FluChip-8G Influenza A+B Assay

K182513 · Indevr, Inc. · OZE · Apr 22, 2019 · Microbiology

Device Facts

• Record ID: K182513
• Device Name: FluChip-8G Influenza A+B Assay
• Applicant: Indevr, Inc.
• Product Code: OZE · Microbiology
• Decision Date: Apr 22, 2019
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3980
• Device Class: Class 2
• Attributes: AI/ML, Real-World Evidence

Real-World Evidence

Submission	Device	Sponsor	RWD Sources	RWE Use Summary	Key Tags
K182513 · Apr 22, 2019	FluChip-8G Influenza A+B Assay	Indevr, Inc.	Archived clinical nasopharyngeal/nasal swab specimens from influenza surveillance programs; Vendor-acquired archived clinical specimens	Retrospective analysis of archived clinical specimens collected during routine influenza surveillance was used to establish clinical performance characteristics (sensitivity/specificity) of the FluChip-8G assay.	Retrospective clinical study; Surveillance specimens; Clinical performance validation

Clinical Evidence

Study Design	Population	Comparator	Key Endpoints
Prospective Clinical Study (Surveillance Cohort); Retrospective analysis of prospectively collected surveillance specimens; Follow-up/Duration: 2013-2017; Study Period: June 2017 - July 2018 (testing period)	Patients meeting febrile respiratory illness (FRI) case definition; Sample Size: 984 evaluable specimens; Number of Sites: 7	FDA-cleared influenza molecular assay	Sensitivity and specificity for influenza A and B detection
Supplemental Clinical Study; Retrospective analysis of vendor-acquired archived specimens; Follow-up/Duration: 2014, 2016	Patients meeting FRI case definition; Sample Size: 500 NPS specimens; Number of Sites: 3	FDA-cleared influenza molecular assay	Sensitivity and specificity for influenza A and B detection

AI Performance

Output	Algorithm	Acceptance	Observed	Dev DS	Dev Readers	Test DS	Test Readers
Influenza A/H1N1pdm09 detection	Neural network-based pattern recognition	—	Sensitivity: 100.0% (95% CI: 43.9–100.0); Specificity: 99.4% (95% CI: 98.7–99.7)	Training set: 3,372 microarray images (3,064 trained, 308 excluded)	—	Prospective Clinical Study: 984 specimens; Supplemental Clinical Study: 500 specimens; Retrospective Clinical Study #1: 97 specimens; Retrospective Clinical Study #2: 108 specimens	—
Influenza A/H3N2 detection	Neural network-based pattern recognition	—	Sensitivity: 87.2% (95% CI: 76.0–93.7); Specificity: 99.5% (95% CI: 98.7–99.8)	Training set: 3,372 microarray images (3,064 trained, 308 excluded)	—	Prospective Clinical Study: 984 specimens; Supplemental Clinical Study: 500 specimens; Retrospective Clinical Study #1: 97 specimens; Retrospective Clinical Study #2: 108 specimens	—
Non-seasonal Influenza A detection	Neural network-based pattern recognition	—	Sensitivity: 99% (95% CI: 94.6–99.8); Specificity: 100% (95% CI: 96.7–100.0)	Training set: 3,372 microarray images (3,064 trained, 308 excluded)	—	Non-Seasonal Influenza A Virus Challenge Study: 213 contrived specimens	—
Influenza B/Victoria detection	Neural network-based pattern recognition	—	Sensitivity: 100.0% (95% CI: 78.5–100.0); Specificity: 100.0% (95% CI: 99.6–100.0)	Training set: 3,372 microarray images (3,064 trained, 308 excluded)	—	Prospective Clinical Study: 984 specimens; Supplemental Clinical Study: 500 specimens; Retrospective Clinical Study #1: 97 specimens; Retrospective Clinical Study #2: 108 specimens	—
Influenza B/Yamagata detection	Neural network-based pattern recognition	—	Sensitivity: 100.0% (95% CI: 67.6–100.0); Specificity: 100.0% (95% CI: 99.6–100.0)	Training set: 3,372 microarray images (3,064 trained, 308 excluded)	—	Prospective Clinical Study: 984 specimens; Supplemental Clinical Study: 500 specimens; Retrospective Clinical Study #1: 97 specimens; Retrospective Clinical Study #2: 108 specimens	—

Indications for Use

The FluChip-8G Influenza A+B Assay is a multiplex RT-PCR in vitro diagnostic test intended for the qualitative detection and differentiation of seasonal influenza A/H3N2, seasonal influenza A/H1N1pdm09, and “non-seasonal” influenza A subtypes other than seasonal H1N1pdm09 or H3N2. The assay is also intended for the qualitative detection and differentiation of the genetic lineage of human influenza B viruses as B/Victoria or B/Yamagata. The assay is designed for use on influenza nucleic acids isolated and purified from nasopharyngeal swab and nasal swab specimens from human patients with signs and symptoms of respiratory infection in conjunction with clinical and epidemiological risk factors. This assay amplifies the hemagglutinin (HA) gene segment, neuraminidase (NA) gene segment, matrix (M) gene segment, non-structural (NS) gene segment, and nucleoprotein (NP) gene segment for detection and discrimination of influenza A, and amplifies the hemagglutinin (HA) gene segment and neuraminidase (NA) gene segment for detection and discrimination of influenza B. This assay is not intended to detect influenza C viruses. FluChip-8G Influenza A+B Assay “non-seasonal” influenza A positive results are for the presumptive detection of influenza A subtypes other than seasonal influenza A/H1N1pdm09 or A/H3N2. The definitive identification of a “non-seasonal” influenza A case requires additional testing and confirmation procedures in consultation with the appropriate public health authorities (e.g., local or state public health departments, etc.) for whom reporting is necessary. Negative results do not preclude influenza virus infection. FluChip-8G Influenza A+B Assay “non-seasonal” influenza A negative results, even in the context of a FluChip-8G Influenza A+B Assay positive result for seasonal influenza A/H1N1pdm09 or A/H3N2, or influenza B, do not preclude “non-seasonal” influenza A infection and should not be used as the sole basis for patient management decisions. Performance characteristics of the FluChip-8G Influenza A+B Assay for detecting and differentiating seasonal influenza A viruses were established when seasonal influenza A/H3N2 was the predominant influenza A virus circulating in the United States. Performance characteristics may vary with other emerging seasonal influenza A viruses. Performance characteristics of the FluChip-8G Influenza A+B Assay for detecting and differentiating human influenza B genetic lineages were established when influenza B/Victoria was the predominant influenza B virus circulating in the United States. Due to low prevalence of “non-seasonal” influenza A viruses, performance characteristics of the FluChip-8G Influenza A+B Assay for detecting “non-seasonal” influenza A viruses and distinguishing “non-seasonal” influenza A from seasonal influenza A H1N1pdm09 and H3N2 were assessed exclusively by conducting cross-validation on a total of 759 microarray images generated from bench testing contrived samples consisting of 352 unique “non-seasonal” influenza A strains representing 62 subtypes, and by bench testing contrived samples and surrogate clinical specimens consisting of 133 unique non-seasonal influenza A strains representing 46 subtypes. FluChip-8G Influenza A+B Assay performance may vary when testing “non-seasonal” influenza A strains not represented in the performance assessment. If infection with a novel influenza A virus strain 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 local or state health department(s) for testing. Viral culture should not be attempted unless a BSL 3E facility is available to receive and culture specimens.

Device Story

System performs multiplex RT-PCR on extracted viral RNA from nasal/nasopharyngeal swabs; amplifies HA, NA, M, NS, NP gene segments for Influenza A and HA, NA for Influenza B. Biotinylated amplicons are heat-fragmented and hybridized to a microarray slide containing 464 capture oligonucleotides. Fluorescently labeled products are imaged by the FluChip-8G Imaging System. Software uses a neural network-based pattern recognition algorithm to analyze microarray signal intensities; identifies influenza types/subtypes/lineages. Used in clinical labs by trained personnel. Output provides diagnostic results to clinicians for patient management and public health reporting. Benefits include rapid differentiation of seasonal vs. non-seasonal influenza strains.

Clinical Evidence

Clinical performance evaluated in prospective (n=984) and supplemental (n=500) studies using nasal/nasopharyngeal swabs. Comparator was an FDA-cleared influenza molecular assay. Overall sensitivity for Influenza A was 87.9-92.6% and specificity 98.8-98.9%. Influenza B sensitivity was 98.1-100% and specificity 99.3-99.8%. Retrospective studies (n=97 and n=108) and a non-seasonal influenza A challenge study (n=213) further supported performance across diverse subtypes.

Technological Characteristics

System uses multiplex RT-PCR amplification followed by microarray-based hybridization and fluorescence imaging. Reagents include biotinylated dUTP for labeling. Microarray slide (75mm x 25mm) contains 464 synthetic DNA capture oligonucleotides. Instrumentation: BioRad T100 thermal cycler and FluChip-8G Imaging System. Software utilizes a neural network-based pattern recognition algorithm. Sterilization: Not applicable (reagents). Connectivity: Standalone imaging system.

Indications for Use

Indicated for qualitative detection/differentiation of seasonal influenza A (H3N2, H1N1pdm09), non-seasonal influenza A subtypes, and influenza B lineages (B/Victoria, B/Yamagata) in human nasopharyngeal/nasal swab specimens from patients with respiratory infection symptoms and clinical/epidemiological risk factors.

Regulatory Classification

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.

Special Controls

*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.

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION
DECISION SUMMARY

A. 510(k) Number:

K182513

B. Purpose for Submission:

New device 510(k) clearance for the FluChip-8G Influenza A+B Assay

C. Measurand:

Influenza A and influenza B viral nucleic acids.

D. Type of Test:

Qualitative multiplex one-step RT-PCR followed by downstream microarray-based hybridization, imaging, and subsequent influenza virus detection and characterization using a pattern recognition-based algorithm

E. Applicant:

InDevR, Inc.

F. Proprietary and Established Names:

FluChip-8G Influenza A+B Assay (FC8G assay)

G. Regulatory Information:

1. Regulation section:

21 CFR 866.3980, Respiratory viral panel multiplex nucleic acid assay

2. Classification:

Class II

3. Product code(s):

OZE - Influenza A and influenza B multiplex nucleic acid assay (Primary)

NSU - Instrumentation for clinical multiplex test systems (Subsequent)

OEP - Influenza A virus subtype differentiation nucleic acid assay (Subsequent)

OQW - 2009 H1N1 influenza virus (swine origin), nucleic acid or antigen, detection and

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identification (Subsequent)

# 4. Panel:

Microbiology (83)

# H. Intended Use:

# 1. Intended use(s):

The FluChip-8G Influenza A+B Assay is a multiplex RT-PCR in vitro diagnostic test intended for the qualitative detection and differentiation of seasonal influenza A/H3N2, seasonal influenza A/H1N1pdm09, and “non-seasonal” influenza A subtypes other than seasonal H1N1pdm09 or H3N2. The assay is also intended for the qualitative detection and differentiation of the genetic lineage of human influenza B viruses as B/Victoria or B/Yamagata. The assay is designed for use on influenza nucleic acids isolated and purified from nasopharyngeal swab and nasal swab specimens from human patients with signs and symptoms of respiratory infection in conjunction with clinical and epidemiological risk factors.

This assay amplifies the hemagglutinin (HA) gene segment, neuraminidase (NA) gene segment, matrix (M) gene segment, non-structural (NS) gene segment, and nucleoprotein (NP) gene segment for detection and discrimination of influenza A, and amplifies the hemagglutinin (HA) gene segment and neuraminidase (NA) gene segment for detection and discrimination of influenza B. This assay is not intended to detect influenza C viruses.

FluChip-8G Influenza A+B Assay “non-seasonal” influenza A positive results are for the presumptive detection of influenza A subtypes other than seasonal influenza A/H1N1pdm09 or A/H3N2. The definitive identification of a “non-seasonal” influenza A case requires additional testing and confirmation procedures in consultation with the appropriate public health authorities (e.g., local or state public health departments, etc.) for whom reporting is necessary.

Negative results do not preclude influenza virus infection. FluChip-8G Influenza A+B Assay “non-seasonal” influenza A negative results, even in the context of a FluChip-8G Influenza A+B Assay positive result for seasonal influenza A/H1N1pdm09 or A/H3N2, or influenza B, do not preclude “non-seasonal” influenza A infection and should not be used as the sole basis for patient management decisions.

Performance characteristics of the FluChip-8G Influenza A+B Assay for detecting and differentiating seasonal influenza A viruses were established when seasonal influenza A/H3N2 was the predominant influenza A virus circulating in the United States. Performance characteristics may vary with other emerging seasonal influenza A viruses. Performance characteristics of the FluChip-8G Influenza A+B Assay for detecting and differentiating human influenza B genetic lineages were established when influenza

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B/Victoria was the predominant influenza B virus circulating in the United States.

Due to low prevalence of “non-seasonal” influenza A viruses, performance characteristics of the FluChip-8G Influenza A+B Assay for detecting “non-seasonal” influenza A viruses and distinguishing “non-seasonal” influenza A from seasonal influenza A H1N1pdm09 and H3N2 were assessed exclusively by conducting cross-validation on a total of 759 microarray images generated from bench testing contrived samples consisting of 352 unique “non-seasonal” influenza A strains representing 62 subtypes, and by bench testing contrived samples and surrogate clinical specimens consisting of 133 unique non-seasonal influenza A strains representing 46 subtypes. FluChip-8G Influenza A+B Assay performance may vary when testing “non-seasonal” influenza A strains not represented in the performance assessment.

If infection with a novel influenza A virus strain 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 local or state health department(s) for testing. Viral culture should not be attempted unless a BSL 3E facility is available to receive and culture specimens.

2. Indication(s) for use:

Same as Intended Use(s)

3. Special conditions for use statement(s):

For prescription use only

4. Special instrument requirements:

- BioRad T100 endpoint thermal cycler
- FluChip-8G Imaging System with FluChip-8G Software v1.0.9.0 or newer

# I. Device Description:

# Overview

The FluChip-8G Influenza A+B Assay system is a molecular assay system for the detection and differentiation of influenza viruses in which a multiplexed one-step RT-PCR amplification is coupled with downstream microarray-based hybridization, imaging, and subsequent influenza virus detection and characterization using a pattern recognition-based algorithm.

The system consists of the following:

1) A reagent kit comprising the reagents required to conduct RT-PCR and for performing

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post-PCR sample processing including PCR product fragmentation, microarray hybridization, washing, fluorescent labeling, and drying.

2) An accessory kit comprising a variety of laboratory equipment and supplies for facilitating execution of the assay.
3) Other general laboratory equipment and supplies required but not provided.
4) The FluChip-8G Imaging System with pre-installed FluChip-8G Software including an image processing module, an underlying neural network-based pattern recognition algorithm for identifying patterns in microarray signals representative of certain influenza target virus groups, and a user interface to facilitate data entry/imaging and provide result reports to the end user.

## Materials Provided

FluChip-8G Influenza A+B Assay Reagent Kit (FC-6101)

| Reagents | Description | Units Per Kit | Assays Per Unit | Volume Per Unit | Cat. # |
| --- | --- | --- | --- | --- | --- |
| **FC8G Amplification Reagents:** | | | | | **FC-6101A** |
| FC8G Polymerase Mix | Mixture of components necessary for PCR amplification | 6 vials | 16 | 500 μL | FC-5015 |
| FC8G RT Enzyme | Enzyme necessary to perform reverse transcription step | 6 vials | 16 | 35 μL | FC-5014 |
| FC8G Primer Mix | Mixture of oligonucleotide primers containing sodium azide (0.02%) as a preservative | 6 vials | 16 | 40 μL | FC-5004 |
| FC8G Biotin dUTP | Lyophilized biotinylated dUTP | 6 vials | 16 | 50 nmol | FC-5008 |
| **FC8G Microarray Processing Reagents:** | | | | | **FC-6101B** |
| FluChip-8G Microarray Slide | Microarray slide with 16-well silicone chamber. Each well contains a single oligonucleotide microarray | 6 slides | 16 | N/A | FC-5006 |
| Wash Buffer 1 (20x) | Concentrated wash buffer containing sodium azide (0.05%) as a preservative | 2 bottles | 48 | 50 mL | MI-5014 |
| Wash Buffer 2 (20x) | Concentrated wash buffer containing sodium azide (0.05%) as a preservative | 2 bottles | 48 | 50 mL | MI-5015 |
| FC8G Binding Buffer | Concentrated binding buffer containing sodium azide (0.02%) as a preservative | 6 vials | 16 | 1850 μL | FC-5001 |
| FC8G Hyb Mix | Protein-based hybridization solution containing hybridization positive control oligonucleotides | 6 vials | 16 | 192 μL | FC-5013 |

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| FC8G Label | Lyophilized fluorophore- streptavidin label containing sodium azide (1.5 ng) as a preservative | 6 vials | 16 | 15 μg | FC-5002 |
| --- | --- | --- | --- | --- | --- |
| FC8G Diluent | Diluent containing sodium azide (0.025%) as a preservative | 6 vials | 16 | 1500 μL | FC-5012 |
| Slide Drying Sheets | Absorbent sheets to remove wash buffer from the FluChip- 8G Microarray Slide | 20 sheets | N/A | N/A | FC-4005 |
| Desiccant Pouches | Desiccant provided to aid in slide drying steps | 18 pouches | N/A | N/A | FC-4013 |

## Materials Required but Not Provided

Plasticware and Consumables

| Material | Cat. # |
| --- | --- |
| FluChip-8G Influenza A+B Assay Accessory Kit | FC-4000 |
| Humidity Chamber (available in the FluChip-8G Influenza A+B Accessory Kit FC-4000) | MI-4001 |
| Four (4) Wash Bins (available in the FluChip-8G Influenza A+B Accessory Kit FC-4000) | FC-4001 |
| Two (2) Rinse Bottles (available in the FluChip-8G Influenza A+B Accessory Kit FC-4000) | MI-4004 |
| Plastic Forceps (available in the FluChip-8G Influenza A+B Accessory Kit FC-4000) | FC-4002 |
| Slide Drying Box (available in the FluChip-8G Influenza A+B Accessory Kit FC-4000) | FC-4006 |
| | |
| Sterile flocked swabs (Copan FLOQSwabs available through VWR, Fisher Healthcare, and Cardinal Healthcare) | -- |
| Universal Transport Media (UTM) consisting of: Hanks' Balanced Salts, Bovine Serum Albumin, L-Cysteine, Gelatin, Sucrose, L-Glutamic Acid, HEPES Buffer, Vancomycin, Amphotericin B, Colistin, and Phenol Red at pH 7.3 ± 0.2 @ 25 °C | -- |
| Bio-Rad 0.2 mL 8-tube PCR strips | TBS0201 (Bio-Rad) |
| Bio-Rad optical flat 8-cap strips | TCS0803 (Bio-Rad) |
| Qiagen Collection Tubes | 19201 (QIAGEN) |
| Ice/Ice Bucket or Cold Block | -- |
| Sterile RNase/DNase-free filter micropipettor tips (5 – 1000 μL) | -- |
| Standard micropipettor tips (5 – 1000 μL) | -- |
| Nuclease-free Polypropylene Tubes (1.5 mL) | -- |
| Lint free tissue wipes | -- |
| Clean 1 Liter bottles (2) with lids | -- |

Reagents and Controls

| Material | Cat. # |
| --- | --- |
| QIAamp DSP Virus Spin Kit | 61704 (QIAGEN) |
| Purified (18 MΩ) water for preparation of wash buffers | -- |
| Nuclease-free/PCR grade water to be used for no-template negative control(s) | -- |
| Ethanol (96 – 100% purity) | -- |
| External positive control (previously characterized influenza positive clinical specimen) | -- |

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| External negative control (previously characterized influenza negative clinical specimen) | -- |
| --- | --- |

# **Instrumentation/Equipment**

| Material | Cat. # |
| --- | --- |
| FluChip-8G Imaging System (FluChip-8G Software Package pre-installed) | FC-6000 |
| T100 Thermal Cycler | 1861096 (Bio-Rad) |
| Standard pipettes (5 – 1000 μL) | -- |
| Orbital Shaker (at least 8.5” x 7” stage) - capable of no tilt horizontal movement at 80- 140 RPM | -- |
| Vortex Mixer | -- |
| Centrifuge with rotor for 1.5 and 2 mL tubes capable of achieving 20,000 g (14,000 rpm) | -- |
| Microcentrifuge with rotor adaptor for 0.2 mL 8-tube strips | -- |
| Heating block for lysing samples at 56°C in 1.5 and 2 ml tubes | -- |
| Timer, capable of timing 1 minute intervals over the range from 1 to 30 min | -- |

# **Optional Materials Not Provided**

| Material | Cat. # |
| --- | --- |
| Barcode Reader | 9029247015 (Zebra) |
| Slide Storage Box | FC-4024 |

# **Quality Control**

# **Assay Controls**

- **Internal Control:** Sequences corresponding to portions of the 18s rRNA gene are included on each microarray to monitor adequate specimen collection, and in the absence of influenza, also to indicate the absence of reaction inhibition. Primers to amplify the 18s internal control are included in the Primer Mix so that the sequence complement to these microarray markers is amplified during RT-PCR and subsequently hybridized to the microarray if sufficient human material is present in the collected specimen. This control is expected to fail in samples containing no human cellular material. This control is evaluated in the absence of an influenza positive result and is expected to result in “No Call: Assay Failure (Internal Control)” in samples containing no human cellular material or poorly collected specimens.
- **Hybridization Control:** Oligonucleotides are included on each microarray to monitor the integrity of the hybridization procedure. The complementary sequences are fluorescently tagged single-stranded synthetic DNA sequences included in the Hyb Mix solution that bind specifically to the hybridization control sequences on the microarray. When the hybridization process fails, the FC8G assay result will be “No Call: Assay Failure (Hybridization Control)”.
- **Labeling Control:** Each microarray includes a biotin-terminated capture oligonucleotide that bind to the fluorophore-coupled streptavidin solution present in the label applied during the labeling step. This control serves as an indicator of successful microarray labeling. When the labeling process fails, the FC8G assay result will be “No Call: Assay Failure (Labeling Control)”.

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- **Image Processing Control:** Direct fluorescently tagged sequences are included on each microarray at specific locations. The image processing controls serve as points of reference for microarray placement used by the FluChip-8G software during microarray image analysis. When the FluChip-8G Microarray Slide is improperly positioned in the FluChip-8G Imaging system, the FC8G assay result will be “Error: Image processing failure”. A failure of this control may indicate an issue with the microarray quality, placement of the FluChip-8G Microarray Slide in the FluChip-8G Imaging System, or a focus issue of the FluChip-8G Imaging System.

### External Quality Control (QC)

- **External Batch Positive Control:** A previously characterized influenza-positive clinical specimen is recommended for inclusion as part of good laboratory practices. This control is user-supplied.
- **External Batch Negative Control:** A previously characterized influenza-negative clinical specimen is recommended for inclusion as part of good laboratory practices. This control is user supplied.
- **Extraction Negative Control:** This control is recommended for inclusion in each testing batch, and is composed of nuclease-free water added by the end user in place of specimen during the nucleic acid extraction step.
- **RT-PCR No Template Control:** This control is recommended for inclusion in each testing batch, and is composed of nuclease-free water added by the end user in place of extracted nucleic acid template during RT-PCR reaction setup.

### Workflow

The FluChip-8G Influenza A+B Assay is comprised of the following workflow:

1) Nucleic acid extraction

First, nucleic acids are extracted from human nasal swab or nasopharyngeal swab specimen(s) stabilized in Universal Transport Media (UTM) employing the QIAamp DSP Virus Spin Kit (catalog #61704). During the nucleic acid extraction step, both DNA and RNA are co-eluted from starting material, and any influenza viral RNA as well as the endogenous 18s rRNA internal control are made accessible for downstream amplification. Extracted nucleic acid is then eluted and utilized as template for downstream amplification.

2) Multiplexed reverse transcription polymerase chain reaction (RT-PCR) amplification followed by heat fragmentation

The RT-PCR amplification primers designed for the FluChip-8G Influenza A+B Assay (provided as FC8G Primer Mix) provide simultaneous (multiplexed) amplification of several full-length gene segments of the influenza A virus genome (HA, NA, M, NS, and NP), two full length gene segments of the influenza B virus genome (HA, NA), and a segment of the 18s rRNA found in eukaryotic cells as an internal process control. The

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FC8G Primer Mix provides amplification of all targeted gene segments for all influenza A and B viruses, regardless of subtype or lineage, within a single reaction. A biotinylated dUTP is supplied, and is used during RT-PCR to incorporate biotin molecules into the amplified product. The FC8G Biotin dUTP supplements the standard dNTPs already present in the FC8G Polymerase Mix. These biotin moieties are utilized for downstream fluorescence labeling with a streptavidin-coupled fluorophore (FC8G Label) after the microarray hybridization step. Post-amplification, RT-PCR products are heat-fragmented for 10 min at 95°C to fragment the amplicons into shorter pieces for more efficient downstream hybridization to the FluChip-8G Microarray Slide.

3) Microarray processing (pre-wash slides, hybridization, post-hybridization slide wash, labeling, and post-label wash)

The FluChip-8G Microarray Slide consists of a 75 mm x 25 mm glass microscope slide onto which 16 replicate microarrays (labeled A1 through H2) are printed. The Microarray Slide is also labeled with a slide number at the top and a barcode at the bottom. Within each replicate microarray, 464 unique synthetic DNA oligonucleotides (capture oligonucleotide sequences) are printed that act as either influenza capture sequences (458, each printed in triplicate) or assay controls (six control sequences). A heat-fragmented post-amplification mixture is added to the microarray and incubated to allow for hybridization between the amplified nucleic acid and the microarray capture sequences. Once hybridization is complete, the microarray is washed to remove any unbound material. The microarray is then labeled with a streptavidin-coupled fluorophore to enable downstream fluorescence imaging.

4) Microarray imaging and data analysis

FluChip-8G Imaging System consists of: a) an optical imaging subsystem used to obtain fluorescence images of FluChip-8G Microarray Slides processed via the FC8G Influenza A+B Assay, b) instrument control software and application programming interface (API), and c) FC8G Software that automates image acquisition, controls the settings and instrument features available to end users through the user interface (UI), and interprets the FluChip-8G Microarray Slide data using a neural network module to provide results to the end users. Image processing algorithms within the FC8G Software locate the microarray pattern within the image and determine the fluorescence intensity of individual oligonucleotide captures on the microarray. Specific captures on the microarray that are intended as controls verify that specific assay processing steps were successfully completed. Trained neural networks are used to recognize the patterns of intensities of the influenza-targeted captures on the microarray as belonging to specific influenza types or subtypes/lineages. The neural network module utilizes seven individual neural networks, with the microarray signals from the 458 influenza capture sequences utilized as inputs. The output of each neural network is a value between 0 and 1, and a cutoff (optimized subsequent to neural network training) is applied to each to determine if each is positive or negative for its trained target. A deterministic decision tree then uses the binary outputs of the controls and neural networks to generate a diagnostic output for each clinical sample. The diagnostic output is then displayed to the

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end user in un-editable format for printing and/or saving.

## Neural Networks Training and Verification

Neural networks training is an interactive process and includes the following elements:

a) Read in the library of results from well-characterized known samples corresponding to all target virus groups for training.
b) Determine which result files contain sufficient signal to be used for training. Training set inclusion is based on key capture sequences identified for different target virus groups.
c) Use the Fast Artificial Neural Network (FANN) open source library functions to train individual networks. Each individual neural network uses a single hidden layer neuron and a single output neuron trained via supervised learning using backpropagation.
d) Determine thresholds that optimize network performance in terms of positive percent agreement (PPA) and negative percent agreement (NPA) with the known results of a clinically-representative set of naïve samples.
e) Save network files with threshold values for verification and inclusion in FC8G Software.

The database of assay results available for training the neural networks consisted of 3372 microarray images. Inclusion criteria were applied to ensure that the influenza-positive results had detectable signals on key captures of the microarray. This prevented result files with undetectable signal levels from being trained as positives. The final count of microarray images used for training by virus target group is presented in Table 1 below. This includes:

- 478 results from RT-PCR No Template Controls and Extraction Negative Controls.
- Results from 850 unique clinical specimens containing influenza, clinical specimens negative for influenza, and contrived influenza positive samples.
- Results from 1736 replicate analyses from the 850 unique sample strains noted above (primarily from dilution series testing).

Table 1: Neural Network Training Set Composition and Results Excluded from Training

| Category | # Trained Images | # Excluded Images | Total Images |
| --- | --- | --- | --- |
| Influenza A, H1N1pdm09 | 446 | 60 | 506 |
| Influenza A, Seasonal H3N2 | 378 | 30 | 408 |
| Non-Seasonal Influenza A | 759 | 75 | 834 |
| Influenza B, Victoria lineage | 287 | 112 | 399 |
| Influenza B, Yamagata lineage | 244 | 31 | 275 |
| Clinical Negative | 472 | 0 | 472 |
| Negative Controls | 478 | 0 | 478 |
| Overall Totals | 3064 | 308 | 3372 |

Regarding training the non-seasonal influenza A neural network, a total of 363 unique non-seasonal influenza A strains (representing 62 subtypes) were used to prepare contrived samples that upon testing generated a total of 834 microarray images. Of these 834 images,

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759 microarray images were included in training, and 75 microarray images were excluded from training due to failure to meet the inclusion criteria. Table 2 below lists all 352 unique non-seasonal influenza A strains that were used to prepare contrived samples that upon testing generated the total of 759 microarray images that were included in the training, stratified by subtypes.

Table 2: Unique Non-Seasonal Influenza A Strains that Were Used to Prepare Contrived Samples to Generate the Microarray Images Included in Training the Non-Seasonal Influenza A Neural Network

| Strain | Subtype | # of Unique Strain in Subtype | # of Replicate Analyses per Strain | # of Microarray Images per Subtype |
| --- | --- | --- | --- | --- |
| A/Arizona/07/2007 | H1N1 (pre 2009) | 12 | 1 | 188 |
| A/Virginia/01/2006 | | | 2 | |
| A/Santiago/5248/2008 | | | 1 | |
| A/Chelyabinsk/01/2006 | | | 1 | |
| A/Denver/1/57 | | | 5 | |
| A/Texas/35/2008, E3 (03/12/2009) | | | 2 | |
| A/Mississippi/3/2001 wt 275H | | | 1 | |
| A/Mississippi/3/2001 mut H275Y | | | 1 | |
| Clinical Specimen #1 | | | 5 | |
| Clinical Specimen #2 | | | 3 | |
| A/New Caledonia/20/1999 | | | 74 | |
| A/Brisbane/59/2007 | | | 92 | |
| A/Teal/Egypt/677/2004 | H1N1 Avian | 10 | 2 | 24 |
| Unknown #1- from CDC 2014 | | | 2 | |
| Unknown #2- from CDC 2014 | | | 2 | |
| A/Mallard/Republic of Georgia/4/2010 | | | 3 | |
| A/Duck/Alberta/35/76 | | | 2 | |
| A/Mallard/Alberta/21/2014 | | | 3 | |
| A/Redheaded Duck/MN/SG-00123/2007 | | | 2 | |
| A/Gull/DE/428/2009 | | | 1 | |
| A/Ruddy Turnstone/DE/274/2009 | | | 3 | |
| A/Aquatic Bird/Hong Kong/D125/2002 | | | 4 | |
| A/Swine/1976/1931 | H1N1 Swine | 3 | 2 | 5 |
| A/Swine/TN/26/1977 | | | 2 | |
| A/Swine/NC/18162/2002 | | | 1 | |
| Swine Origin Influenza Virus (soiv) | H1N2 | 5 | 2 | 10 |
| A/Teal/Egypt/431/2003 | | | 2 | |
| Unknown #3- from CDC 2014 | | | 2 | |
| A/Swine/Ohio/09SW1484E/2009 | | | 2 | |
| A/Swine/Ohio/09SW1477/2009 | | | 2 | |
| A/Shorebird/Delaware Bay/211/1994 | H1N3 | 1 | 2 | 2 |
| A/Red Knot/Delaware Bay/240/1994 | H1N8 | 1 | 2 | 2 |
| A/Japan/305/57 | H2N2 | 2 | 1 | 3 |
| Unknown #4- from CDC 2014 | | | 2 | |
| A/Duck/Germany/1215/73 | H2N3 | 1 | 2 | 2 |
| Blue-winged Teal Texas | H2N9 | 2 | 2 | 4 |
| A/Pintail/ALB/293/77 | | | 2 | |
| A/Swine/North Carolina/88708/2000, CX/C1 (10/17/2011) | H3N2 Swine | 14 | 2 | 23 |
| A/Swine/North Carolina/32760/2007, CX/C1 (10/13/2011) | | | 2 | |

10

{10}

| A/Swine/North Carolina/44897/2009, C2/C1 (10/13/2011) | | | 2 | |
| --- | --- | --- | --- | --- |
| A/Swine/North Carolina/52796/2006, CX/C1 (10/13/2011) | | | 2 | |
| A/Swine/Ohio/09SW73E/2009 | | | 2 | |
| A/Swine/Ohio/09SW83E/2009 | | | 2 | |
| A/Swine/Ohio/11SW87/2011 | | | 2 | |
| A/Swine/Ohio/09SW79M/2009 | | | 2 | |
| A/Swine/Texas/4199-2/1998 | | | 1 | |
| A/Swine/NC/0668/2011 | | | 2 | |
| A/Swine/NC/0666/2011 | | | 1 | |
| 4D-0114-P29 swine swab | | | 1 | |
| 4D-0114-P21 swine swab | | | 1 | |
| 4D-0114-P15 swine swab | | | 1 | |
| A/Ohio/20/2012 | H3N2v | 14 | 2 | 28 |
| A/Indiana/16/2012 | | | 2 | |
| A/Indiana/14/2012 | | | 2 | |
| A/Indiana/65/2012 | | | 2 | |
| A/Michigan/20/2012 | | | 2 | |
| A/Pennsylvania/17/2012 | | | 2 | |
| A/Wisconsin/28/2012 | | | 1 | |
| A/Ohio/44/2012 | | | 2 | |
| A/Ohio/47/2012 | | | 2 | |
| A/Ohio/56/2012 | | | 1 | |
| A/Ohio/83/2012 | | | 2 | |
| A/Ohio/36/2012 | | | 2 | |
| A/Indiana/8/2011 | | | 1 | |
| A/Swine/MN/3908-2/11 | | | 5 | |
| A/Blue-winged Teal/Texas/G77/2007 | H3N6 | 3 | 2 | 6 |
| A/Blue-Winged Teal/Illinois/10OS1546/2010 | | | 2 | |
| A/Redhead/Alberta/192/2002 | | | 2 | |
| A/Equine/TX/2004 | H3N8 | 21 | 1 | 62 |
| A/Equine/New York/452/2003 | | | 2 | |
| A/Canine/New York/100525,1/06 | | | 2 | |
| A/Canine/Florida/89911,2/06 | | | 2 | |
| A/Canine/VA/93653/09 | | | 2 | |
| A/Canine/New York/4732/2012 | | | 2 | |
| A/Duck/Bangladesh/1293/2008 | | | 2 | |
| A/Duck/Bangladesh/1575/2009 | | | 2 | |
| Unknown #5- from CDC 2014 | | | 2 | |
| Unknown #6- from CDC 2014 | | | 2 | |
| A/Camel/Mongolia/335/2012 | | | 2 | |
| A/Equine-2/Miami/1963 | | | 1 | |
| A/Blue-Winged Teal/Iowa/10OS2411/2010 | | | 2 | |
| A/Equine/Pennsylvania/1/2007 | | | 2 | |
| A/Duck/Ukraine/63 | | | 4 | |
| A/Duck/Chabarovsk/1610/72 | | | 2 | |
| A/Equine/KY/4/2011 | | | 5 | |
| A/Mallard/Alberta/435/2013 | | | 10 | |
| A/Mallard/Alberta/307/2012 | | | 8 | |
| A/Laughing Gull/DE/45/2005 | | | 5 | |
| A/Songbird/Hong Kong/SB18/2001 | 2 | | | |
| A/Mallard/Alberta/54/1993 | H3N9 | 1 | 2 | 2 |
| A/Duck/Bangladesh/1746/2010 | H4N2 | 1 | 2 | 2 |
| A/Blue-Winged Teal/Alberta/346/2007 | H4N3 | 1 | 2 | 2 |

11

{11}

| A/Duck/Bangladesh/1283/2008 | H4N6 | 5 | 2 | 10 |
| --- | --- | --- | --- | --- |
| Unknown #7- from CDC 2014 | 2 |
| A/Blue-Winged Teal/Illinois/10OS1563/2010 | 2 |
| A/Red Knot/Delaware/541/1988 | 2 |
| A/Duck/Czech/56 | 2 |
| Unknown #8- from CDC 2014 | H4N8 | 1 | 2 | 2 |
| A/Vietnam/JP20-2/2005 | H5N1 | 43 | 1 | 63 |
| A/Chicken/Vietnam/NCVDCDC33/2005 | 2 |
| A/Chicken/Vietnam/NCVDCDC42/2005 | 1 |
| A/Chicken/Vietnam/NCVDCDC37/2005 | 2 |
| A/Chicken/Vietnam/NCVDCDC52/2005 | 2 |
| A/Chicken/Vietnam/NCVDCDC36/2005 | 1 |
| A/Environment/Vietnam/NCVDCDC53/2005 | 1 |
| A/Environment/Vietnam/NCVDCDC54/2005 | 2 |
| A/Anhui/2/2005 | 2 |
| A/Duck/Kulon Progo/Bb Vet/IX/2004 | 1 |
| A/Turkey/VA/505477-18/07 | 2 |
| A/Mallard/PA/454069-9/2006 | 9 |
| A/Ruddy Turnstone/DE/105/2007 | 3 |
| A/Duck/MN/1525/81 | 5 |
| A/Chicken/Egypt/A10543A/2015 | 1 |
| A/Chicken/Egypt/D10565A/2015 | 1 |
| A/Chicken/Bangladesh/18247/12 | 1 |
| A/Quail/Bangladesh/19250/13 | 1 |
| A/Hong Kong/156/97 | 1 |
| A/Vietnam/1203/2004 | 1 |
| A/Japanese White-Eye/Hong Kong/1038/2006 | 1 |
| A/Chicken/Hunan/2246/2006 | 1 |
| A/Chicken/Qalubia-Egypt/1/2006 | 1 |
| A/Turkey/Egypt/7/2007 | 1 |
| A/Egret/Egypt/1162-NAMRU3/2006 | 1 |
| A/Common Magpie/Hong Kong/5052/2007 | 1 |
| A/Common Buzzard/Bulgaria/38WB/2010 | 1 |
| A/Chicken/Egypt/Q1089E/2010 | 1 |
| A/Goose/Egypt/M2794A/2011 | 1 |
| A/Goose/Hong Kong/631/2009 | 1 |
| A/Large-Billed Crow/Hong Kong/497/2011 | 1 |
| A/Chicken/Egypt/S3806B/2011 | 1 |
| A/Crested Myna/Hong Kong/8381/2012 | 1 |
| A/Black Headed Gull/Hong Kong/84/2012 | 1 |
| A/Turkey/Egypt/S6405C/2012 | 1 |
| A/Chicken/Egypt/M7217B/2013 | 1 |
| A/Chicken/Egypt/B9040A/2013 | 1 |
| A/Chicken/Egypt/F9514A/2014 | 1 |
| A/Chicken/Bangladesh/23974/2014 | 1 |
| A/Goose/Bangladesh/25169/2015 | 1 |
| A/Chicken/Egypt/A10758D/2015 | 1 |
| A/Chicken/Egypt/Q10937B/2015 | 1 |
| A/Shorebird/DE/472/2007 | 1 |
| A/Duck/Bangladesh/1559/2009 | H5N2 | 18 | 2 | 26 |
| A/Mallard/MN/346250/00 | 2 |
| Unknown #9- from CDC 2014 | 1 |
| A/Mule Duck/Bulgaria/237/2011 | 3 |

12

{12}

| A/Ruddy Turnstone/DE/431/2011 | | | 2 | |
| --- | --- | --- | --- | --- |
| A/Mallard/AR/1C/2001 | | | 4 | |
| A/Turkey/ND/11419-1/15 | | | 1 | |
| A/Turkey/MN/11668-1/2015 | | | 1 | |
| A/Turkey/MN/10777/15 | | | 1 | |
| A/Turkey/MN/110915-1/15 | | | 1 | |
| A/Northern Pintail/WA/40564/14 | | | 1 | |
| A/Chicken/Italy/312/97 | | | 1 | |
| A/Northern Pintail/Washington/40964/2014 | | | 1 | |
| A/Snow Goose/CC15-84B/2015 | | | 1 | |
| A/White-Faced Whistling Duck/Colombia/1/2011 | | | 1 | |
| A/Shorebird/DE/318/2011 | | | 1 | |
| A/Northern Pintail/CA/44242-758/2006 | | | 1 | |
| A/Mallard/OH/14OS2758/2014 | | | 1 | |
| Unknown #10- from CDC 2014 | | | 2 | |
| A/Duck/Hokkaido/69/2000 | H5N3 | 3 | 1 | 4 |
| A/Ruddy Turnstone/DE/215/91 | | | 1 | |
| A/Mallard/Italy/80/93 | H5N4 | 2 | 1 | 2 |
| A/Shorebird/DE/230/2000 | | | 1 | |
| A/Mallard/Alberta/383/2009 | H5N5 | 1 | 1 | 1 |
| A/Peregrine Falcon/Hong Kong/4955/2015 | H5N6 | 1 | 1 | 1 |
| A/Shorebird/DE/75/2004 | H5N7 | 1 | 1 | 1 |
| A/Mule Duck/Bulgaria/328/2011 | | | 1 | |
| A/Quail/CA/K1400794/2014 | | | 1 | |
| A/Ruddy Turnstone/DE/237/91 | | | 1 | |
| A/Shorebird/DE/192/98 | H5N8 | 7 | 4 | 13 |
| A/Laughing Gull/DE/156/2004 | | | 4 | |
| A/Mallard/Korea/W452/14 | | | 1 | |
| A/Gyrfalcon/WA/41088-6/14 | | | 1 | |
| A/Chicken/Italy/9097/97 | H5N9 | 2 | 1 | 2 |
| A/Ruddy Turnstone/DE/117/2011 | | | 1 | |
| Unknown #1- from CDC 2013 | | | 2 | |
| Unknown #11- from CDC 2014 | H6N1 | 3 | 2 | 6 |
| A/Shorebird/Delaware Bay/230/2009 | | | 2 | |
| Unknown #12- from CDC 2014 | H6N2 | 2 | 1 | 3 |
| A/Turkey/Massachusetts/3740/65 | | | 2 | |
| A/Mallard/Alberta/58/1989 | H6N4 | 1 | 2 | 2 |
| Blue-winged Teal Texas | | | 2 | |
| A/Mallard/Alberta/203/1992 | H6N5 | 3 | 2 | 6 |
| A/Shearwater/Australia/1/73 | | | 2 | |
| Unknown #13- from CDC 2014 | H6N8 | 1 | 1 | 1 |
| A/Shovele/Egypt/597/2004 | | | 2 | |
| Unknown #14- from CDC 2014 | | | 1 | |
| Unknown #15- from CDC 2014 | | | 1 | |
| A/Turkey/Italy/6423-1/99 | H7N1 | 8 | 1 | 9 |
| A/Parakeet/Netherlands/267497/94 | | | 1 | |
| A/Ostrich/Zimbabwe/222/96 | | | 1 | |
| A/Mallard/Alberta/34/2001 | | | 1 | |
| A/Chicken/Italy/1285/2000 | | | 1 | |
| A/Avian/New York/11678-4/2005 | | | 1 | |
| A/Shorebird/DE/282/2011 | H7N2 | 9 | 3 | 19 |
| A/Chicken/NY/116124/2003 | | | 3 | |
| A/Pheasant/NJ/30739-9/2000 | | | 3 | |

13

{13}

| A/Ruddy Turnstone/DE/130/99 | | | 3 | |
| --- | --- | --- | --- | --- |
| A/Australian Shelduck/Western Australia/1756/83 | | | 3 | |
| A/Mallard/Ramon/79/14T | | | 1 | |
| A/Lesser Noddy/Western Australia/2371/83 | | | 1 | |
| A/Ruddy Turnstone/DE/282/2011 | | | 1 | |
| A/Mallard/Netherlands/12/2000 | | | 1 | |
| A/Sanderling/DE/280/2015 | | | 1 | |
| A/Mallard/Alberta/27/2001 | | | 1 | |
| A/Mallard/Alberta/243/2006 | | | 1 | |
| A/Shorebird/DE/552/2006 | | | 1 | |
| A/Dunlin/DE/281/2015 | | | 1 | |
| A/Red Knot/DE/269/2015 | | | 1 | |
| A/Ruddy Turnstone/DE/115/2011 | | | 1 | |
| A/Mallard/Alberta/341/2012 | | | 1 | |
| A/Ruddy Turnstone/DE/124/2007 | | | 1 | |
| A/Ruddy Turnstone/DE/503/2011 | | | 1 | |
| A/Ruddy Turnstone/DE/282/2006 | | | 1 | |
| A/Ruddy Turnstone/DE/108/2007 | | | 1 | |
| A/Shorebird/DE/53/2002 | | | 1 | |
| A/Mallard/Alberta/167/2010 | H7N3 | 28 | 1 | 28 |
| A/Red Knot/DE/239/2015 | | | 1 | |
| A/Turkey/CA/K1500529/2015 | | | 1 | |
| A/Mallard/Alberta/747/2015 | | | 1 | |
| A/Mallard/Alberta/174/2010 | | | 1 | |
| A/Shorebird/DE/218/2015 | | | 1 | |
| A/Laughing Gull/DE/22/2002 | | | 1 | |
| A/Chicken/Chile/176822/2002 | | | 1 | |
| A/Mallard/Netherlands/12/2000 | | | 1 | |
| A/Mallard/OH/14OS823/2015 | | | 1 | |
| A/Chicken/Chile/184240-4957/2002 | | | 1 | |
| A/Canada/RV444/2004 | | | 1 | |
| A/Chicken/Karachi(Pakistan)/NARC-100/2004 | | | 1 | |
| A/Chicken/Saskatchewan/HR10/2007 | | | 1 | |
| A/Duck/Nanchang/1944/93 | H7N4 | 2 | 1 | 2 |
| A/Ruddy Turnstone/DE/284/2006 | | | 1 | |
| A/Ruddy Turnstone/DE/262/2006 | H7N5 | 2 | 1 | 2 |
| A/Mallard/OH/14OS0821/2015 | | | 1 | |
| A/Chilean Teal/Chile/9/2013 | H7N6 | 1 | 1 | 1 |
| A/Teal/Egypt/835/2004 | | | 2 | |
| Unknown #16- from CDC 2014 | | | 2 | |
| A/Equine/Prague/56 | | | 2 | |
| A/Ruddy Turnstone/NJ/AI11-1678/2011 | | | 3 | |
| A/Ruddy Turnstone/DE/134/99 | H7N7 | 10 | 5 | 23 |
| A/Duck/Potsdam/S28716/88 | | | 2 | |
| A/Red Knot/DE/259/94 | | | 3 | |
| A/Seal/MA/1/80 | | | 2 | |
| A/Turkey/Ireland/PV74/95 | | | 1 | |
| A/Netherlands/219/2003 | | | 1 | |
| A/Mallard/Alberta/579/2010 | H7N8 | 2 | 1 | 2 |
| A/Turkey/IN/1573-2/2016 | | | 1 | |
| A/Anhui/1/2013 | | | 1 | |
| A/Turkey/MN/037767/2009 | H7N9 | 9 | 3 | 22 |
| A/Mallard/Alberta/177/2004 | | | 3 | |

14

{14}

| A/Mallard/Alberta/114/99 | | | 4 | |
| --- | --- | --- | --- | --- |
| A/Shorebird/DE/28/95 | | | 3 | |
| A/Turkey/MN/1/88 | | | 2 | |
| A/Hong Kong/5942/2013 | | | 2 | |
| A/Taiwan/T1.4/2013 | | | 2 | |
| A/Shanghai/1/2013 | | | 2 | |
| A/Turkey/Ontario/6118/68 | H8N4 | 1 | 2 | 2 |
| A/Shorebird/DE/133/2002 | | | 1 | |
| A/Laughing Gull/DE/5/2003 | H9N1 | 3 | 1 | 3 |
| A/Ruddy Turnstone/NJ/A107-283/2007 | | | 1 | |
| A/Chicken/Hong Kong/g9/1997 | | | 1 | |
| A/Hong Kong/1073/99 | | | 1 | |
| A/Shorebird/Delaware Bay/127/2003 | | | 4 | |
| A/Turkey/Wisconsin/1/66 | | | 1 | |
| A/Pheasant/UAE/D1307.B/2011 | | | 4 | |
| A/Quail/Lebanon/272/2010 | | | 4 | |
| A/Laughing Gull/DE/12/2006 | | | 2 | |
| A/Environment/Bangladesh/10306/11 (quail cage) | | | 1 | |
| A/Chicken/Bangladesh/10450/11 | | | 1 | |
| A/Chicken/Beijing/1/94 | | | 1 | |
| A/ Quail /Hong Kong/G1/97 | | | 1 | |
| A/Duck/Hong Kong/Y280/97 | | | 1 | |
| A/Sanderling/DE/449/2006 | | | 1 | |
| A/Hong Kong/69955/2008 | | | 1 | |
| A/Quail/Lebanon/273/2010 | | | 1 | |
| A/Hong Kong/33982/2009 | | | 1 | |
| A/Shorebird/DE/464/2011 | | | 1 | |
| A/Chukar/Shantou/22116/2005 | | | 1 | |
| A/Chicken/Hong Kong/NT10/2011 | | | 1 | |
| A/Chicken/Hong Kong/CRA45/2010 | H9N2 | 40 | 1 | 50 |
| A/Chicken/Hong Kong/YU341/2008 | | | 1 | |
| A/American Oystercatcher/Chile/C1307/2105 | | | 1 | |
| A/Chicken/Egypt/S4454B/2011 | | | 1 | |
| A/Chicken/Bangladesh/25947/2015 | | | 1 | |
| A/Chicken/Egypt/S4456B/2011 | | | 1 | |
| A/Chicken/Egypt/S5018B/2012 | | | 1 | |
| A/Chicken/Egypt/D7663A/2013 | | | 1 | |
| A/Duck/Egypt/C9787/2014 | | | 1 | |
| A/Quail/Egypt/D9842/2014 | | | 1 | |
| A/Chicken/Egypt/S10489C/2015 | | | 1 | |
| A/Chicken/Egypt/F10533D/2015 | | | 1 | |
| A/Chicken/Egypt/D10553A/2015 | | | 1 | |
| A/Chicken/Egypt/D10561/2015 | | | 1 | |
| A/Chicken/Egypt/A1093D/2015 | | | 1 | |
| A/Chicken/Egypt/S10598E/2015 | | | 1 | |
| A/Chicken/Egypt/D10705/2015 | | | 1 | |
| A/Chicken/Egypt/D10975/2015 | | | 1 | |
| A/Pheasant/Shantou/2785/2015 | | | 1 | |
| A/Chukar/Shantou/2777/2015 | | | 1 | |
| A/Pigeon/Shantou/3577/2015 | | | 1 | |
| A/Mallard/Ireland/PV46B/93 | H9N3 | 1 | 1 | 1 |
| A/Shorebird/DE/231/2003 | H9N4 | 1 | 1 | 1 |
| A/Shorebird/DE/261/2003 | H9N5 | 2 | 1 | 2 |

15

{15}

| A/Mallard/Alberta/162/2007 | | | 1 | |
| --- | --- | --- | --- | --- |
| A/Ruddy Turnstone/DE/268/2011 | H9N6 | 1 | 1 | 1 |
| A/Shorebird/Delaware Bay/31/1996 | H9N7 | 3 | 2 | 4 |
| A/Shorebird/DE/277/2000 | | | 1 | |
| A/Ruddy Turnstone/DE/253/2011 | | | 1 | |
| A/Ruddy Turnstone/DE/116/98 | H9N8 | 1 | 1 | 1 |
| A/Pheasant/WA/373/49/85 | H9N9 | 4 | 1 | 4 |
| A/Ruddy Turnstone/Virginia/2297/1988 | | | 1 | |
| A/Shorebird/DE/141/2002 | | | 1 | |
| A/Shorebird/DE/554/2007 | | | 1 | |
| A/Teal/Egypt/0457/2003 | H10N1 | 4 | 2 | 7 |
| Unknown #17- from CDC 2014 | | | 2 | |
| A/Mallard/Wisconsin/4230/2009 | | | 2 | |
| A/Shorebird/Delaware Bay/338/2009 | | | 1 | |
| A/Shorebird/Delaware Bay/63/1996 | H10N2 | 1 | 2 | 2 |
| Unknown #1- from CDC 2012 | H10N7 | 8 | 1 | 15 |
| Green-winged Teal Texas | | | 2 | |
| A/Shoveler/Egypt/0600/2004 | | | 2 | |
| A/Shoveler/Egypt/845/2004 | | | 2 | |
| Unknown #18- from CDC 2014 | | | 2 | |
| Unknown #19- from CDC 2014 | | | 2 | |
| A/Mallard/Illinois/10OS4334/2010 | | | 2 | |
| A/Chicken/Germany/N/49 | | | 2 | |
| A/Quail/Italy/1117/65 | H10N8 | 1 | 2 | 2 |
| Unknown #20- from CDC 2014 | H11N1 | 1 | 2 | 2 |
| A/Shorebird/Delaware Bay/216/1999 | H11N2 | 2 | 2 | 4 |
| A/Laughing Gull/Delaware Bay/94/1995 | | | 2 | |
| A/Duck/Bangladesh/1052/2007 | H11N3 | 2 | 2 | 4 |
| A/Ruddy Turnstone/Delaware Bay/39/1994 | | | 2 | |
| A/Mallard/Alberta/125/1999 | H11N6 | 2 | 2 | 4 |
| A/Duck/England/56 | | | 2 | |
| Unknown #21- from CDC 2014 | H11N9 | 5 | 2 | 11 |
| Unknown #22- from CDC 2014 | | | 2 | |
| A/American Green-Winged Teal/Mississippi/300/2010 | | | 2 | |
| A/Common Goldeneye/Iowa/3192/2009 | | | 2 | |
| A/Duck/Memphis/546/74 | | | 3 | |
| A/Mallard/Wisconsin/4218/2009 | H12N5 | 5 | 2 | 10 |
| A/Mallard/Ohio/1688/2009 | | | 2 | |
| A/Northern Shoveler/Mississippi/09OS025/2009 | | | 2 | |
| A/Northern Pintail/Missouri/319/2009 | | | 2 | |
| A/Duck/Alberta/60/76 | | | 2 | |
| A/Black-Legged Kittiwake/Quebec/02838-1/2009 | H13N6 | 3 | 2 | 5 |
| A/Ring-Billed Gull/Quebec/02434-1/2009 | | | 2 | |
| A/Gull/Maryland/704/77 | | | 1 | |
| Unknown #23- from CDC 2014 | H14N5 | 2 | 2 | 4 |
| A/Mallard/Astrakhan/263/82 | | | 2 | |
| A/Shearwater/Australia/2576/79 | H15N9 | 1 | 2 | 2 |
| A/Shorebird/Delaware/172/2006 | H16N3 | 1 | 2 | 2 |
| **Total** | **62 Subtypes** | **352** | **759** | **759** |

Note: H1N1 (pre 2009), H1N1 Avian, and H1N1 Swine are counted as one subtype; H3N2 Swine and H3N2v are counted as one subtype.

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Performance of the neural network algorithm was verified utilizing the technique of k-fold cross-validation (k=10) that allows assessment of how the neural network algorithm will generalize to a completely independent dataset. This mathematical approach was accomplished by dividing the training set randomly into 10 groups. All groups but one are used to train the neural network algorithm, and the algorithm is tested on the remaining group. Then, the groups used for training are rotated until all combinations of nine training groups/one test group have been used. The performance of each test group was then compiled to estimate performance expected on an independent dataset. Therefore, while viruses in the training set were not technically naïve to the training, this approach allowed an estimation of performance as if each portion of the training set had been naïve prior to validation activities.

## Postmarket Surveillance and Software Update Plan

InDevR developed a plan to monitor genetic changes in the circulating strains of influenza annually in order to assess the impact of those changes on the results generated by the FC8G assay. InDevR will test the CDC human influenza panel (includes seasonal influenza A and influenza B strains) and the CDC animal influenza panel (includes select, important non-seasonal strains with pandemic potential) annually, if appropriate and available. If the CDC human and animal influenza panels are not available or not appropriate for testing with the FC8G assay, e.g., virus samples inactivated with Betapropiolactone (BPL) are not compatible with the FC8G assay, etc. InDevR will conduct similar experiments on similar panels compiled from other sources (such as commercial sources and/or collaborators). Such a human influenza panel would consist of two strains of each target virus group, and include strains genetically similar to those included in the annual quadrivalent vaccine formulation for the four components in addition to two pre-2009 H1N1 strains. Composition of such an animal influenza panel will be based on recommendations from the CDC and other surveillance laboratories regarding selecting important non-seasonal strains with pandemic potential, and will be subject to availability limitations. In addition, if testing and reporting from CDC or other surveillance laboratory suggests that a newly emerging strain of influenza is on the rise in the US and it is not represented in the CDC panels, then InDevR will work with CDC and/or surveillance laboratories to test the newly emerging strain with the FC8G assay to determine its reactivity with the newly emerging strain.

If the annual reactivity testing indicates that the assay is not reactive to one or more of the strains tested, InDevR will further investigate by testing other similar strains to confirm that a systematic non-reactivity exists and that this failure to detect the virus was not due to an isolated assay failure or issue. If a change in reactivity is confirmed, the discovery will be reported to the FDA. An update to the training database and re-optimization of the neural network parameters would likely be necessary.

Once data have been generated that indicate an update to the training database is necessary, InDevR will obtain as many strains representing the non-reactive group as possible. If the virus(es) exhibiting low reactivity were initially emerging, it may be difficult to obtain a large number of strains. Even with a relatively low number of strains, it may be possible to

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create a robust collection of images by analyzing strains in replicates, as well as analyzing each additional strain at a variety of concentrations by preparing a contrived panel spiked at a variety of concentrations into pooled clinical negative material. These samples will then be analyzed to generate FC8G assay images. Once FC8G images are obtained for these samples, InDevR will utilize the same criteria currently in place for determining suitability for inclusion in the training database. Once the training database update is complete, InDevR will follow the same general procedure used during the initial software development to optimize the neural network parameters and finalize the software update. In brief, InDevR will utilize the FC8G Data Tools software developed for neural network training to optimize the neural network parameters using a k-fold cross-validation approach. Once the neural network parameters have been optimized, InDevR will optimize the thresholds applied to each neural network and sub-net (assay cutoff) to ensure the proper balance of sensitivity and specificity. After the assay thresholds have been optimized, the FC8G Software code would be updated accordingly. Final validation of the updated software with the new neural network parameters and cutoffs will also require bench testing of training-naïve samples of the strain(s) in question.

Once validation of the newly updated software is completed, InDevR will submit a 510(k) to the FDA for the modified device.

## Results Interpretation

### Interpretation of External Quality Control (QC) Results

- • **External Batch Positive Control** (user supplied influenza positive specimen):
Interpret the External Batch Positive Control results (if included) to determine whether the expected result of influenza positive was obtained and if the batch of samples is valid. Failure to obtain the expected influenza positive result should invalidate the batch, and all samples included in that batch should be retested.
- • **External Batch Negative Control** (user supplied influenza negative clinical specimen):
Interpret the External Batch Negative Control results (if included) to determine whether the expected result of “Influenza Not Detected” was obtained and if the batch of samples is valid. Failure to obtain the expected “Influenza Not Detected” result should invalidate the batch, and all samples included in that batch should be retested.
- • **Extraction Negative Control** (nuclease-free water used in place of specimen during extraction):
Interpret the Extraction Negative Control results to determine whether the expected result of either “Influenza Not Detected” or “No Call: Assay Failure (Internal Control)” was obtained and if the batch of samples are valid. Failure to obtain either “Influenza Not Detected” or “No Call: Assay Failure (Internal Control)” result should invalidate the batch, and all samples included in that batch should be retested.
- • **RT-PCR No Template Control** (nuclease-free water used in place of template during RT-PCR reaction setup):

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Interpret the RT-PCR No Template Control results to determine whether the expected result of either “Influenza Not Detected” or “No Call: Assay Failure (Internal Control)” was obtained and if the batch of samples are valid. Failure to obtain either “Influenza Not Detected” or “No Call: Assay Failure (Internal Control)” result should invalidate the batch, and all samples included in that batch should be retested.

### Interpretation of Assay Results

The table below lists the expected results for the FluChip-8G A+B Assay. The FluChip-8G Software automatically determines the sample result and qualified testing personnel should interpret the sample results as follows:

| If Result is: | Explanation | Interpretation of Results |
| --- | --- | --- |
| **Influenza not Detected** | Valid negative result. | **Report Result** |
| **Influenza A Detected, Seasonal H1N1 (2009)** | Valid positive result. | |
| **Influenza A Detected, Seasonal H3N2** | | |
| **Influenza B Detected, Victoria Lineage** | | |
| **Influenza B Detected, Yamagata Lineage** | Valid positive result. | |
| **Influenza B Detected** | Low virus titers can result in detection of influenza B without an associated lineage. | |
| **Influenza A Detected: Equivocal Result (no subtyping)** | Low virus titers can result in detection of influenza A without an associated subtype. Detection of influenza A without a subtype can also in rare cases indicate the presence of a novel strain. | **Retest Specimen to Confirm Result** If the retest provides a different result, test the sample a third time to ensure the accuracy of the result. In the rare instance that all 3 results conflict, the result should be considered inconclusive and a new specimen should be collected and tested if possible. If an A/equivocal result is confirmed through retesting described above, contact appropriate public health authorities for confirmatory testing. |
| **Influenza A Detected, Non-Seasonal** | This result indicates the presumptive presence of a novel influenza A virus other than the annually circulating A/H1N1pdm09 and A/H3N2 viruses. The A/H1N1 virus circulating prior to the appearance of A/H1N1pdm09 is expected to be identified as “Influenza A Detected, Non-Seasonal”. | **Retest Specimen to Confirm Result** If the retest provides a different result, test the sample a third time to ensure the accuracy of the result. In the rare instance that all 3 results conflict, the result should be considered inconclusive and a new specimen should be collected and tested if possible. If an A/non-seasonal result is confirmed through retesting described above, contact the appropriate public health authorities for confirmatory testing. |

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| **Multiple Infections Detected (any combination of the individual results above)** | This result indicates co-infection with multiple influenza virus types and/or subtypes. Multiple infections are possible but rare. This result may also occur in patients that have recently received an attenuated live intranasal influenza vaccine. If one of the targets detected is 'Influenza A Detected, Non-Seasonal', this result indicates the presumptive presence of a novel influenza A virus other than the annually circulating A/H1N1pdm09 and A/H3N2 viruses. | **Retest Specimen to Confirm Result** If the retest provides a different result, test the sample a third time to ensure the accuracy of the result. In the rare instance that all 3 results conflict, the result should be considered inconclusive and a new specimen should be collected and tested if possible. If a confirmed multiple infection result includes an A/equivocal or A/non-seasonal result, contact appropriate public health authorities for confirmatory testing. |
| --- | --- | --- |
| **No Call: Assay Failure (Internal Control)** | This result indicates the internal control failed to be detected. This result typically indicates poor specimen collection, specimen degradation, or user error in the specimen extraction or RT-PCR steps. | **Retest Specimen** If upon retest a valid result is generated, follow the recommended actions for that result. |
| **No Call: Assay Failure (Hybridization Control)** | This result is likely due to an issue in the hybridization step. | **Retest Specimen** If upon retest a valid result is generated, follow the recommended actions for that result. |
| **No Call: Assay Failure (Labeling Control)** | This result is likely due to an issue in the post- hybridization labeling step. | |
| **Error: Image Processing Failure** | This may indicate an error in microarray processing (See final wash step in assay procedure), in the placement of the FluChip-8G Microarray Slide within the FluChip-8G Imaging System, or the FluChip-8G Imaging System focus setting. | Visually inspect the back of the slide for artifacts such as dust or salt precipitates. Using a damp lint-free cloth, wipe the back of the slide and **Rescan the Entire Slide**. Check for correct slide and slide holder orientation, correct any orientation errors and **Rescan the Entire Slide**. See FluChip-8G Imaging System Operation Manual for instructions on confirming system focus, **Rescan the Entire Slide**. If upon rescan a valid result is generated for the sample that produced the error, follow the recommended actions for that result. For samples that did not produce the error upon initial scan the result from the 1st scan should be used. If upon rescan a valid result is not generated, retest the specimen. |
| **Not Analyzed** | Microarray designated as 'EMPTY' during sample type selection in FluChip-8G software. The microarray will not be analyzed and no result will be displayed | If the position was unintentionally left empty, rescan the entire slide using the correct sample ID selection. For samples that did not produce the result of 'Not Analyzed' in the initial scan the result from the 1st scan should be used. |

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### J. Substantial Equivalence Information:

1. Predicate device name(s):

CDC Human Influenza Virus Real-Time PCR Diagnostic Panel

2. Predicate 510(k) number(s):

K172091

3. Comparison with predicate:

| Similarities and Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| | FluChip-8G Influenza A+B Assay (K182513) | CDC Human Influenza Virus Real-Time PCR Diagnostic Panel (K172091) |
| Measurand | Influenza RNA | Same |
| Viruses Detected | Influenza A and B viruses | Same |
| Influenza A subtype Differentiation | Seasonal influenza A/H1N1pdm09Seasonal influenza A/H3N2“Non-seasonal” influenza A | SameSameInfluenza A/H5 (Asian Lineage) |
| Influenza B lineage Differentiation | Influenza B/VictoriaInfluenza B/Yamagata | Same |
| Assay Type | Qualitative | Same |
| CLIA Complexity | High | Same |
| Sample Type | Nasal swab and nasopharyngeal swab specimens | Upper respiratory tract specimens, including nasal swabs and nasopharyngeal swabs; lower respiratory tract specimens |
| Sample Preparation Method | Nucleic Acid Extraction | Same |
| Detection Technology | Fluorescence | Same |
| Technological Principles | RT-PCR followed by endpoint detection by microarray via nucleic acid probes and fluorescence | Real-time RT-PCR via nucleic acid probes and fluorescence |
| Nucleic Acid Extraction | QIAamp DSP Virus Spin Kit | QIAamp Viral RNA Mini Kit, QiagenQIAcube with QIAamp Viral RNA Mini KitMagNA Pure Compact—Total Nucleic Acid Kit, RocheMagNA Pure Compact—RNA Isolation Kit, RocheMagNA Pure LC—RNA Isolation Kit II, RocheNucliSENS easyMAG, bioMerieux |
| Controls | Internal Control in each specimenExternal positive control processed with each batchExternal negative control processed with each batch | Same |
| Amplification Reagents | Quanta Biosciences qScript One-Step RT-PCR Kit | Quanta Biosciences qScript One-Step qRT-PCR Kit, low ROXInvitrogen SuperScript III Platinum One-Step Quantitative RT-PCR Kit |

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| Similarities and Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| | FluChip-8G Influenza A+B Assay (K182513) | CDC Human Influenza Virus Real-Time PCR Diagnostic Panel (K172091) |
| **Results Interpretation** | Automated test interpretation via neural network-based algorithm using fluorescence intensities | Manual test interpretation via C_{t} value determined from fluorescence intensities |
| **Instrumentation** | FluChip-8G Imaging System | Applied Biosystems 7500 Dx Real-Time PCR Instrument |

## K. Standard/Guidance Document Referenced (if applicable):

None

## L. Test Principle:

The FluChip-8G Influenza A+B Assay (FC8G assay) includes a multiplex primer mix to amplify the hemagglutinin (HA), neuraminidase (NA), matrix (M), non-structural (NS), and nucleoprotein (NP) gene segments of all influenza A viruses, as well as the hemagglutinin (HA) and neuraminidase (NA) gene segments of all influenza B viruses, and a portion of the 18s gene present in eukaryotic cells as an endogenous internal control. During a one-step RT-PCR reaction, the primers and RT-PCR enzymes and reagents are utilized to amplify nucleic acids extracted from human nasal specimens while incorporating biotin for downstream fluorescence labeling. The biotinylated amplified products are then heat-fragmented and applied to the FC8G microarray for hybridization, labeling, and detection using the FluChip-8G Imaging System. The system software is trained to identify the patterns of the influenza target virus groups using a large database of thousands of results from viruses of known characterization, and a simple result in the form of a report is returned to the end user via the software user interface.

## M. Performance Characteristics (if/when applicable):

### 1. Analytical performance:

#### a. Precision/Reproducibility:

Two independent studies: within-laboratory repeatability and external multi-site user-to-user reproducibility, were carried out to evaluate the precision and reproducibility of the FluChip-8G Influenza A+B Assay.

A 45-member panel was used for the two studies. The panel was made with five viral strains that represent the five target influenza virus groups: A/New Caledonia/20/1999 (A/non-seasonal), A/California/07/2009 (A/H1N1pdm09), A/Victoria/361/2011 (A/H3N2), B/Wisconsin/01/2010 (B/Yamagata), and B/Florida/02/2006 (B/Victoria). Each strain was diluted into pooled influenza

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negative clinical material at three different target levels: Moderate Positive (MP) (approximately 3xLoD), Low Positive (LP) (approximately 1xLoD), and High Negative (HN) (approximately 0.1xLoD). Influenza negative clinical material consisted of clinical nasopharyngeal swab samples stabilized in universal transport medium (UTM) that were determined to be negative for the presence of influenza virus via an FDA-cleared influenza molecular assay.

## External Multi-Site User-to-User Reproducibility Study

Two operators at each of the three separate laboratory sites performed the FluChip-8G Influenza A+B Assay (FC8G assay) according to the assay instructions for use. Performance was evaluated on six unique testing points at the three separate laboratory sites for a total of 18 testing points. Each testing point corresponds to a unique run/execution of the FC8G assay, where each member of the blinded 45-member panel of contrived samples was tested in triplicate. Two unique lots of FC8G assay reagents were evenly divided between the three sites. At each site, each of the two operators executed three testing points. Operators at each site alternated testing until all six test points/site were completed. Each testing point was separated by a minimum of one full day in which no FC8G assay processing for this study occurred.

A total of 810 data points (45 samples/testing point x 18 testing points) were generated in this study. Performance was evaluated by determining the agreement with the expected results, where the MP and LP concentrations were expected to be positive for the intended target, and the HN was expected to be negative.

Table 3 below shows the results of the multi-site user-to-user reproducibility study.

Table 3: Multi-Site User-to-User Reproducibility Study Results

| | Site A | | | Site B | | | Site C | | | Overall % Agreement and 95% Confidence Interval by Sample (%) | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | Count and % Agreement | | 95% Confidence Interval (%) | Count and % Agreement | | 95% Confidence Interval (%) | Count and % Agreement | | | | | 95% Confidence Interval (%) |
| A/H1N1 (pre-2009) (A/non-seasonal) | MP (3x LoD) | 18/18 | 100% | 82.4 - 100 | 18/18 | 100% | 82.4 - 100 | 17/18^{a} | 94% | 74.2 - 99.0 | 53/54 | 98% | 90.2 - 99.7 |
| | LP (1x LoD) | 18/18 | 100% | 82.4 - 100 | 18/18 | 100% | 82.4 - 100 | 18/18 | 100% | 82.4 - 100 | 54/54 | 100% | 93.4 - 100 |
| | HN (0.1x LoD) | 7/18^{b} | 39% | 20.3 - 61.4 | 11/18^{c} | 61% | 38.6 - 79.7 | 13/18^{d} | 72% | 49.1 - 87.5 | 31/54 | 57% | 44.2 - 69.7 |
| A/H1N1pdm09 | MP (3x LoD) | 18/18 | 100% | 82.4 - 100 | 18/18 | 100% | 82.4 - 100 | 18/18 | 100% | 82.4 - 100 | 54/54 | 100% | 93.4 - 100 |
| | LP (1x LoD) | 18/18 | 100% | 82.4 - 100 | 14/18^{e} | 78% | 54.8 - 91.0 | 17/18^{f} | 94% | 74.2 - 99.0 | 49/54 | 91% | 80.1 - 96.0 |
| | HN (0.1x LoD) | 9/18^{g} | 50% | 29.0 - 71.0 | 11/18^{h} | 61% | 38.6 - 79.7 | 7/18^{i} | 39% | 20.3 - 61.4 | 27/54 | 50% | 37.1 - 62.9 |
| A/H3N2 | MP (3x LoD) | 18/18 | 100% | 82.4 - 100 | 18/18 | 100% | 82.4 - 100 | 18/18 | 100% | 82.4 - 100 | 54/54 | 100% | 93.4 - 100 |
| | LP (1x LoD) | 18/18 | 100% | 82.4 - 100 | 18/18 | 100% | 82.4 - 100 | 18/18 | 100% | 82.4 - 100 | 54/54 | 100% | 93.4 - 100 |
| | HN (0.1x LoD) | 17/18^{j} | 94% | 74.2 - 99.0 | 14/18^{m} | 78% | 54.8 - 91.0 | 16/18^{n} | 89% | 67.2 - 96.9 | 47/54 | 87% | 75.6 - 93.6 |
| B/Yamagata | MP (3x LoD) | 18/18 | 100% | 82.4 - 100 | 18/18 | 100% | 82.4 - 100 | 18/18 | 100% | 82.4 - 100 | 54/54 | 100% | 93.4 - 100 |
| | LP (1x LoD) | 18/18 | 100% | 82.4 - 100 | 17/18^{o} | 94% | 74.2 - 99.0 | 18/18 | 100% | 82.4 - 100 | 53/54 | 98% | 90.2 - 99.7 |
| | HN (0.1x LoD) | 12/18^{p} | 67% | 43.7 - 83.7 | 17/18^{q} | 94% | 74.2 - 99.0 | 15/18^{r} | 83% | 60.8 - 94.2 | 44/54 | 81% | 69.2 - 89.6 |
| | MP (3x LoD) | 18/18 | 100% | 82.4 - 100 | 18/18 | 100% | 82.4 - 100 | 18/18 | 100% | 82.4 - 100 | 54/54 | 100% | 93.4 - 100 |

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| **B/Victoria** | LP (1x LoD) | 17/18^{a} | 94% | 74.2 - 99.0 | 18/18 | 100% | 82.4 - 100 | 18/18 | 100% | 82.4 - 100 | 53/54 | 98% | 90.2 - 99.7 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | HN (0.1x LoD) | 7/18^{b} | 39% | 20.3 - 61.4 | 13/18^{c} | 72% | 49.1 - 87.5 | 6/18^{d} | 33% | 16.3 - 56.3 | 26/54 | 48% | 35.4 - 61.1 |
| **Overall** | **Moderate Positive (MP) (3x LoD)** | | | | | | | | | | **269/270** | **100%** | **97.9 - 99.9** |
| | **Low Positive (LP) (1x LoD)** | | | | | | | | | | **263/270** | **97%** | **94.7 - 98.7** |
| | **High Negative (HN) (0.1x LoD)** | | | | | | | | | | **175/270** | **65%** | **58.9 - 70.3** |

$^{a}$ 1/18 MP replicates was No Call: Assay Failure (Process Control)

$^{b}$ 7/18 HN replicates were Flu A non-seasonal; 4/18 HN replicates were Flu A Equivocal

$^{c}$ 5/18 HN replicates were Flu A non-seasonal; 2/18 HN replicates were Flu A Equivocal

$^{d}$ 4/18 HN replicates were Flu A non-seasonal; 1/18 HN replicates was Flu A Equivocal

$^{e}$ 3/18 LP replicates were Flu A/H1N1pdm09 and Flu A non-seasonal dual positive; 1/18 LP replicates was Flu A Equivocal

$^{f}$ 1/18 LP replicates were Flu A/H1N1pdm09 and Flu A non-seasonal dual positive

$^{g}$ 6/18 HN replicates were Flu A/H1N1pdm09; 3/18 HN replicates were Flu A Equivocal

$^{h}$ 2/18 HN replicates were Flu A/H1N1pdm09; 3/18 HN replicates were Flu A Equivocal; 2/18 HN replicates were Flu A non-seasonal

$^{i}$ 5/18 HN replicates were Flu A/H1N1pdm09; 5/18 HN replicates were Flu A Equivocal; 1/18 HN replicates was Flu A non-seasonal

$^{j}$ 1/18 HN replicates was Flu A/H3N2

$^{m}$ 1/18 HN replicates was Flu A/H3N2; 1/18 HN replicates was Flu A Equivocal; 2/18 HN replicates were Flu A/H3N2 and Flu A non-seasonal dual positive

$^{n}$ 2/18 HN replicates were Flu A/H3N2

$^{o}$ 1/18 LP replicates was Influenza Not Detected

$^{p}$ 2/18 HN replicates were Flu B; 2/18 HN were Flu B Yamagata; 2/18 HN were Flu B Victoria

$^{q}$ 1/18 HN replicates was Flu B

$^{r}$ 1/18 HN replicates was Flu B; 2/18 HN were Flu B Yamagata

$^{s}$ 1/18 LP replicates was Error: Image Processing Failure

$^{t}$ 1/18 HN replicates was Flu B; 8/18 HN were Flu B Victoria; 2/18 HN were Flu B Yamagata

$^{u}$ 1/18 HN replicates was Flu B; 4/18 HN were Flu B Victoria

$^{v}$ 12/18 HN replicates were Flu B Victoria

In addition, site-to-site, operator-to-operator, run-to-run, and reagent lot-to-lot reproducibility performance were also assessed using the data generated from this multi-site reproducibility study. There were no statistically significant differences in the observed performance between sites, between operators, between runs, or between reagent lots, as indicated by overlapping confidence intervals for all samples expected to be positive.

### Within-Laboratory Precision Study

Two operators at a manufacturer's internal laboratory site performed testing in the within-laboratory precision study. The same panel of 45 contrived samples described previously was utilized to conduct a total of 12 test points, with six test points conducted by each operator. Each testing point was separated by a minimum of one full day in which no FC8G assay processing for this study occurred. The same lot of FC8G reagents, including the QIAamp DSP Virus Spin Kit, and the same imaging system was utilized by both operators for testing.

A total of 540 data points (45 samples/testing point x 6 testing points/operator x 2 operators) were generated in this study. Performance was evaluated by determining the agreement with the expected results, where the MP and LP concentrations were expected to be positive for the intended target, and the HN was expected to be negative.

Table 4 below shows the results of the within-laboratory precision study.

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**Table 4: Within-Laboratory Precision Study Results**

| | Operator 1 | | Operator 2 | | Overall % Agreement and 95% Confidence Interval by Sample (%) | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | Count and % Agreement | 95% Confidence Interval (%) | Count and % Agreement | | | 95% Confidence Interval (%) |
| A/H1N1 (pre-2009) (A/non-seasonal) | MP (3x LoD) | 18/18 100% | 82.4 - 100 | 18/18 100% | 82.4 - 100 | 36/36 100% | 90.4 - 100 |
| | LP (1x LoD) | 18/18 100% | 82.4 - 100 | 18/18 100% | 82.4 - 100 | 36/36 100% | 90.4 - 100 |
| | HN (0.1x LoD) | 7/18 39% | 20.3 - 61.4 | 4/18 22% | 9.0 - 45.2 | 11/36 31% | 18.0 - 46.9 |
| A/H1N1pdm09 | MP (3x LoD) | 18/18 100% | 82.4 - 100 | 18/18 100% | 82.4 - 100 | 36/36 100% | 90.4 - 100 |
| | LP (1x LoD) | 18/18 100% | 82.4 - 100 | 12/18^{a} 67% | 43.7 - 83.7 | 30/36 83% | 68.1 - 92.1 |
| | HN (0.1x LoD) | 3/18 17% | 5.8 - 39.2 | 10/18 56% | 33.7 - 75.4 | 13/36^{b} 36% | 22.5 - 52.4 |
| A/H3N2 | MP (3x LoD) | 18/18 100% | 82.4 - 100 | 18/18 100% | 82.4 - 100 | 36/36 100% | 90.4 - 100 |
| | LP (1x LoD) | 18/18 100% | 82.4 - 100 | 18/18 100% | 82.4 - 100 | 36/36 100% | 90.4 - 100 |
| | HN (0.1x LoD) | 10/18 56% | 33.7 - 75.4 | 9/18 50% | 29.0 - 71.0 | 19/36^{c} 53% | 37.0 - 68.0 |
| B/Yamagata | MP (3x LoD) | 18/18 100% | 82.4 - 100 | 18/18 100% | 82.4 - 100 | 36/36 100% | 90.4 - 100 |
| | LP (1x LoD) | 18/18 100% | 82.4 - 100 | 17/18 94% | 74.2 - 99.0 | 35/36 97% | 85.8 - 99.5 |
| | HN (0.1x LoD) | 9/18 50% | 29.0 - 71.0 | 17/18 94% | 74.2 - 99.0 | 26/36 72% | 56.0 - 84.2 |
| B/Victoria | MP (3x LoD) | 18/18 100% | 82.4 - 100 | 18/18 100% | 82.4 - 100 | 36/36 100% | 90.4 - 100 |
| | LP (1x LoD) | 18/18 100% | 82.4 - 100 | 18/18 100% | 82.4 - 100 | 36/36 100% | 90.4 - 100 |
| | HN (0.1x LoD) | 13/18 72% | 49.1 - 87.5 | 18/18 100% | 82.4 - 100 | 31/36 86% | 71.3 - 93.9 |
| Overall | Moderate Positive (MP) (3x LoD) | | | | | 180/180 100% | 97.9 - 100 |
| | Low Positive (LP) (1x LoD) | | | | | 173/180 97% | 92.2 - 98.1 |
| | High Negative (HN) (0.1x LoD) | | | | | 100/180 56% | 48.3 - 62.6 |

$^{a}$ 6/18 LP replicates were Flu A/H1N1pdm09 and Flu A non-seasonal dual positive on initial testing. All six samples generated the expected Flu A/H1N1pdm09 positive results upon repeat testing per the Instructions for Use (IFU).

$^{b}$ 5/36 HN replicates were Flu A non-seasonal on initial testing. Upon repeat testing per the IFU, 2 of 5 samples generated the expected Flu A/H1N1pdm09 positive results, 2 of 5 samples generated Inconclusive results, and 1 of 5 samples generated an Influenza Not Detected result.

$^{c}$ 2/36 HN replicates were Flu A/H1N1pdm09 and Flu A non-seasonal dual positive on initial testing. All two samples generated Inconclusive results upon repeat testing per the IFU.

$^{d}$ 1/36 replicates was Flu A Equivocal and Flu A non-seasonal on initial testing. This sample generated an Inconclusive result upon repeat testing per the IFU

$^{e}$ 2/36 HN replicates were Flu A non-seasonal on initial testing. Upon repeat testing per the IFU, 1 of 2 samples generated an Inconclusive result, and 1 of 2 samples generated an Influenza Not Detected result.

# *b. Linearity/assay reportable range:*

Not applicable

# *c. Traceability, Stability, Expected values (controls, calibrators, or methods):*

# External Quality Control (QC)

The following external controls are recommended to be included in each testing batch, but are not provided in the test kit.

- External Batch Positive Control: A previously characterized influenza-positive clinical specimen is recommended for inclusion as part of good laboratory practices. This control is user-supplied.

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- External Batch Negative Control: A previously characterized influenza-negative clinical specimen is recommended for inclusion as part of good laboratory practices. This control is user supplied.
- Extraction Negative Control: This control is recommended for inclusion in each testing batch, and is composed of nuclease-free water added by the end user in place of specimen during the nucleic acid extraction step.
- RT-PCR No Template Control (NTC): This control is recommended for inclusion in each testing batch, and is composed of nuclease-free water added by the end user in place of extracted nucleic acid template during RT-PCR reaction setup.

The following language, pertaining to the external controls, is included in the Instructions for Use (IFU):

"Good laboratory practice recommends that both an external positive control and external negative control are included alongside clinical specimens in each batch of extractions. These external positive and negative controls should be treated as samples under analysis and interpreted as such. It is also recommended that an extraction negative control (consisting of nuclease-free water) and an RT-PCR No Template Control be run alongside each batch of specimens. These controls should be carried through the entire assay alongside the clinical specimens under analysis."

External batch positive controls were prepared for inclusion in each batch of samples analyzed during the prospective clinical study testing. The following four viruses were utilized to prepare the external batch positive controls: Influenza A/Netherlands/2290/2009 (A/H1N1pdm09), Influenza A/Victoria/361/2011 (A/H3N2), Influenza B/Florida/02/2006 (B/Victoria), and Influenza B/Florida/04/2006 (B/Yamagata). Three dilutions of each of the viruses were prepared in viral transport media (VTM) in large volumes to produce twelve overall external positive controls representing high, medium, and low influenza concentrations in each sample, as determined by an influenza molecular assay. Replicate aliquots of each control were prepared and provided to the clinical sites for use in testing. During the prospective clinical study, each site included one of the four external batch positive controls in each batch of specimens tested on a rotating basis.

Replicate aliquots of pooled influenza negative clinical materials that were determined to be negative for the presence of influenza virus via an FDA-cleared influenza molecular assay were also provided to the clinical study sites. During the prospective clinical study, each site included one aliquot of the provided external batch negative control in each batch of specimens tested.

In addition, an Extraction Negative Control and a No Template Control were also included in each batch of specimens tested at each site.

During the prospective clinical study between June 2017 and July 2018 at three sites, upon initial testing, there were one failed external batch negative control, one failed

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extraction negative control, and one failed external batch positive control. All controls with initial failed results generated expected results upon repeat testing. The failure rate upon initial testing (including the failed and the invalid results) of the external quality control materials was 1.7% (3/176), with 95% CI: (0.6% - 4.9%).

# Nasal and Nasopharyngeal Swab in UTM Specimens Stability

An analytical study was performed to evaluate the stability of clinical nasal swab (NS) and nasopharyngeal swab (NPS) in UTM specimens, when stored at refrigerator temperature (2° - 8°C). Contrived samples consisting of one strain representing each FC8G target virus group (A/H1N1pdm09, A/H3N2, A/non-seasonal, B/Victoria, and B/Yamagata) were prepared in pooled influenza negative clinical matrix at an influenza concentration of approximately 3x LoD. Triplicate extractions of the contrived sample were performed following storage at 10°C ± 2°C for each of the following time points: 0 hour, 24 hours, 36 hours, 48 hours, and 96 hours. For all storage time points tested in this analytical study, an overall agreement of 100% (95%CI, 79.6 – 100) with the expected result was obtained. This data supports a stability claim of 72 hours at refrigerator temperature (2° - 8°C) for clinical nasal swab (NS) and nasopharyngeal swab (NPS) in UTM specimens.

While a formal analytical study was not conducted to determine specimen stability at ≤ -70°C, data from the external controls performed during the clinical study were analyzed to assess stability over time when stored at ≤ -70°C for 427 days. The samples evaluated were those used as external batch controls executed weekly during the course of the clinical study at the InDevR clinical site. Influenza positives in the testing panel were stock influenza viruses spiked into UTM. Influenza negatives in the testing panel were pooled influenza negative clinical material. Based on the results of this analysis, it appears that the nasal swab and nasopharyngeal swab in UTM specimens are stable for up to 14 months when stored at ≤ -70°C.

# Nasal and Nasopharyngeal Swab in UTM Specimens Freeze and Thaw Study

An analytical study was conducted to assess the performance of the FC8G Assay on contrived samples that underwent repeated freeze/thaw cycles to establish the equivalency of samples subjected to freeze/thaw and support the clinical study design of testing archived frozen specimens. A panel of 60 contrived influenza samples were utilized in this testing, consisting of 12 unique strains for each of the five target groups (A/H1N1pdm2009, A/H3N2, A/non-seasonal, B/Yamagata, and B/Victoria). Samples were contrived by spiking stock whole virus into pooled influenza negative clinical material at a final concentration of approximately 3x LoD (ranging from 1x to 9x LoD, depending on strain), and five replicate aliquots of each prepared sample were made. The first aliquot of each strain was tested without re-freezing (immediately after preparation), with one aliquot of each strain tested after each of four consecutive freeze-thaw cycles to assess % agreement with expected result. Data from this study demonstrated that the FC8G Assay performance was not affected by up to four freeze and thaw cycles of the specimens.

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### Extracted RNA Specimens Stability

An analytical evaluation was performed to evaluate the stability of extracted RNA specimens, when stored at $\leq -70^{\circ}\text{C}$. A large volume of a representative A/H3N2 stock virus, A/Victoria/361/2011, was utilized to perform this testing by spiking stock virus into pooled influenza-negative clinical material at approximately 3x LoD and creating individual homogeneous aliquots for downstream testing. Sixteen aliquots of the prepared A/H3N2 contrived sample were extracted according to the Instructions for Use (IFU), subsequently pooled to create a homogeneous material, and aliquoted into five aliquots. Each aliquot was of sufficient volume to enable 14 replicate tests. One aliquot was utilized to assess the assay performance at "Day 0', and the remaining four aliquots were stored at $\leq -70^{\circ}\text{C}$ until tested. Three of the four remaining aliquots were utilized to conduct testing at Days 14, 30, and 40 after storage. The last aliquot was utilized to test the cumulative "worst case" stability of the assay intermediates, including Extracted RNA Specimen, RT-PCR Product, Fragmented RT-PCR product, and Labeled Microarray.

Extracted RNA specimens stored at $\leq -70^{\circ}\text{C}$ showed 100% agreement with "Day 0' results for the duration of the testing schedule of 40 days, supporting a stability claim of 30 days at $\leq -70^{\circ}\text{C}$.

### Intact and Heat-Fragmentated RT-PCR Products Stability

An analytical evaluation was performed to evaluate the stability of intact and heat-fragmented RT-PCR products when stored at either $+10^{\circ}\text{C}$ or $-10^{\circ}\text{C}$.

A large volume of a representative A/H3N2 stock virus, A/Victoria/361/2011, was utilized to perform this testing by spiking stock virus into pooled influenza-negative clinical material at approximately 3x LoD and creating individual homogeneous aliquots for downstream testing. Thirty-five (35) aliquots of the prepared A/H3N2 sample were extracted according to the IFU, and subsequently pooled to create a homogeneous material. This extracted material was then used as template in 254 replicate RT-PCR amplification reactions conducted according to the IFU. Once amplified, all of the replicates of the amplified A/H3N2 sample were pooled to create a homogeneous RT-PCR product which was subsequently aliquoted into approximately 220 individual PCR tubes for downstream processing by the remainder of the FC8G assay.

For the intact (un-fragmented) RT-PCR product testing, 14 replicate aliquots of the amplified A/H3N2 sample were processed through the remainder of the FC8G assay according to the IFU at "Day 0'. Eighty-four (84) replicate aliquots of the amplified A/H3N2 sample were divided evenly at two different storage temperatures: 42 aliquots at $+10^{\circ}\text{C}$ and 42 aliquots at $-10^{\circ}\text{C}$. For the $+10^{\circ}\text{C}$ stored products, 14 aliquots were processed through the remainder of the FC8G assay according to the IFU at Day 3, Day 7, and Day 14 after storage. For the $-10^{\circ}\text{C}$ stored products, 14

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aliquots were processed through the remainder of the assay at Days 7, 14, and 30 after storage.

Intact (un-fragmented) RT-PCR products stored at +10°C showed 100% agreement with “Day 0” results for the duration of the testing schedule of 14 days, supporting a stability claim of 7 days at ≤ 8°C. Intact (un-fragmented) RT-PCR products stored at -10°C showed 100% agreement with “Day 0” results for the duration of the testing schedule of 30 days, supporting a stability claim of 14 days at ≤ -15°C.

For the heat-fragmented RT-PCR product testing, 14 replicate aliquots of the amplified A/H3N2 sample were heat-fragmented and processed through the remainder of the FC8G assay according to the IFU at “Day 0”. The remaining replicate RT-PCR product aliquots were heat-fragmented according to the IFU, and the fragmented products were then immediately pooled to create a homogeneous material. The homogeneous material was then re-aliquoted. These aliquots were evenly divided between two different storage temperatures, +10°C and -10°C. For the +10°C stored products, aliquots were tested on Days 1, Day 3, Day 7, and Day 14 after storage. For the aliquots stored at -10°C, aliquots were tested on Days 3, 7, 14, and 30 after storage.

Heat-fragmented RT-PCR products stored at +10°C showed 100% agreement with “Day 0” results for the duration of the testing schedule of 14 days, supporting a stability claim of 7 days at ≤ 8°C. Heat-fragmented RT-PCR products stored at -10°C showed 100% agreement with “Day 0” results for the duration of the testing schedule of 30 days, supporting a stability claim of 14 days at ≤ -15°C.

#### Labeled Microarray Stability

An analytical evaluation was performed to evaluate the stability of labeled microarrays when stored at room temperature (18°C - 25°C).

Two microarray slides processed for other aspe…