2/3(+) | 0/3(-) | 2/3(+) |
| 101.4 | 2/3(+) | 0/3(-) | 0/3(-) | 1/3(+) | 0/3(-) | 1/3(+) | 0/3(-) | 2/3(+) |
| 100.7 | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) |
| 100.0 | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) |
Table 8-6: B/Yamagata Assay LOD Equivalency- VER 1.1 and VER 2 Designs - B/Texas/06/2011
- Indicates number of positive replicates out of three total.
| Table 8-7: B/Yamagata Assay LOD Equivalency- VER 1.1 and VER 2 Designs - B/Texas/81/2016 | ||
|---|---|---|
| InfluenzaB/Texas/81/2016Titer (EID50/mL) | Invitrogen SuperscriptTM | Quanta qScriptTM | ||||||
|---|---|---|---|---|---|---|---|---|
| InfBIVD | YAMIVD | YAMVER 1.1 | YAMVER 2 | InfBIVD | YAMIVD | YAMVER 1.1 | YAMVER 2 | |
| 104.3 | 3/3*(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| 103.6 | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| 102.9 | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| 102.2 | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| 101.5 | 3/3(+) | 2/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) | 1/3(+) | 2/3(+) |
| 100.8 | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) | 1/3(+) | 0/3(-) | 0/3(-) |
| 100.1 | 1/3(+) | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) | 2/3(+) | 0/3(-) | 0/3(-) |
| 10-0.6 | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) | 0/3(-) |
- Indicates number of positive replicates out of three total.
{11}------------------------------------------------
A confirmation of the LOD for each modified or new assay was performed using a current strain of the corresponding influenza B/Victoria or B/Yamagata lineage. The VER 1.1 and VER 2 VIC and YAM primer and probe sets were tested alongside the currently cleared InfB assay as required for interpretation by the routine testing algorithm. The confirmatory LOD testing for each primer and probe set was performed on extraction replicates (n=20) of each dilution. The lowest virus concentration where InfB and VIC or InfB and YAM primer and probes sets demonstrated ≥ 95% detection is reported as the LOD for each virus (Table 8-8).
| Influenza B Lineage | Influenza Strain Designation | Assay Design | LOD (EID50/mL) | |
|---|---|---|---|---|
| Invitrogen SuperScriptTM | Quanta qScriptTM | |||
| Victoria | B/Maryland/15/2016 | VER 1.1 | 101.7 | 101.7 |
| VER 2 | 101.7 | 101.7 | ||
| Yamagata | B/Texas/81/2016 | VER 1.1 | 102.2 | 101.5 |
| VER 2 | 102.2 | 101.5 |
Table 8-8: LOD Confirmation Summary
Analytical Sensitivity - Inclusivity
Inclusivity testing was performed to demonstrate the capability of the modified VER 1.1 and VER 2 VIC and YAM primer and probe sets to detect strains of the corresponding influenza B lineage at or near the established LOD. Ten influenza B viruses of each lineage and representative of different geographic locations and phylogenetic clades were selected. Characterized stocks were serially diluted to concentrations near the LOD of the assays and extracted using the Roche MagNA Pure Compact RNA Isolation Kit. Samples were tested in triplicate with the VER 2 VIC and YAM assays with both enzyme systems cleared for use with the CDC Human Influenza Real-Time RT-PCR Diagnostic Panel. The inclusivity results are summarized in Tables 8-9 and 8-10.
{12}------------------------------------------------
| Influenza VirusStrain Designation | EID50/mL orTCID50/mL | InvitrogenSuperScript™ | Quanta qScript™ | ||
|---|---|---|---|---|---|
| VICVER 1.1 | VICVER 2 | VICVER 1.1 | VICVER 2 | ||
| B/Hong Kong/259/2010 | 10 1.2 | 3/3*(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| B/Bolivia/1526/2010 | 10 2.4 | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| B/Laos/89/2011 | 10 1.2 | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| B/Michigan/09/2011 | 10 1.5 | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| B/New Jersey/01/2012 | 10 1.9 | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| B/Montana/05/2012 | 10 2.4 | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| B/Texas/02/2013 | 10 2.1 | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| B/Florida/103/2016 | 10 0.3 | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| B/Florida/76/2016 | 10 1.5 | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| B/Hong Kong/269/2017 | 10 -0.8 | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) |
Table 8-9: B/Victoria Assay Inclusivity VER 1.1 and VER 2 Designs
- Indicates number of positive replicates out of three total.
{13}------------------------------------------------
| Influenza VirusStrain Designation | EID50 /mLorTCID50/mL | InvitrogenSuperScript™ | Quanta qScript™ | ||
|---|---|---|---|---|---|
| YAMVER 1.1 | YAMVER 2 | YAMVER 1.1 | YAMVER 2 | ||
| B/Brisbane/03/2007 | 10 2.4 | 3/3*(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| B/Pennsylvania/07/2007 | 10 2.2 | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| B/Hubei-Wujiagang/158/2009 | 10 2.2 | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| B/Wisconsin/01/2010 | 10 2.9 | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| B/Finland/39/2010 | 10 2.1 | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| B/Estonia/55669/2011 | 10 2.8 | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| B/Taiwan/1242/2011 | 10 3.9 | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| B/Massachusetts/02/2012 | 10 2.2 | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| B/Phuket/3073/2013 | 10 2.9 | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) |
| B/Guangdong-Liwan/1133/2014 | 10 3.9 | 3/3(+) | 3/3(+) | 3/3(+) | 3/3(+) |
Table 8-10: B/Yamagata Assay Inclusivity VER 1.1 and VER 2 Designs
Indicates number of positive replicates out of three total.
Analytical Specificity – Cross-Reactivity
Cross-reactivity of the modified VER 1.1 and VER 2 VIC and YAM primer and probe sets was evaluated by testing each set with influenza B viruses of the opposite lineage and from diverse geographic locations. Samples were extracted from high titer preparations of viruses (≥ 106 EID50 or TCID50/mL) using the Roche MagNA Pure Compact RNA Isolation Kit. Cross-reactivity was evaluated with both enzyme systems cleared for use with the CDC Human Influenza Real-Time RT-PCR Diagnostic Panel. No cross-reactivity was detected with either primer and probe set design (Tables 8-11 and 8-12).
|--|
| Influenza VirusStrain DesignationB/Yamagata Lineage | EID50/mLorTCID50/mL | InvitrogenSuperScript™ | Quanta qScript™ | ||
|---|---|---|---|---|---|
| VICVER 1.1 | VICVER 2 | VICVER 1.1 | VICVER 2 | ||
| B/Brisbane/03/2007 | 10 8.2 | - | - | - | - |
| B/Pennsylvania/07/2007 | 10 8.4 | - | - | - | - |
| B/Hubei-Wujiagang/158/2009 | 10 6.2 | - | - | - | - |
| B/Wisconsin/01/2010 | 10 8.5 | - | - | - | - |
| B/Finland/39/2010 | 10 8.9 | - | - | - | - |
| B/Estonia/55669/2011 | 10 8.4 | - | - | - | - |
| B/Taiwan/1242/2011 | 10 9.1 | - | - | - | - |
| B/Massachusetts/02/2012 | 10 6.3 | - | - | - | - |
| B/Phuket/3073/2013 | 10 6.5 | - | - | - | - |
| B/Guangdong-Liwan/1133/2014 | 10 4.2 | - | - | - | - |
{14}------------------------------------------------
| Influenza VirusStrain DesignationB/Victoria Lineage | EID50/mLorTCID50/mL | InvitrogenSuperScript™ | QuantaqScript™ | ||
|---|---|---|---|---|---|
| YAMVER 1.1 | YAMVER 2 | YAMVER 1.1 | YAMVER 2 | ||
| B/Hong Kong/259/2010 | 10 $8.4$ | - | - | - | - |
| B/Bolivia/1526/2010 | 10 $8.2$ | - | - | - | - |
| B/Laos/89/2011 | 10 $8.2$ | - | - | - | - |
| B/Michigan/09/2011 | 10 $8.9$ | - | - | - | - |
| B/New Jersey/01/2012 | 10 $8.1$ | - | - | - | - |
| B/Montana/05/2012 | 10 $7.8$ | - | - | - | - |
| B/Texas/02/2013 | 10 $9.9$ | - | - | - | - |
| B/Florida/103/2016 | 10 $9.2$ | - | - | - | - |
| B/Florida/76/2016 | 10 $9.9$ | - | - | - | - |
| B/Hong Kong/269/2017 | 10 $9.9$ | - | - | - | - |
Table 8-12: B/Yamagata Assay Cross-Reactivity VER 1.1 and VER 2 Designs
Analytical Specificity - Exclusivity
Exclusivity with Influenza A Viruses
Exclusivity of the VER 1.1 and VER 2 VIC and YAM assays was examined with influenza A viruses of various subtypes that circulate in humans and from animal origin that infect humans (Table 8-13 and 8-14). Samples were prepared from characterized, high titer stocks (≥106 TCID50 or EID50/mL) by extracting RNA using the Roche MagNA Pure Compact RNA Isolation Kit. Testing was performed using both of the currently cleared enzyme systems.
| Table 8-13: Exclusivity with Influenza A Viruses- VER 1.1 and VER 2 -Invitrogen SuperScript™ | ||||
|---|---|---|---|---|
| Influenza VirusStrain Designation | Origin | Subtype | EID50/mLorTCID50/mL | Invitrogen SuperScript™ | |||
|---|---|---|---|---|---|---|---|
| VICVER 1.1 | VICVER 2 | YAMVER 1.1 | YAMVER 2 | ||||
| A/Michigan/45/2015 | Human | A(H1N1)pdm09 | $10^{8.3}$ | - | - | - | - |
| A/Hong Kong/4801/2014 | Human | A(H3N2) | $10^{7.9}$ | - | - | - | - |
| A/Ohio/35/2017 | Swine | A(H1N2)v | $10^{6.9}$ | - | - | - | - |
| A/Ohio/13/2017 | Swine | A(H3N2)v | $10^{6.9}$ | - | - | - | - |
| A/gyrfalcon/Washington/41088-6/2014 | Avian | A(H5N8) | $10^{9.75}$ | - | - | - | - |
| A/Northern pintail/Washington/40964/2014 | Avian | A(H5N2) | $10^{9.4}$ | - | - | - | - |
| A/Bangladesh/0994/2011 | Avian | A(H9N2) | $10^{10.5}$ | - | - | - | - |
| A/Anhui/01/2013 | Avian | A(H7N9) | $10^{10.9}$ | - | - | - | - |
{15}------------------------------------------------
| Influenza VirusStrain Designation | Origin | Subtype | EID50/mLorTCID50/mL | Quanta qScript™ | |||
|---|---|---|---|---|---|---|---|
| VICVER 1.1 | VICVER 2 | YAMVER 1.1 | YAMVER 2 | ||||
| A/Michigan/45/2015 | Human | A(H1N1)pdm09 | 108.3 | - | - | - | - |
| A/Hong Kong/4801/2014 | Human | A(H3N2) | 107.9 | - | - | - | - |
| A/Ohio/35/2017 | Swine | A(H1N2)v | 106.9 | - | - | - | - |
| A/Ohio/13/2017 | Swine | A(H3N2)v | 106.9 | - | - | - | - |
| A/gyrfalcon/Washington/41088-6/2014 | Avian | A(H5N8) | 109.75 | - | - | - | - |
| A/Northern pintail/Washington/40964/2014 | Avian | A(H5N2) | 109.4 | - | - | - | - |
| A/Bangladesh/0994/2011 | Avian | A(H9N2) | 1010.5 | - | - | - | - |
| A/Anhui/01/2013 | Avian | A(H7N9) | 1010.9 | - | - | - | - |
Table 8-14: Exclusivity with Influenza A Viruses- VER 1.1 and VER 2 -Quanta qScript™
Exclusivity with Non-Influenza Respiratory Pathogens
Exclusivity of the VER 2 VIC and YAM assays was examined with non-influenza human respiratory viruses, bacteria, and yeast (Table 8-15). Nucleic acids were extracted using the Roche MagNA Pure Compact RNA Isolation Kit from 36 organisms (16 viruses, 19 bacteria, and 1 yeast) representing common respiratory pathogens or flora commonly present in specimens collected from the nasopharynx region. All bacteria, yeast, and non-influenza viruses were from CDC repositories or acquired from American Type Culture Collection (ATCC, Manassas, VA) and tested at high titers, typically ≥ 100 TCID50 or EID50/mL, ≥ 10° CFU/mL, or as high as culture allowed. Testing was performed using both of the currently cleared enzyme systems.
{16}------------------------------------------------
| Organism Tested | SuperScript | qScript | ||||
|---|---|---|---|---|---|---|
| Bacteria and Yeast | Strain | cfu / mL | VICVER2 | YAMVER2 | VICVER2 | YAMVER2 |
| Bordetella pertussis | A639 | 10 8.3 | - | - | - | - |
| Candida albicans | 2001-21-196 | 10 8.8 | - | - | - | - |
| Chlamydia pneumoniae1 | TW183 | 40 IFU/mL | - | - | - | - |
| Corynebacterium diphtheriae | NA2 | 10 10 | - | - | - | - |
| Escherichia coli | K12 | 10 9.6 | - | - | - | - |
| Haemophilus influenzae | M15709 | 10 6.4 | - | - | - | - |
| Lactobacillus plantarum | NA | 10 8.8 | - | - | - | - |
| Legionella pneumophila | NA | 10 10.3 | - | - | - | - |
| Moraxella catarrhalis | M15757 | 10 9.5 | - | - | - | - |
| Mycobacterium tuberculosis3 | H37Rv | 95 ng/ µL | - | - | - | - |
| Mycoplasma pneumoniae | MI-29 | 10 7.7 | - | - | - | - |
| Neisseria elongata | NA | 10 8.6 | - | - | - | - |
| Neisseria meningitidis | M2578 | 10 7.9 | - | - | - | - |
| Pseudomonas aeruginosa | NA | 10 10.5 | - | - | - | - |
| Staphylococcus epidermidis | NA | 10 10.5 | - | - | - | - |
| Staphylococcus aureus | NA | 10 10.7 | - | - | - | - |
| Streptococcus pneumoniae | 249-06(Thailand) | 10 6.6 | - | - | - | - |
| Streptococcus pyogenes | 7790-06 | 10 7.5 | - | - | - | - |
| Streptococcus salivarius | SS1672 | 10 8.4 | - | - | - | - |
| Viruses | Strain | EID50/mLorTCID50/mL | VICVER2 | YAMVER2 | VICVER2 | YAMVER2 |
| Enterovirus | Echo 6 | 10 6.9 | - | - | - | - |
| Human Adenovirus, type 1 | Ad.71 | 10 9.2 | - | - | - | - |
| Human Adenovirus, type 7a | S-1058 | 10 7.1 | - | - | - | - |
| Human Coronavirus virus3 | OC43 | 50.4 ng/µL | - | - | - | - |
| Human Coronavirus virus3 | 299E | 31.6 ng/µL | - | - | - | - |
| Human Rhinovirus A | 1A | 10 5.8 | - | - | - | - |
| Human Parainfluenza 1 virus3 | NA | 3.0 ng/ µL | - | - | - | - |
| Human Parainfluenza 2 virus | Greer | 10 3.1 | - | - | - | - |
| Human Parainfluenza 3 virus | C-243 | 10 7.9 | - | - | - | - |
| Respiratory Syncytial virus | CH93-18b | 10 6.8 | - | - | - | - |
| Herpes Simplex Virus | KOS | 10 8.4 | - | - | - | - |
| Varicella-zoster Virus | AV92-3 | 10 4.4 | - | - | - | - |
| Epstein Barr Virus3 | B95-8 | 1.7 ng/µL | - | - | - | - |
| Measles Virus | Edmonston | 10 5.2 | - | - | - | - |
| Mumps Virus | Enders | 10 7.2 | - | - | - | - |
| Cytomegalovirus | AD-169 | 10 6.9 | - | - | - | - |
Table 8-15: Assay Exclusivity with Respiratory Viruses, Bacteria, and Yeast- VER 2 Design
1 Organism quantified by Infectious Forming Units (IFU)
² NA = not applicable
3 Organism quantified by spectrophotometry (ng/μL)
{17}------------------------------------------------
CLINICAL PERFORMANCE EVALUATION IX.
Influenza B Assay -Retrospective Study
The clinical performance of the InfB oligonucleotide probe quenched with ZEN was evaluated using retrospective clinical samples collected during the 2011-2012 influenza season that were previously determined to be positive or negative for influenza B virus. A total of 30 positive and 50 negative upper respiratory tract samples were evaluated with the cleared InfB assay using either the existing oligonucleotide probe quenched with BHQ or the investigational probe quenched with ZEN. Testing was performed with both enzymes cleared for use with the CDC Human Influenza Real-Time RT-PCR Diagnostic Panel and one of the currently cleared extraction methods. Result interpretation followed the instructions of the Package Insert for the cleared Influenza B Lineage Genotyping Kit. The InfB assay containing the investigational probe quenched with ZEN demonstrated 100% positive and negative agreement with the cleared oligonucleotide probe quenched with BHQ (Tables 8-16 and 8-17).
| Invitrogen SuperScript™ | Quanta qScript™ | |||
|---|---|---|---|---|
| Specimen Type | # ofPositives1 | % Positive Agreement(95% CI) | # ofPositives1 | % Positive Agreement(95% CI) |
| NPS, NS | 18/18 | 100.0 (82.4-100.0) | 18/18 | 100.0 (82.4-100.0) |
| NPS/TS | 11/11 | 100.0 (74.1-100.0) | 11/11 | 100.0 (74.1-100.0) |
| NW | 1/1 | 100.0 (20.7-100.0) | 1/1 | 100.0 (20.7-100.0) |
| Total | 30/30 | 100.0 (88.7-100.0) | 30/30 | 100.0 (88.7-100.0) |
Table 8-16: InfB Assay-Retrospective Positive Clinical Study Results
1 Proportion of positive samples correctly identified versus the comparator.
Table 8-17: InfB Assay-Retrospective Negative Clinical Study Results
| Invitrogen SuperScript™ | Quanta qScript™ | |||
|---|---|---|---|---|
| Specimen Type | # ofNegatives1 | % Negative Agreement(95% CI) | # ofNegatives1 | % Negative Agreement(95% CI) |
| NPS, NS | 48/48 | 100.0 (92.6-100.0) | 48/48 | 100.0 (92.6-100.0) |
| TS | 2/2 | 100.0 (34.2-100.0) | 2/2 | 100.0 (34.2-100.0) |
| Total | 50/50 | 100.0 (92.9-100.0) | 50/50 | 100.0 (92.9-100.0) |
1 Proportion of negative samples correctly identified versus the comparator.
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Influenza B Lineage Genotyping Kit (VER 2) - Prospective Study
A prospective clinical investigation was conducted at 3 U.S. public health laboratories using upper respiratory tract specimens collected during the 2016-2017 influenza season. Samples were taken from specimens collected for routine influenza testing at each site from individuals symptomatic for influenza-like illness. The range of patient ages and specimen types for the total of 592 samples collected are represented in Table 8-18. A total of 13 were excluded for reasons of unspecified specimen type, inconclusive result of the comparator, or technician testing error. A total of 579 upper respiratory tract specimens were included in the data analysis.
| Patient Age | Totals |
|---|---|
| 0-16 | 149 |
| 17-54 | 192 |
| ≥55 | 249 |
| Not Reported | 2 |
| Specimen Type1 | Totals |
| NPS | 374 |
| NPS/TS | 44 |
| NA | 12 |
| NW | 34 |
| TS | 13 |
| NS | 114 |
| Not Reported | 1 |
Table 8-18: Specimen Information
1 NPS=nasopharyngeal swab, NPS/TS=dual nasopharyngeal and throat swab, NA=nasal aspirate, NW=nasal wash, TS=throat swab, NS=nasal swab
Specimens were tested with assays from the FDA-cleared CDC Human Influenza Real-Time RT-PCR Diagnostic Panel and the investigational Influenza B Lineage Genotyping Kit (VER 2). The performance is summarized in Tables 8-19 and 8-20. Due to the low prevalence of influenza B/Victoria lineage viruses in specimens collected during the prospective study, the performance of the Influenza B Lineage Genotyping Kit (VER 2) was also evaluated in a retrospective study.
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| Specimen Type1 | # ofPositives2 | % Sensitivity (95% CI) | # of Negatives3 | % Specificity (95%CI) |
|---|---|---|---|---|
| NPS, NS | 5/5 | 100.0 (56.6 – 100.0) | 472/472 | 100.0 (99.2 - 100.0) |
| NPS/TS | 0/0 | NA4 | 44/44 | 100.0 (92.0 - 100.0) |
| TS | 0/0 | NA | 13/13 | 100.0 (77.2 – 100.0) |
| NA, NW | 0/0 | NA | 45/45 | 100.0 (92.1 – 100.0) |
| Total5 | 5/5 | 100.0 (56.6 – 100.0) | 574/574 | 100.0 (99.3 – 100.0) |
Table 8-19: B/Victoria Assay - VER 2 Design- Prospective Study Results
1 NPS=nasopharyngeal swab. NPS/TS=dual nasopharyngeal and throat swab. NA=nasal wash. TS=throat swab, NS=nasal swab
2Proportion of positive samples correctly identified versus the comparator.
3 Proportion of negative samples correctly identified versus the comparator.
4NA=not applicable
| Specimen Type1 | # ofPositives2 | % Sensitivity (95% CI) | # of Negatives3 | % Specificity (95%CI) |
|---|---|---|---|---|
| NPS, NS | 31/31 | 100.0 (89.0 – 100.0) | 446/446 | 100.0 (99.1 - 100.0) |
| NPS/TS | 2/2 | 100.0 (34.2 – 100.0) | 42/42 | 100.0 (91.6 - 100.0) |
| TS | 0/0 | NA4 | 13/13 | 100.0 (77.2 - 100.0) |
| NA, NW | 5/5 | 100.0 (56.6 - 100.0) | 39/40 | 97.5 (87.1 - 99.6) |
| Total5 | 38/38 | 100.0 (90.8 - 100.0) | 540/541 | 99.8 (99.0 - 100.0) |
Table 8-20: B/Yamagata Assay - VER 2 Design - Prospective Study Results
1 NPS=nasopharyngeal swab, NPS/TS=dual nasopharyngeal and throat swab, NA=nasal wash, TS=throat swab, NS=nasal swab
2Proportion of positive samples correctly identified versus the comparator.
3 Proportion of negative samples correctly identified versus the comparator.
4NA=not applicable
Influenza B Lineage Genotyping Kit VER 1.1 and VER 2 - Retrospective Study Results
A retrospective study was performed using the VER 1.1 and YER 2 VIC and Y AM assays to evaluate their clinical performance. Upper respiratory tract clinical samples were collected during the 2017 and 2017-2018 influenza seasons and determined to be positive for influenza B/Victoria or B/Yamagata viruses using the FDA-cleared Influenza B Lineage Genotyping Kit. In the current study, specimens were tested using the cleared InfB assay and the investigative VER 1.1 and VER 2 VIC and Y AM assays. A total of 126 specimens positive for either influenza B/Victoria or influenza B/Yamagata viruses and 61 specimens negative for influenza B viruses were tested. Four lung tissue specimens were included in the testing, but were used for informational purposes only and not included in calculations of assay performance (Tables 8-21 to 8-24). Result interpretation followed the instructions of the Package Insert for the cleared Influenza B Lineage Genotyping Kit.
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| Specimen Type | # ofPositives1 | % Positive Agreement(95% CI) | # ofNegatives2 | % Negative Agreement(95% CI) |
|---|---|---|---|---|
| NPS, NS | 29/29 | 100.0 (88.3 - 100.0) | 156/156 | 100.0 (97.6 - 100.0) |
| TS | 1/1 | 100.0 (20.7 - 100.0) | 1/1 | 100.0 (20.7 - 100.0) |
| Total | 30/30 | 100.0 (88.7 - 100.0) | 157/157 | 100.0 (97.6 - 100.0) |
Table 8-21: VIC VER 1.1 Design-Retrospective Study Results
1Proportion of positive samples correctly identified versus the comparator.
2Proportion of negative samples correctly identified versus the comparator.
| Specimen Type | # ofPositives1 | % Positive Agreement(95% CI) | # ofNegatives2 | % Negative Agreement(95% CI) |
|---|---|---|---|---|
| NPS, NS | 95/95 | 100.0 (96.1 – 100.0) | 90/90 | 100.0 (95.9 – 100.0) |
| TS | 1/1 | 100.0 (20.7 - 100.0) | 1/1 | 100.0 (20.7 - 100.0) |
| Total | 96/96 | 100.0 (96.2 - 100.0) | 91/91 | 100.0 (96.0 – 100.0) |
Table 8-22: YAM VER 1.1 Design-Retrospective Study Results
1Proportion of positive samples correctly identified versus the comparator.
2Proportion of negative samples correctly identified versus the comparator.
| Specimen Type | # ofPositives1 | % Positive Agreement(95% CI) | # ofNegatives2 | % Negative Agreement(95% CI) |
|---|---|---|---|---|
| NPS, NS | 29/29 | 100.0 (88.3 – 100.0) | 156/156 | 100.0 (97.6 – 100.0) |
| TS | 1/1 | 100.0 (20.7 - 100.0) | 1/1 | 100.0 (20.7 - 100.0) |
| Total | 30/30 | 100.0 (88.7 – 100.0) | 157/157 | 100.0 (97.6 – 100.0) |
Table 8-23: VIC VER 2 Design-Retrospective Study Results
1Proportion of positive samples correctly identified versus the comparator.
2Proportion of negative samples correctly identified versus the comparator.
| Specimen Type | # ofPositives1 | % Positive Agreement(95% CI) | # ofNegatives2 | % Negative Agreement(95% CI) |
|---|---|---|---|---|
| NPS, NS | 95/95 | 100.0 (96.1 – 100.0) | 90/90 | 100.0 (95.9 – 100.0) |
| TS | 1/1 | 100.0 (20.7 - 100.0) | 1/1 | 100.0 (20.7 - 100.0) |
| Total | 96/96 | 100.0 (96.2 - 100.0) | 91/91 | 100.0 (96.0 – 100.0) |
Table 8-24: YAM VER 2 Design-Retrospective Study Results
1Proportion of positive samples correctly identified versus the comparator.
2Proportion of negative samples correctly identified versus the comparator.
X. CONCLUSION
The modification of the Influenza B Lineage Genotyping Kit of the CDC Human Influenza Virus rRT-PCR Diagnostic Panel to ensure comprehensive detection of influenza B viruses does not
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change the fundamental scientific technology of the device. Analytical and clinical data demonstrate that the performance of the modified device to detect and characterize influenza B viruses is accomplished with high positive and negative percent agreement in a manner substantially equivalent to the predicate. The indications for use remain the same.
§ 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.