← Product Code [OCC](/submissions/MI/subpart-d%E2%80%94serological-reagents/OCC) · K112172

# QUIDEL MOLECULAR INFLUENZA A + B ASSAY (K112172)

_Quidel Corp. · OCC · Dec 22, 2011 · Microbiology · SESE_

**Canonical URL:** https://fda.innolitics.com/submissions/MI/subpart-d%E2%80%94serological-reagents/OCC/K112172

## Device Facts

- **Applicant:** Quidel Corp.
- **Product Code:** [OCC](/submissions/MI/subpart-d%E2%80%94serological-reagents/OCC.md)
- **Decision Date:** Dec 22, 2011
- **Decision:** SESE
- **Submission Type:** Traditional
- **Regulation:** 21 CFR 866.3980
- **Device Class:** Class 2
- **Review Panel:** Microbiology

## Indications for Use

The Quidel® Molecular Influenza A + B Assay is a multiplex Real Time RT-PCR assay for the in vitro qualitative detection and differentiation of influenza A and influenza B viral RNA in nasal and nasopharyngeal swabs from patients with signs and symptoms of respiratory infection. The test is intended for use as an aid in the differential diagnosis of influenza A and influenza B viral infections in humans in conjunction with clinical and epidemiological risk factors. The assay does not detect the presence of influenza C virus. Negative results do not preclude influenza virus infection and should not be used as the sole basis for diagnosis, treatment or other patient management decisions. Performance characteristics for influenza A were established during the 2010-2011 influenza season when influenza A/H3 and 2009 H1N1 influenza were the predominant influenza A viruses in circulation. When other influenza A viruses are emerging, performance characteristics may vary. If infection with a novel influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent Influenza viruses and sent to state or local health department for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.

## Device Story

Multiplex RT-PCR assay; detects influenza A (matrix protein gene) and influenza B (neuraminidase gene) viral RNA. Input: nasal/nasopharyngeal swabs; RNA extracted via bioMérieux NucliSENS easyMAG. Amplification/detection performed on Applied Biosystems 7500 Fast Dx instrument. Uses TaqMan chemistry; enzyme with reverse transcriptase, DNA polymerase, and 5'-3' exonuclease activity. Output: qualitative detection/differentiation of influenza A and B. Used in clinical laboratory settings by trained personnel. Results aid differential diagnosis alongside clinical/epidemiological factors. Benefits: rapid, sensitive molecular identification of influenza viruses to guide patient management.

## Clinical Evidence

Prospective study (N=668) and retrospective study (N=372) compared subject device to FDA-cleared molecular comparator. Prospective Influenza A: 100% PPA, 98.5% NPA. Prospective Influenza B: 95.5% PPA, 97.8% NPA. Retrospective Influenza A: 100% PPA, 100% NPA. Retrospective Influenza B: 97.4% PPA, 99.4% NPA. Discordant analysis performed via sequencing.

## Technological Characteristics

Multiplex Real-Time RT-PCR; TaqMan chemistry. Targets: Influenza A matrix gene, Influenza B neuraminidase gene. Instrumentation: Applied Biosystems 7500 Fast Dx (software v1.4). Extraction: bioMérieux NucliSENS easyMAG (software v2.0). Reagents: lyophilized Master Mix (primers, probes, dNTPs, enzymes). Detection: FAM (Flu A), CAL Fluor Orange 560 (Flu B), Quasar 670 (Process Control).

## Regulatory 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.

## Predicate Devices

- Prodesse ProFlu+ (k073029, k092500, k081030)

## Submission Summary (Full Text)

> This content was OCRed from public FDA records by [Innolitics](https://innolitics.com). If you use, quote, summarize, crawl, or train on this content, cite Innolitics at https://innolitics.com.
>
> Innolitics is a medical-device software consultancy. We help companies design, build, and clear FDA-regulated software and AI/ML devices, including [a 510(k)](https://innolitics.com/services/510ks/), [a De Novo](https://innolitics.com/services/regulatory/), [a SaMD](https://innolitics.com/services/end-to-end-samd/), [an AI/ML medical device](https://innolitics.com/services/medical-imaging-ai-development/), or [an FDA regulatory strategy](https://innolitics.com/services/regulatory/).

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

A. 510(k) Number:

k112172

B. Purpose for Submission:

This is a new 510(k) application for a qualitative Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) assay used with the Applied Biosystems 7500 Fast Dx Real-Time PCR System for the *in vitro* qualitative detection and differentiation of influenza A and influenza B viral RNA in nasal swabs (NS) and nasopharyngeal swabs (NPS) from symptomatic human patients.

C. Measurand:

Target RNA sequences for the highly conserved regions of the matrix protein gene of influenza A virus and the neuraminidase gene of influenza B virus.

D. Type of Test:

Multiplex Real-Time RT-PCR assay for the qualitative detection and differentiation of influenza A and influenza B viral RNA from nasal and nasopharyngeal swab specimens using nucleic acid isolation and amplification. The isolation and purification of the viral RNA is performed using the NucliSENS® easyMAG™ System (bioMérieux) and the Automated Magnetic Extraction Reagents (bioMérieux). The amplification and detection is performed on the Applied Biosystems 7500 Fast Dx Real-Time PCR Instrument with the SDS Software version 1.4.

E. Applicant:

Quidel® Corporation

F. Proprietary and Established Names:

Quidel® Molecular Influenza A+B Assay

G. Regulatory Information:

1. Regulation section:

21 CFR 866.3980, Respiratory viral panel multiplex nucleic acid assay

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2. Classification:
Class II

3. Product codes:
OCC, OOI

4. Panel:
Microbiology (83)

H. Intended Use:

1. Intended use(s):

The Quidel® Molecular Influenza A + B Assay is a multiplex Real Time RT-PCR assay for the in vitro qualitative detection and differentiation of influenza A and influenza B viral RNA in nasal and nasopharyngeal swabs from patients with signs and symptoms of respiratory infection. The test is intended for use as an aid in the differential diagnosis of influenza A and influenza B viral infections in humans in conjunction with clinical and epidemiological risk factors. The assay does not detect the presence of influenza C virus.

Negative results do not preclude influenza virus infection and should not be used as the sole basis for diagnosis, treatment or other patient management decisions.

Performance characteristics for influenza A were established during the 2010-2011 influenza season when influenza A/H3 and 2009 H1N1 influenza were the predominant influenza A viruses in circulation. When other influenza A viruses are emerging, performance characteristics may vary.

If infection with a novel influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent Influenza viruses and sent to state or local health department for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.

2. Indication(s) for use:
Same as Intended Use

3. Special conditions for use statement(s):
For prescription use only

2

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4. Special instrument requirements:

bioMérieux NucliSENS easyMAG System (software version 2.0)

Applied Biosystems (ABI) 7500 FAST Dx (software version 1.4)

# I. Device Description:

The assay detects influenza A and influenza B viral RNA that has been extracted from a patient sample using the NucliSENS® easyMAG® automated extraction platform. A multiplex RT-PCR reaction is carried out under optimized conditions in a single tube generating amplicons for each of the target viruses present in the sample. This reaction is performed utilizing the Applied Biosystems® 7500 Fast Dx platform. Identification of influenza A occurs by the use of target specific primers and a fluorescent- labeled probe that hybridizes to a conserved region within the matrix protein gene. Identification of influenza B occurs by the use of target specific primers and a fluorescent-labeled probe that hybridizes to a conserved influenza B sequence within the neuraminidase gene.

|  Quidel Molecular Probe Labels  |   |
| --- | --- |
|  Target | Dye  |
|  Influenza A | FAM  |
|  Influenza B | CAL Fluor Orange 560  |
|  Process Control | Quasar 670  |

The following is a summary of the procedure:

1. Sample Collection: Obtain nasal swabs or nasopharyngeal swabs using standard techniques from symptomatic patients. These specimens are transported, stored, and processed according to established laboratory procedures.
2. Nucleic Acid Extraction: Extract viral RNA from the specimens with the NucliSENS easyMAG System following the manufacturer's instructions and using the appropriate reagents. Prior to the extraction procedure add  $20~\mu \mathrm{L}$  of the Process Control (PRC) to each  $180~\mu \mathrm{L}$  aliquot of specimen. The PRC serves to monitor inhibitors in the extracted specimen, assures that adequate amplification has taken place and confirms that the nucleic acid extraction was sufficient.
3. Rehydration of Master Mix: Rehydrate the lyophilized Master Mix using the Rehydration Solution. The Master Mix contains oligonucleotide primers, fluorophore and quencher-labeled probes targeting highly conserved regions of the influenza A and influenza B viruses as well as the PRC sequence. The primers are complementary to highly specific and conserved regions in the genome of these viruses. The probes are dual labeled with a reporter dye attached to the  $5^{\prime}$  end and a quencher attached to the  $3^{\prime}$  end.

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4. Nucleic Acid Amplification and Detection: Add  $15~\mu \mathrm{L}$  of the rehydrated Master Mix to each plate well.  $5\mu \mathrm{L}$  of extracted nucleic acids (specimen with PRC) is then added to the plate well. Place the plate into the ABI 7500 Fast Dx instrument. Once the plate is added to the instrument, the assay protocol is initiated. This protocol initiates reverse transcription of the RNA targets generating complementary DNA, and the subsequent amplification of the target sequences occurs. The Quidel Molecular Influenza A+B assay is based on TaqMan chemistry, and uses an enzyme with reverse transcriptase, DNA polymerase, and  $5^{\prime} - 3^{\prime}$  exonuclease activities. During DNA amplification, this enzyme cleaves the probe bound to the complementary DNA sequence, separating the quencher dye from the reporter dye. This step generates an increase in fluorescent signal upon excitation by a light source of the appropriate wavelength. With each cycle, additional dye molecules are separated from their quenchers resulting in additional signal. If sufficient fluorescence is achieved by 35 cycles during the data collection stage of amplification, the sample is reported as positive for the detected target sequence.

# Materials Provided

SKU # M100

Detection Kit (96 Reactions) - Store at  $2^{\circ}$  to  $8^{\circ}\mathrm{C}$

|  # | Component | Quantity  |
| --- | --- | --- |
|  1 | Rehydration Solution Part M5003 | 1 vial/kit 1.9 mL  |
|  2 | Quidel Molecular Influenza A+B Master Mix Part M5004Lyophilized Contents:DNA polymerase enzyme with reverse transcriptase activityOligonucleotide primer pairs; Oligonucleotide probes dNTPs (dATP, dCTP, dGTP, dUTP, dTTP)Stabilizers | 12 vials/kit,8 reactions/vial  |
|  CONTROL | Process Control Part M5005 | 1 vial/kit 2.0 mL  |
|   | Instructions for Use (English) M0002 | 1/kit  |

# Optional Materials

Positive controls for influenza A and influenza B (Quidel Molecular Influenza A/B Control Set #M106 which serves as an external processing and extraction control

# Materials Required But Not Provided

- Micropipettors (range between 1 to  $10~\mu \mathrm{L}$  and 100 to  $1000~\mu \mathrm{L}$ )
Non-aerosol pipette tips

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- Applied Biosystems 7500Fast Dx software version 1.4
- Applied Biosystems 7500Fast Dx 96 well PCR plate
- Applied Biosystems optical plate films
- Plate centrifuge for ABI 96 well plate
- bioMerieux NucliSENS easyMAG software version 2.0
- bioMerieux NucliSENS easyMAG Buffers 1, 2, 3
- bioMerieux NucliSENS easyMAG Lysis Buffer
- bioMerieux NucliSENS easyMAG Silica Magnetic Beads

## Interpretation of Results using the ABI 7500 Fast Dx Thermocycler

|  Interpretation of the Quidel Molecular Influenza A+B Assay Results on the ABI 7500 Fast Dx Thermocycler  |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- |
|  Assay Result | Detector: Influenza A | Detector: Influenza B | Detector: Process Control | Warning / Error Code | Interpretation of Results  |
|  Negative | Ct<5.0 or Ct>35.0 | Ct<5.0 or Ct>35.0 | 5.0≤ Ct ≤35.0 | None | No influenza A or influenza B viral RNA detected; PRC Detected  |
|  Influenza A Positive | 5.0≤ Ct ≤35.0 | Ct<5.0 or Ct>35.0 | NA* | None | Influenza A viral RNA detected  |
|  Influenza B Positive | Ct<5.0 or Ct>35.0 | 5.0≤ Ct ≤35.0 | NA* | None | Influenza B viral RNA detected  |
|  Influenza A and B Positive | 5.0≤ Ct ≤35.0 | 5.0≤ Ct ≤35.0 | NA* | None | Influenza A and Influenza B viral RNA detected**  |
|  Invalid | Ct<5.0 or Ct>35.0 | Ct<5.0 or Ct>35.0 | Ct<5.0 or Ct>35.0 | None | No Influenza A or Influenza B and no PRC viral RNA detected; invalid test. Retest the same purified sample. If the test is also invalid, re-extract and re-test another aliquot of the same sample or obtain a new sample and retest.  |
|  Invalid | Undetermined | Undetermined | Undetermined | 4598 | Not Determined. Retest the same purified sample. If the test is also invalid, re-extract and retest another aliquot of the same sample or obtain a new sample and retest.  |

*No Ct value is required for the Process Control to make a positive call.
** Dual infections are rare. Repeat testing using the purified sample. If the retest confirms this result, collect and test a new specimen. Contact Quidel if multiple samples provide this result

## J. Substantial Equivalence Information:

1. Predicate device name(s):

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Prodesse ProFlu+

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

k073029, k092500 and k081030

3. Comparison with predicate:

|  Item | Subject Device: Quidel Molecular Influenza A+B Assay | Predicate Device: Prodesse ProFlu+  |
| --- | --- | --- |
|  Intended Use | The Quidel Molecular Influenza A+B assay is a multiplex Real Time RT-PCR assay for the in vitro qualitative detection and differentiation of influenza A and influenza B viral RNA in nasal and nasopharyngeal swabs from patients with signs and symptoms of respiratory infection. This test is intended for use as an aid in the differential diagnosis of influenza A and influenza B viral infections in humans in conjunction with clinical and epidemiological risk factors. The assay does not detect the presence of influenza C virus. Negative results do not preclude Influenza virus infection and should not be used as the sole basis for diagnosis, treatment or other patient management decisions. Performance characteristics for influenza A were established during the 2010-2011 influenza season when influenza A/H3 and 2009 H1N1 influenza were the predominant influenza A viruses in circulation. When other influenza A viruses are emerging, performance characteristics may vary. If infection with a novel influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent Influenza viruses and sent to state or local health department for testing. Viral culture should not be | The ProFluTM+ Assay is a multiplex Real-Time PCR (RT-PCR) in vitro diagnostic test for the rapid and qualitative detection and discrimination of Influenza A Virus, Influenza B Virus, and Respiratory Syncytial Virus (RSV) nucleic acids isolated and purified from nasopharyngeal (NP) swab specimens obtained from symptomatic patients. This test is intended for use to aid in the differential diagnosis of Influenza A, Influenza B and RSV viral infections in humans and is not intended to detect Influenza C. Negative results do not preclude influenza or RSV virus infection and should not be used as the sole basis for treatment or other management decisions. It is recommended that negative RSV results be confirmed by culture. Performance characteristics for Influenza A Virus were established when Influenza A/H3 and A/H1 were the predominant Influenza A viruses in circulation. When other Influenza A viruses are emerging, performance characteristics may vary. If infection with a novel Influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent Influenza viruses and sent to state or local health department for testing. Viral culture should not be  |

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|  Item | Subject Device: Quidel Molecular Influenza A+B Assay | Predicate Device: Prodesse ProFlu+  |
| --- | --- | --- |
|   | attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens. | sent to state or local health department for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.  |
|  Assay Target | Influenza A virus, influenza B virus | Influenza A virus, influenza B virus, respiratory syncytial virus  |
|  Sample Types | nasal swab and nasopharyngeal swab | nasopharyngeal swab  |
|  Extraction Methods | bioMérieux easyMAG Automated Magnetic Extraction Reagents | Roche MagNA Pure LC Total Nucleic Acid Isolation Kit or the bioMérieux easyMAG Automated Magnetic Extraction Reagents  |
|  Assay Methodology | PCR-based system for detecting the presence or absence of viral RNA in clinical specimens | PCR-based system for detecting the presence or absence of viral RNA in clinical specimens  |
|  Detection Techniques | Multiplex assay using different reporter dyes for each target | Multiplex assay using different reporter dyes for each target  |
|  Viral Targets | Influenza A: Matrix Gene; Influenza B: conserved influenza B sequence within the neuraminidase gene | Influenza A: Matrix Gene; Influenza B: Non-structural NS1 and NS2  |

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

Class II Special Controls Guidance Document: Reagents for Detection of Specific Novel Influenza A Viruses (March 2006) http://www.fda.gov/cdrh/oivd/guidance/1596.pdf.

Guidance on In Vitro Diagnostic Devices to Detect Influenza A Viruses: Labeling and Regulatory Path (April 2006) http://www.fda.gov/cdrh/oivd/guidance/1594.pdf.

Guidance on Informed Consent for In Vitro Diagnostic Device Studies Leftover Human Specimens that are Not Individually Identifiable (April 2006) http://www.fda.gov/cdrh/oivd/guidance/1588.pdf.

Draft Guidance on Nucleic Acid Based In Vitro Diagnostic Devices for Detection of Microbial Pathogens (Dec 2005) http://www.fda.gov/cdrh/oivd/guidance/1560..

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CLSI EP17-A: Guidance for Protocols for Determination of Limits of Detection and Limits of Quantitations (Vol. 2, No. 34) (Oct 2004).

CLSI MM13-A: Guidance for the Collection, Transport, Preparation and Storage of Specimens for Molecular Methods (Vol. 25, No. 31) (Dec 2005).

CLSI EP7-A2: Guidance for Interference Testing in Clinical Chemistry (Vol. 25, No.27 Second Ed) (Nov 2005).

CLSI EP12-A: Guidance for User Protocol for Evaluation of Qualitative Test Performance (Vol. 22, No. 14) (Sept 2002).

CLSI MM6-A: Guidance for the Quantitative Molecular Methods for Infectious Diseases (Vol. 23, No.28) (Oct 2003).

CLSI EP5-A2: Guidance for Evaluation of Precision Performance of Quantitative Measurement Methods (Vol. 24, No. 25 Second Ed.) (Aug 2004).

Establishing Performance Characteristics of In Vitro Diagnostic Devices for Detection or Detection and Differentiation of Influenza Viruses. Document issued on February 15, 2008. Docket number FDA-2008-D-0095.

Guidance for Industry, FDA Reviewers and Compliance on Off-The-Shelf Software Use in Medical Devices. Document issued on September 9, 1999. Docket number FDA-1999-D-585

L. Test Principle:

The real-time RT-PCR process simultaneously amplifies and detects nucleic acid targets in a single closed-tube reaction. Detection of influenza A, B and the Process Control (PRC) is based on three processes: nucleic acid isolation, reverse transcription and real time PCR amplification/detection. Human respiratory specimens (nasal swabs and nasopharyngeal swabs) from symptomatic patients are processed initially to isolate and purify viral nucleic acid from the cellular specimen matrix. After initial reverse transcription of RNA into complementary DNA (cDNA), amplification proceeds during which the probe anneals specifically to a region of the template between the forward and reverse primers. As primer extension and amplification occurs, the exonuclease activity of the Taq polymerase cleaves the probe separating the reporter dye away from the quencher. This generates an increase in fluorescent signal upon excitation from a light source of appropriate wavelength. With each cycle, additional reporter dye molecules are cleaved from their respective probes, yielding increased fluorescence signal. The amount of fluorescence at any given cycle is dependent on the amount of PCR product (amplicons) present at that time. Fluorescent intensity is monitored at each PCR cycle by fluorescent detection modules within the real-time instrument.

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M. Performance Characteristics (if/when applicable):

1. Analytical performance:

a. Precision/Reproducibility:

The reproducibility of the Quidel Molecular Influenza A and B assay was evaluated at three laboratory sites. The reproducibility panel and controls were tested at each site by two operators for five days in triplicate (2 operators x 5 days x triplicate testing x 3 sites = 90 results per sample). The panels and controls were extracted using the bioMérieux easyMAG system and tested on the ABI 7500 Fast Dx.

The reproducibility panel was composed of four simulated samples each for influenza A and influenza B, made by diluting Influenza A H1N1 A/Mexico/4108/2009 or Influenza B Florida into negative nasal matrix. The panel included a medium positive (5x LoD) influenza A sample, a low positive (2x LoD) influenza A sample, a high negative (0.3x LoD) influenza A sample, and an influenza A negative sample. The panel also included a medium positive (5x LoD) influenza B sample, a low positive (2x LoD) influenza B sample, a high negative (0.3x LoD) influenza B sample, and an influenza B negative sample.

|  Reproducibility Results  |   |   |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  Panel Member ID | Site 1 |   |   | Site 2 |   |   | Site 3 |   |   | Total Results  |
|   |  Results | AVE Ct | %CV | Results | AVE Ct | %CV | Results | AVE Ct | %CV  |   |
|  Influenza A High Negative (1.44E+01 TCID_{50}/mL) | 4/30 | 34.03* | 2.0 | 8/30 | 34.07 | 1.9 | 0/30 | N/A | N/A | 12/90  |
|  Influenza A Low Positive (9.6E+01 TCID_{50}/mL) | 30/30 | 27.3 | 3.5 | 30/30 | 27.3 | 6.3 | 30/30 | 29.2 | 7.0 | 90/90  |
|  Influenza A Med Positive (2.4E+02 TCID_{50}/mL) | 30/30 | 25.3 | 2.9 | 30/30 | 25.2 | 5.1 | 30/30 | 26.8 | 5.5 | 90/90  |
|  Influenza A Negative | 0/30 | N/A | N/A | 0/30 | N/A | N/A | 0/30 | N/A | N/A | 0/90  |
|  Influenza B High Negative (1.3E+01 TCID_{50}/mL) | 0/30 | N/A | N/A | 3/30 | 34.2 | 1.2 | 0/30 | N/A | N/A | 3/90  |
|  Influenza B |  |  |  |  |  |  |  |  |  |   |

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|  Reproducibility Results  |   |   |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  Panel Member ID | Site 1 |   |   | Site 2 |   |   | Site 3 |   |   | Total Results  |
|   |  Results | AVE Ct | %CV | Results | AVE Ct | %CV | Results | AVE Ct | %CV  |   |
|  Low Positive (8.6E+01 TCID_{50}/mL) | 30/30 | 24.7 | 2.6 | 30/30 | 24.6 | 4.8 | 30/30 | 25.7 | 5.1 | 90/90  |
|  Influenza B Med Positive (2.2E+02 TCID_{50}/mL) | 30/30 | 22.9 | 2.0 | 30/30 | 22.7 | 2.6 | 30/30 | 23.5 | 2.9 | 90/90  |
|  Influenza B Negative | 0/30 | N/A | N/A | 0/30 | N/A | N/A | 0/30 | N/A | N/A | 0/90  |
|  Influenza A Positive Control | 30/30 | 12.4 | 1.6 | 30/30 | 11.8 | 2.2 | 30/30 | 12.1 | 1.1 | 90/90  |
|  Influenza B Positive Control | 30/30 | 15.2 | 2.6 | 30/30 | 14.9 | 3.1 | 30/30 | 15.1 | 1.4 | 90/90  |
|  Negative Control | 0/30 | N/A | N/A | 0/30 | N/A | N/A | 0/30 | N/A | N/A | 0/90  |

* CV of positive results

The data from all the sites indicates that the Quidel Molecular assay generates reproducible results for both influenza A and influenza B viruses when tested with the ABI 7500 Fast Dx.

b. Linearity/assay reportable range:

Not applicable

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

Freeze-Thaw Equivalency

The impact of using fresh clinical samples versus frozen clinical specimens on the Quidel Molecular Influenza A+B Assay’s ability to detect influenza A and influenza B was evaluated. Briefly, 220 clinical specimens were aliquoted and processed fresh (&lt; 72-hours post collection) and frozen (a minimum of 7-days storage at -70°C). Six (6) samples were discarded from analysis due to invalid results. The invalid rate of the fresh samples was 4/220 (1.8%) and the invalid rate of the frozen samples was 6/220 (2.7%). Four of the invalids overlapped between fresh and frozen. There were two influenza A and influenza B positive samples that did not overlap between fresh and frozen in the comparison study. The positive samples evaluated in the study spanned virus concentrations detected from 6 Ct to 35 Ct for influenza A and 8 Ct to 33 Ct for influenza B. The results are summarized in the 2x2 table below and indicate that the use of fresh specimens and frozen specimens, as defined above, are equivalent.

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|  Influenza A  |   |   |   |
| --- | --- | --- | --- |
|  Nasopharyngeal Swab | Fresh Specimen  |   |   |
|  Frozen Specimen | Positive | Negative | Total  |
|  Positive | 41 | 1 | 42  |
|  Negative | 1 | 171 | 172  |
|  Total | 42 | 172 | 214  |
|  |   |   |   |
|  PPA | 97.6% |  |   |
|  NPA | 99.4% |  |   |
|  Influenza B  |   |   |   |
| --- | --- | --- | --- |
|  Nasopharyngeal Swab | Fresh Specimen  |   |   |
|  Frozen Specimen | Positive | Negative | Total  |
|  Positive | 35 | 1 | 27  |
|  Negative | 2 | 176 | 181  |
|  Total | 37 | 177 | 214  |
|  |   |   |   |
|  PPA | 94.6% |  |   |
|  NPA | 99.4% |  |   |

## Kit Stability

An evaluation of real-time stability using 3 lots of the Quidel Molecular Influenza A+B Assay kits at 2°C - 8°C and room temperature was done over the course of 61 days, with additional real time results to be collected for 460 days (15 months). The following information summarizes the acceptance criteria; furthermore these criteria will also be used throughout the stability study:

1. Quidel Molecular Influenza A+B Assay kit stability demonstrates 20 months real time stability at 2°C to 8°C.
2. Quidel Molecular Influenza A+B Assay kit stability demonstrates 15 months equivalent accelerated stability at 2°C to 8°C.
3. Quidel Molecular Influenza A+B Assay kit stability demonstrates 15 months real time stability at room temperature. (RT)
4. Influenza A and influenza B test positive at each time point tested, 3x, 100x and 1000x LoD (≥ 5 cycles and ≤35 cycles for 7500 Dx).
5. The NTCs test negative at each time point tested (undetermined).

No significant failure of the assay was detected at any of the experimental conditions to date (real time stability data collected to day 61) when compared

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to the  $t = 0$  time point. Also, all NTCs tested negative at each time point tested.

# Master Mix Stability after Rehydration

An evaluation of master mix stability after rehydration was carried out over a 5 day period using various storage conditions, including  $-20^{\circ}\mathrm{C}$ ,  $4^{\circ}\mathrm{C}$ , and  $25^{\circ}\mathrm{C}$  (room temperature). Mock specimens at  $100\mathrm{X}$ ,  $10\mathrm{X}$ , and  $3\mathrm{X}$  LoD were used in the analysis. Results show that the rehydrated master mix can be stored for up to 24-hours at room temperature without any significant affect on the Ct values for influenza A, influenza B or the PRC. The data demonstrates that for times longer than 24-hours the rehydrated master mix can be stored at  $-20^{\circ}\mathrm{C}$  for up to two weeks. Storage of the rehydrated master mix at  $-4^{\circ}\mathrm{C}$  is not recommended.

# Extracted Specimen Stability

An evaluation of the stability of extracted specimens was carried out using the easyMAG extraction system followed by real-time RT-PCR analysis. Parallel aliquots of samples that were influenza A (3X LoD) and influenza B (3X LoD) positive were used for this study. Samples were analyzed at time 0 (immediately after extraction), after 2h at room temperature, after 8h at  $2 - 8^{\circ}\mathrm{C}$ , after 96 hours with up to three freeze thaw cycles, and after 1 month at  $-20^{\circ}\mathrm{C}$ . There was no indication of degradation of the extracted specimen stored at the conditions tested.

# d. Detection limit:

The analytical sensitivity (limit of detection or LoD) of the Quidel Molecular Influenza A+B assay was determined using quantified  $(\mathrm{TCID}_{50} / \mathrm{mL})$  cultures of three influenza A strains (one H1N1, one 2009H1N1 and one H3N2) and three influenza B strains, serially diluted in negative nasopharyngeal matrix. Each dilution was extracted using the NucliSENS easyMAG System in replicates of 21 per concentration of virus and tested on the Applied Biosystems® 7500 Fast Dx platform. Analytical sensitivity (LoD) is defined as the lowest concentration at which  $95\%$  of all replicates tested positive.

LoD Determination

|  Strain | Stock Virus Concentration (TCID50/ml) | Test Concentration (TCID50/ml) | Call Rate | Ct Avg | STDEV  |
| --- | --- | --- | --- | --- | --- |
|  A/Mexico/4108/2009 | 2.89E+08 | 4.80E+01 | 21/21 | 30.76 | 1.00  |
|  A1/Mal/302/54 | 4.19E+08 | 1.60E+01 | 21/21 | 30.91 | 0.93  |
|  A/Victoria/3/75 | 1.10E+08 | 9.20E+01 | 21/21 | 27.12 | 0.76  |
|  B/RCHIN 8/05 | 3.20E+06 | 1.20E+01 | 21/21 | 29.99 | 0.93  |
|  B/Florida/04/2006 | 2.56E+06 | 4.30E+01 | 21/21 | 28.99 | 0.84  |
|  B/Malaysia/25/06/04 | 3.41E+06 | 5.70E+00 | 21/21 | 29.20 | 1.11  |

{12}

# e. Analytical specificity (inclusivity):

The inclusivity of the Quidel Molecular Influenza A+B assay was evaluated against multiple strains of influenza A and influenza B viruses. The clinical panel consisted of 10 influenza A subtype H1N1, two influenza A subtype 2009H1N1, eight influenza A subtype H3N2, two influenza A subtype H5N1, 13 Influenza B strains. An additional panel of non-clinical influenza isolates was also tested. Each panel member was extracted using the NucliSENS easyMAG instrument and tested in triplicate.

The Quidel Molecular Influenza A+B assay detected  $100\%$  of the influenza A (38/38) and influenza B strains (15/15) at 2 to 3x LoD levels including pandemic and avian influenza A strains, and recent circulating influenza B strains.

|  Clinical Panel Influenza A viruses  |   |   |   |   |
| --- | --- | --- | --- | --- |
|  Subtype | Strain | TCID50/mL | (7500 Dx)  |   |
|   |   |   |  A | B  |
|  2009 H1N1 | H1N1 A/California/07/2009 | 1.45E+02 | Positive | Negative  |
|  H1N1 | A/New Caledonia/20/1999 | 1.12E+02 | Positive | Negative  |
|  H1N1 | A/New Jersey/8/76 | 3.80E+02 | Positive | Negative  |
|  H1N1 | A/PR/8/34 | 5.89E+02 | Positive | Negative  |
|  H1N1 | A/NWS/33 | NA | Positive | Negative  |
|  H1N1 | A/Denver/1/57 | 1.26E+02 | Positive | Negative  |
|  H1N1 | A/FM/1/47 | 3.80E+02 | Positive | Negative  |
|  2009 H1N1 | A/Mexico/4108/2009 | 1.40E+02 | Positive | Negative  |
|  H1N1 | A1/Mal/302/54 | 4.19E+02 | Positive | Negative  |
|  H1N1 | A/Taiwan/42/06 | 3.39E+02 | Positive | Negative  |
|  H1N1 | A/Brisbane/59/07 | 7.24E+01 | Positive | Negative  |
|  H1N1 | A/Solomon Islands/3/06 | 1.41E+01 | Positive | Negative  |
|  H3N2 | A/Hong Kong/8/68 | 1.15E+02 | Positive | Negative  |
|  H3N2 | A/Wisconsin/67/2005 | 7.24E+02 | Positive | Negative  |
|  H3N2 | A/Aichi/2/68 | 4.17E+02 | Positive | Negative  |
|  H3N2 | A/Port Chalmers/1/73 | 4.57E+02 | Positive | Negative  |
|  H3N2 | A/Perth/16/2009 | 9.83E+02 | Positive | Negative  |
|  H3N2 | A/Uruguay/7/16/2007 | 1.03E+02 | Positive | Negative  |
|  H3N2 | A/Victoria/3/75 | 2.19E+02 | Positive | Negative  |
|  H3N2 | A/Brisbane/10/07 | 4.17E+02 | Positive | Negative  |
|  Clinical Panel Influenza B viruses  |   |   |   |
| --- | --- | --- | --- |
|  Strain | TCID50/mL | (7500 Dx)  |   |
|   |   |  A | B  |
|  B/HongKong/5/72 | 6.67E+02 | Negative | Positive  |
|  B/Panama/45/90 | 1.02E+02 | Negative | Positive  |
|  B/Florida/02/2006 | 3.16E+02 | Negative | Positive  |
|  B/Florida/04/2006 | 3.80E+02 | Negative | Positive  |
|  B/Florida/07/2004 | 1.26E+02 | Negative | Positive  |
|  B/Malaysia/25/06/04 | 3.41E+02 | Negative | Positive  |

{13}

|  Clinical Panel Influenza B viruses  |   |   |   |
| --- | --- | --- | --- |
|  Strain | TCID50/mL | (7500 Dx)  |   |
|   |   |  A | B  |
|  B/Maryland/1/59 | 1.15E+02 | Negative | Positive  |
|  B/Allen/45 | 4.17E+02 | Negative | Positive  |
|  B/Taiwan/2/62 | 1.51E+02 | Negative | Positive  |
|  B/Russia/69 | 2.19E+02 | Negative | Positive  |
|  B/Mass/3/66 | 1.38E+02 | Negative | Positive  |
|  B/Lee/40 | 1.95E+02 | Negative | Positive  |
|  B/GL/1739/54 | 6.30E+02 | Negative | Positive  |
|  Non-clinical Influenza Viruses  |   |   |   |   |
| --- | --- | --- | --- | --- |
|  Subtype | Strain | TCID50/mL | (7500 Dx)  |   |
|   |   |   |  A | B  |
|  H3N2 | A/WI/629-2/2008 (H3N2) | 2.00E+02 | Positive | Negative  |
|  H1N1 | A/WI/629-S7(D02473)/2009 (H1N1pdm) | 2.00E+02 | Positive | Negative  |
|  H1N1 | A/WI/629-S5 (D02312)/2009 (H1N1pdm) | 2.00E+02 | Positive | Negative  |
|  H2N2 | A/Mallard/NY/6750/78 (H2N2) | 2.00E+02 | Positive | Negative  |
|  H7N3 | A/Chicken/NJ/15086-3/94 (H7N3) | 2.00E+02 | Positive | Negative  |
|  H9N2 | A/Chicken/NJ/12220/97 (H9N2) | 2.00E+02 | Positive | Negative  |
|  H4N8 | A/Mallard/OH/338/86 (H4N8) | 2.00E+02 | Positive | Negative  |
|  H6N2 | A/Chicken/CA/431/00 (H6N2) | 2.00E+02 | Positive | Negative  |
|  H8N4 | A/Blue Winged Teal/LA/B174/86 (H8N4) | 2.00E+02 | Positive | Negative  |
|  H5N1 | A/Anhui/01/2005(H5N1)-PR8-IBCDC-RG5 | 2.00E+02 | Positive | Negative  |
|  H10N7 | A/GWT/LA/169GW/88 (H10N7) | 2.00E+02 | Positive | Negative  |
|  H11N9 | A/Chicken/NJ/15906-9/96 (H11N9) | 2.00E+02 | Positive | Negative  |
|  H12N5 | A/Duck/LA/188D/87 (H12N5) | 2.00E+02 | Positive | Negative  |
|  H13N6 | A/Gull/MD/704/77 (H13N6) | 2.00E+02 | Positive | Negative  |
|  H14N5 | A/Mallard/GurjevRussia/262/82 (H14N5) | 2.00E+02 | Positive | Negative  |
|  H15N9 | A/Shearwater/Australia/2576/79 (H15N9) | 2.00E+02 | Positive | Negative  |
|  H16N3 | A/Shorebird/DE/172/2006(H16N3) | 2.00E+02 | Positive | Negative  |

# f. Analytical specificity (cross-reactivity)

The cross-reactivity of the Quidel Molecular Influenza  $\mathrm{A + B}$  assay was evaluated by testing a panel consisting of 26 viral, 24 bacterial, and one yeast strain representing common respiratory pathogens or flora commonly present in the nasopharynx. Bacteria and yeast were tested at concentrations of  $10^{5}$  to  $10^{10}$  CFU/mL. Viruses were tested at concentrations of  $10^{3}$  to  $10^{6}$  TCID $_{50}$ /mL. Samples were extracted using the NucliSENS easyMAG instrument and tested in triplicate. Analytical specificity of the Quidel Molecular influenza  $\mathrm{A + B}$  assay was  $100\%$ .

|  Cross Reactivity  |   |   |   |
| --- | --- | --- | --- |
|  Organism ID | CFU/mL or TCID50/mL | Influenza A Result | Influenza B Result  |
|  hMPV A1 | 3.70E+04 | Negative | Negative  |
|  hMPV B1 | 2.37E+04 | Negative | Negative  |
|  RSV Long | 4.40E+04 | Negative | Negative  |
|  RSV Long 1 | 2.37E+04 | Negative | Negative  |
|  RSV Long 2 | 2.37E+04 | Negative | Negative  |
|  RSV Long 3 | 2.37E+04 | Negative | Negative  |

{14}

|  Cross Reactivity  |   |   |   |
| --- | --- | --- | --- |
|  Organism ID | CFU/mL or TCID_{50}/mL | Influenza A Result | Influenza B Result  |
|  RSV Washington | 1.75E+03 | Negative | Negative  |
|  Adenovirus 1/Adenoid 71 | 5.67E+04 | Negative | Negative  |
|  Coronavirus 229E | 1.70E+06 | Negative | Negative  |
|  Coronavirus OC43 | 1.67E+06 | Negative | Negative  |
|  Coxsackievirus B4 | 2.43E+06 | Negative | Negative  |
|  Coxsackievirus B5/10/2006 | 2.28E+06 | Negative | Negative  |
|  Cytomegalovirus | 8.76E+05 | Negative | Negative  |
|  Echovirus 7 | 5.38E+08 | Negative | Negative  |
|  Echovirus 9 | 1.50E+06 | Negative | Negative  |
|  Echovirus 6 | 1.05E+08 | Negative | Negative  |
|  Echovirus 11 | 1.50E+05 | Negative | Negative  |
|  Enterovirus 71 | 2.68E+03 | Negative | Negative  |
|  Enterovirus 70 | 1.66E+05 | Negative | Negative  |
|  Epstein Barr Virus | 5,000cp/mL | Negative | Negative  |
|  HSV Type 1 MacIntyre strain | 1.95E+06 | Negative | Negative  |
|  HSV Type 2 G strain | 3.67E+06 | Negative | Negative  |
|  Rubeola virus | 3.78E+05 | Negative | Negative  |
|  Mumps virus | 8.43E+04 | Negative | Negative  |
|  Parainfluenza Type 1 | 2.50E+05 | Negative | Negative  |
|  Parainfluenza Type 2 | 2.20E+04 | Negative | Negative  |
|  Parainfluenza Type 3 | 9.10E+05 | Negative | Negative  |
|  Parainfluenza Type 4 | 9.57E+06 | Negative | Negative  |
|  Varicella Zoster Virus | 7.50E+02 | Negative | Negative  |
|  Bordetella pertussis | 1.04E+07 | Negative | Negative  |
|  Bordetella bronchiseptica | 2.55E+07 | Negative | Negative  |
|  Chlamydia trachomatis | 2.10E+05 | Negative | Negative  |
|  Legionella pneumophila | 2.05E+08 | Negative | Negative  |
|  Mycobacterium intracellulare | 6.90E+08 | Negative | Negative  |
|  Mycobacterium tuberculosis | 6.60E+07 | Negative | Negative  |
|  Mycobacterium avium | 1.36E+10 | Negative | Negative  |
|  Haemophilus influenzae | 5.90E+07 | Negative | Negative  |
|  Pseudomonas aeruginosa | 5.15E+07 | Negative | Negative  |
|  Proteus vulgaris | 2.65E+08 | Negative | Negative  |
|  Proteus mirabilis | 2.75E+07 | Negative | Negative  |
|  Neisseria gonorrhoeae | 2.15E+07 | Negative | Negative  |
|  Neisseria meningitidis | 1.85E+08 | Negative | Negative  |
|  Neisseria mucosa | 1.85E+08 | Negative | Negative  |
|  Klebsiella pneumoniae | 3.30E+07 | Negative | Negative  |
|  Escherichia coli | 6.80E+07 | Negative | Negative  |
|  Moraxella catarrhalis | 5.85E+07 | Negative | Negative  |
|  Corynebacterium diphtheriae | 6.0E+05 | Negative | Negative  |
|  Lactobacillus plantarum | 1.03E+08 | Negative | Negative  |
|  Streptococcus pneumoniae | 4.5E+07 | Negative | Negative  |
|  Streptococcus pyogenes | 2.05E+08 | Negative | Negative  |
|  Streptococcus salivarius | 2.50E+06 | Negative | Negative  |
|  Staphylococcus epidermidis | 2.6E+07 | Negative | Negative  |
|  Staphylococcus aureus | 5.15E+08 | Negative | Negative  |
|  Candida albicans | 1.07E+06 | Negative | Negative  |

15

{15}

# g. Interfering Microorganisms

The role of potentially interfering organisms in the ability of the Quidel Molecular Influenza A+B assay to detect and differentiate influenza A and influenza B viral RNA was evaluated by testing a panel consisting of 26 viral, 24 bacterial, and one yeast strain representing common respiratory pathogens or flora commonly present in the nasopharynx (see Cross-Reactivity table for complete list of organisms). Each organism was evaluated individually in a specimen containing influenza A  $(10^{6}\mathrm{TCID}_{50} / \mathrm{mL})$  and influenza B  $(10^{7}\mathrm{TCID}_{50} / \mathrm{mL})$ . Bacteria and yeast were tested at concentrations of  $10^{5}$  to  $10^{8}$  CFU/mL. Viruses were tested at concentrations of  $10^{2}$  to  $10^{8}$  TCID $_{50} / \mathrm{mL}$ . Samples were extracted using the NucliSENS easyMAG instrument and tested in triplicate. No interference was observed during this study.

# h. Interfering Substances

The performance of Quidel Molecular Influenza A+B assay was evaluated with potentially interfering substances that may be present in nasopharyngeal specimens. The potentially interfering substances were evaluated in influenza A (A/Mexico/4108/2009), and influenza B (B/Florida/04/2006) at concentrations of 3x and 10x LoD. There was no evidence of interference caused by the substances tested below with 3x or 10x LoD viruses.

|  Substance Name | Concentration Tested | Influenza A Result (3x LoD) | Influenza B Result (3x LoD)  |
| --- | --- | --- | --- |
|  Mucin (Bovine Submaxillary Gland, type I-S) | 60 μg/mL | Positive | Positive  |
|  Blood (human), EDTA anticoagulated | 2% (vol/vol) | Positive | Positive  |
|  Neo-Synephrine | 15% (vol/vol) | Positive | Positive  |
|  Afrin Nasal Spray | 15% (vol/vol) | Positive | Positive  |
|  Zicam Homeopathic Non-Drowsy Allergy Relief No Drip Liquid Nasal Gel | 5% (vol/vol) | Positive | Positive  |
|  Saline Nasal Spray | 15% (vol/vol) of dose | Positive | Positive  |
|  Throat Lozenges | 0.68g/mL; 1/18 drop, crushed; active ingredients: 1.7 mg/mL menthol | Positive | Positive  |
|  Zanamivir | 3.3-5 mg/mL | Positive | Positive  |
|  Tobramycin | 4.0 μg/mL | Positive | Positive  |
|  Mupirocin | 6.6-10 mg/mL | Positive | Positive  |
|  Oseltamivir phosphate | 7.5-25 mg/mL | Positive | Positive  |

# i. Assay cut-off

The "cutoff value" represents the fluorescent intensity signal (reported in Relative Fluorescent Units) at which a "positive" reaction reaches a relative

{16}

fluorescent intensity above the background or baseline of a “negative” reaction. If a sample exceeds the threshold in a detection channel during PCR, the sample is considered positive for that channel. If the sample does not exceed the threshold for a detection channel by the last PCR cycle, the sample is considered negative for that channel.

The cut-off for the Quidel Molecular Influenza A+B Assay was determined and confirmed through a phased approach. The preliminary threshold was established using data obtained from the LoD studies and from the analysis of a set of clinical specimens. Data from the analysis of multiple replicates near the LoD of the assay were used to establish the threshold such that sensitivity was maximized. Similarly, the latest Ct value from the LoD data was used to set the cut-off. Using the parameters summarized in the table below, a verification study was done to confirm the threshold and cut-off values.

|  Quidel Molecular Influenza A+B Assay on the ABI 7500 Fast Dx Instrument  |   |   |
| --- | --- | --- |
|  Analyte | Confirmed Threshold (RFU) | Confirmed Ct Cut-Off*  |
|  Influenza A | 1.5e05 | 35  |
|  Influenza B | 1.2e05 | 35  |
|  PRC | 2.7e04 | 35  |

*These values reflect subtraction of the first 10 cycles. Fluorescence is monitored after the first 10 cycles during the data collection stage of amplification.

- Results from an LoD study used to verify the threshold and cut-off values are as follows:
- Influenza A – 119/120 (99.1%)
- Influenza B – 119/120 (99.1%)

These results demonstrated that the preliminary threshold and Ct cut-off settings determined have been confirmed and effective for maximizing sensitivity of the Quidel Molecular Influenza A+B Molecular Assay.

j. Carry-over Contamination Analysis

An evaluation of potential carry-over contamination during extraction and/or amplification was carried out. High negative and positive samples were run in alternating series to evaluate performance of the device and to monitor for potential carry-over contamination. The evaluation included alternating extractions high and low concentration of virus on the indicated platform. During the amplification stage the plate was setup in a checkerboard approach locating negative and positive samples in adjoining wells. No cross contamination was observed in the evaluation of the Quidel Molecular Influenza A+B Assay.

{17}

# k. Comparison of transport media

Analytical performance of the Quidel Molecular Influenza A+B Assay was evaluated with six different transport media. Stocks of influenza A and influenza B were used to evaluate performance across the different transport media at a level close to the LoD (3X). Briefly, dilutions of influenza A and influenza B were made and each of the dilutions was extracted in triplicate. Each extraction was then tested in duplicate on the ABI 7500 Fast Dx using the Quidel Molecular Influenza A+B Assay standard protocol. No differences between the tested transport media were observed and the results are summarized below.

|  Evaluation of Transport Media in the Quidel Molecular Influenza A+B Assay  |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- |
|   |  | Average Ct. |   |   | STDEV  |   |   |
|  Test Media | Template | Influenza A | Influenza B | PRC | Influenza A | Influenza B | PRC  |
|  M4 | 100xLoD Influenza A + 100x LoD Influenza B | 20.04 | 18.23 | 17.62 | 0.13 | 0.11 | 0.12  |
|   |  3x LoD Influenza A + 3x LoD Influenza B | 26.70 | 24.35 | 17.98 | 0.22 | 0.10 | 0.12  |
|   |  Media + PRC | Und | Und | 17.67 | Und | Und | 0.17  |
|  M4-RT | 100xLoD Influenza A + 100x LoD Influenza B | 19.41 | 18.07 | 17.52 | 0.07 | 0.05 | 0.18  |
|   |  3x LoD Influenza A + 3x LoD Influenza B | 26.81 | 24.41 | 17.81 | 0.32 | 0.14 | 0.18  |
|   |  Media + PRC | Und | Und | 17.83 | Und | Und | 0.11  |
|  M5 | 100xLoD Influenza A + 100x LoD Influenza B | 20.11 | 18.30 | 17.56 | 0.40 | 0.06 | 0.18  |
|   |  3x LoD Influenza A + 3x LoD Influenza B | 27.15 | 24.58 | 17.87 | 0.95 | 0.29 | 0.15  |
|   |  Media + PRC | Und | Und | 17.64 | Und | Und | 0.14  |
|  M6 | 100xLoD Influenza A + 100x LoD Influenza B | 19.48 | 18.01 | 17.60 | 0.32 | 0.12 | 0.17  |
|   |  3x LoD Influenza A + 3x LoD Influenza B | 26.70 | 24.58 | 17.89 | 0.68 | 0.12 | 0.21  |
|   |  Media + PRC | Und | Und | 17.73 | Und | Und | 0.04  |

{18}

|  saline | 100xLoD
Influenza A +
100x LoD
Influenza B | 20.33 | 18.36 | 17.85 | 0.45 | 0.12 | 0.18  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|   |  3x LoD
Influenza A +
3x LoD
Influenza B | 27.66 | 25.29 | 18.00 | 0.57 | 0.30 | 0.08  |
|   |  Media + PRC | Und | Und | 18.01 | Und | Und | 0.06  |
|  UTM | 100xLoD
Influenza A +
100x LoD
Influenza B | 19.82 | 18.26 | 18.04 | 0.17 | 0.07 | 0.04  |
|   |  3x LoD
Influenza A +
3x LoD
Influenza B | 27.49 | 25.17 | 18.24 | 0.25 | 0.20 | 0.02  |
|   |  Media + PRC | Und | Und | 18.09 | Und | Und | 0.06  |
|  Overall
Average | 100xLoD | 19.86 | 18.21 |  | 0.25 | 0.09 |   |
|   |  3x LoD | 27.09 | 24.73 |  | 0.50 | 0.19 |   |
|   |  PRC |  |  | 17.83 |  |  | 0.12  |

2. **Comparison studies:**

a. **Method comparison with predicate device:**

Not Applicable

b. **Matrix comparison:**

Not Applicable

3. **Clinical studies:**

a. **Prospective Clinical Studies:**

Performance characteristics of the Quidel Molecular Influenza A+B assay were established in a prospective study during the 2010-2011 respiratory virus season (January to March 2011). Samples used for this study were fresh nasal (373) and nasopharyngeal (313) swab specimens that were collected for routine influenza testing at thirteen (13) sites across the United States. A single specimen was collected per patient and tested within 72-hours of collection at one central location.

A comparator method (a high performance FDA Cleared Influenza A and B molecular test) was used in the evaluation of the Quidel Molecular Influenza A+B assay.

{19}

The gender and age demographics

|  Age and Gender Distribution  |   |   |
| --- | --- | --- |
|  Sex | F | M  |
|  Total | 324 | 362  |
|  |   |   |
|  ≤ 5 years | 164 (50.6%) | 176 (48.6%)  |
|  6 – 21 years | 85 (26.2%) | 113 (31.2%)  |
|  22 – 59 years | 58 (17.9%) | 62 (17.1%)  |
|  ≥ 60 years | 17 (5.2%) | 11 (3.0%)  |
|  Total | 324 | 362  |

Six hundred and eighty six (686) fresh specimens (373 nasal swabs and 313 nasopharyngeal swabs) were tested by the subject and comparator device for influenza A and influenza B viral RNA. Four of these specimens were invalid on initial testing with the subject device (0.6%). Re-testing of the specimens according to the Interpretation algorithm described above also yielded invalid results. Seventeen (17) specimens were invalid on initial and repeat testing (as per the device's PI) on the comparator device (2.5%). Three specimens were invalid in both devices, therefore, a total of 18 invalid specimens were removed from additional analysis. The tables below detail the results for the remaining 668 specimens.

|  Influenza A  |   |   |   |
| --- | --- | --- | --- |
|  Fresh nasal and nasopharyngeal swabs (N=668) | Comparator: FDA Cleared RT-PCR device  |   |   |
|  Quidel Molecular | Positive | Negative | Total  |
|  Positive | 139 | 8* | 147  |
|  Negative | 0 | 521 | 292  |
|  Total | 139 | 529 | 668  |
|  95% CI  |   |   |   |
|  Positive Percent Agreement | 139/139 | 100% | 97.4% to 100%  |
|  Negative Percent Agreement | 521/529 | 98.5% | 97.0% to 99.3%  |

*Seven specimens were negative by FDA Cleared RT-PCR device but positive for influenza A by sequence analysis. One specimen was negative by FDA Cleared RT-PCR device and negative for influenza A by sequence analysis.

|  Influenza B  |   |   |   |
| --- | --- | --- | --- |
|  Fresh nasal and nasopharyngeal swabs (N=668) | Comparator: FDA Cleared RT-PCR device  |   |   |
|  Quidel Molecular | Positive | Negative | Total  |
|  Positive | 105 | 12* | 117  |
|  Negative | 5** | 546 | 551  |
|  Total | 110 | 558 | 668  |
|  95% CI  |   |   |   |

{20}

21

|  Positive Percent Agreement | 105/110 | 95.5% | 89.7% to 98.5%  |
| --- | --- | --- | --- |
|  Negative Percent Agreement | 546/558 | 97.8% | 96.3% to 98.9%  |

*Twelve specimens were negative by FDA Cleared RT-PCR device but positive for influenza B by sequence analysis).
** Five specimens were positive by FDA Cleared RT-PCR device.

The prospective clinical study had a dual infection rate for Influenza A and Influenza B of 1.8% (12/668) using the Quidel Molecular Influenza A + B Assay. Three of these dual infections were concordant with the FDA Cleared RT-PCR device comparator assay. Five of these dual infections were discordant with the Influenza A results from the FDA Cleared RT-PCR device comparator assay. Four of these dual infections were discordant with the Influenza B results from the FDA Cleared RT-PCR device comparator assay.

## b. Retrospective Clinical Studies:

Performance characteristics of the Quidel Molecular Influenza A+B assay were also evaluated during a retrospective study of frozen specimens collected during the 2010-2011 respiratory virus season (January to March of 2011). Samples used for this study were frozen nasopharyngeal (376) swab specimens that were collected for routine influenza testing.

For this study the comparator method was a high performance FDA-Cleared Influenza A and B molecular device.

Three hundred and seventy six (376) frozen nasopharyngeal swabs were tested by the subject and comparator devices for influenza A and influenza B viral RNA. Two of these specimens were invalid on initial testing with the subject device (0.5%). Re-testing of the specimens according to the Interpretation algorithm described above also yielded invalid results. Two specimens were invalid on initial and repeat testing (as per the device's PI) on the comparator device (0.5%). The four invalid specimens were removed from performance analyses. The tables below detail the results for the remaining 372 specimens.

|  Influenza A  |   |   |   |
| --- | --- | --- | --- |
|  Frozen nasopharyngeal swab (N=372) | Comparator: FDA Cleared RT-PCR device  |   |   |
|  Quidel Molecular | Positive | Negative | Total  |
|  Positive | 37 | 0 | 37  |
|  Negative | 0 | 335 | 335  |
|  Total | 37 | 335 | 372  |
|  95% CI  |   |   |   |
|  Positive Percent Agreement | 37/37 | 100% | 90.5% to 100%  |
|  Negative Percent Agreement | 335/335 | 100% | 98.9% to 100%  |

{21}

|  Influenza B  |   |   |   |
| --- | --- | --- | --- |
|  Frozen nasopharyngeal swab (N=372) | Comparator: FDA Cleared RT-PCR device  |   |   |
|  Quidel Molecular | Positive | Negative | Total  |
|  Positive | 37 | 2* | 39  |
|  Negative | 1** | 332 | 333  |
|  Total | 38 | 334 | 372  |
|  95% CI  |   |   |   |
|  Positive Percent Agreement | 37/38 | 97.4% | 86.2% to 99.9%  |
|  Negative Percent Agreement | 332/334 | 99.4% | 97.9% to 99.9%  |

*Two specimens were negative by FDA Cleared RT-PCR device but positive for influenza B by sequence analysis.

4. Clinical cut-off:

Not applicable

5. Expected values/Reference range:

Clinical studies were performed testing prospective specimens collected throughout the United States in the winter of 2011 (January 2011 – March 2011). The number and percentage of positive influenza A cases within this population as determined by the Quidel Molecular Influenza A+B assay was 21.6% (147/682). The number and percentage of positive influenza B cases within this population as determined by the Quidel Molecular Influenza A+B assay was 17.2% (117/682).

N. Proposed Labeling:

The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.

O. Conclusion:

The submitted information in this premarket notification is complete and supports a substantial equivalence decision.

---

**Source:** [https://fda.innolitics.com/submissions/MI/subpart-d%E2%80%94serological-reagents/OCC/K112172](https://fda.innolitics.com/submissions/MI/subpart-d%E2%80%94serological-reagents/OCC/K112172)

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