The Alere™ i Influenza A & B assay performed on the Alere™ i Instrument is a rapid molecular in vitro diagnostic test utilizing an isothermal nucleic acid amplification technology for the qualitative detection and discrimination of influenza A and B viral RNA in direct nasal swabs or nasopharyngeal swabs eluted in viral transport media from patients with signs and symptoms of respiratory infection. It is intended for use as an aid in the differential diagnosis of influenza A and B viral infections in humans in conjunction with clinical and epidemiological risk factors. The assay is not intended to 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 2014-2015 influenza seasons when influenza A/H3 and A/H1N1 pandemic 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 Description

Alere™ i Influenza A & B is a rapid, instrument-based isothermal test for the qualitative detection and differentiation of influenza A and influenza B from nasal or nasopharyngeal swabs eluted in viral transport media from patients presenting with signs and symptoms of respiratory infection. The Alere™ i Influenza A & B System utilizes isothermal nucleic acid amplification technology and is comprised of:

Sample Receiver - single use, disposable containing the elution buffer
. Test Base – single use, disposable comprising two sealed reaction tubes, each containing a lyophilized pellet
Transfer Cartridge – single use, disposable for transfer of the eluted sample to the Test Base, and
Alere™ i Instrument repeat use reader ●

The reaction tubes in the Test Base contain the reagents required for amplification of the target nucleic acid and an internal control. Alere™ i Influenza A & B utilizes a pair of templates (similar to primers) for the specific amplification of RNA from influenza A and B and a fluorescently labeled molecular beacon designed to specifically identify the amplified RNA targets. Alere™ i Influenza A & B is performed within the confinement of the Test Base, and no other part of the Alere™ i Instrument has contact with the sample during the amplification process. This minimizes the risk of instrument contamination and sample carry-over between measurements.

To perform the assay, the Sample Receiver and Test Base are inserted into the Alere™ i Instrument and the elution buffer is automatically heated by the instrument. The sample is added to the Sample Receiver and transferred via the Transfer Cartridge to the Test Base, resuspending the lyophilized pellet contained within the Test Base and initiating target amplification. Heating mixing and detection by fluorescence is provided by the instrument, with results automatically reported.

Results are displayed by the Alere™ i Instrument separately for influenza B. Results are also stored in an on-board archive and are assigned to a sample ID that has been entered into the Alere™ i Instrument by the operator, and the date/time the test was performed. Data can be retrieved and downloaded by the operator at any time after testing. An external Alere™ Universal Printer can be attached via USB to the Alere™ i Instrument to print test results.

AI/ML Overview

Here's a summary of the acceptance criteria and the study that demonstrates the device's performance, extracted from the provided text:

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state "acceptance criteria" with specific numerical targets for sensitivity and specificity. Instead, it presents the device's performance characteristics in a clinical study and analytical studies which implicitly serve as the basis for its substantial equivalence claim. I will present the clinical performance as the "Reported Device Performance."

Performance Metric	Specific Target (Implicit Acceptance Criteria - based on what was demonstrated)	Reported Device Performance (vs. Comparator Method)
Influenza A
Sensitivity	High	97.8% (95% CI: 94.9%-99.1%)
Specificity	High	96.6% (95% CI: 95.3%-97.5%)
Influenza B
Sensitivity	High	92.9% (95% CI: 86.1%-96.5%)
Specificity	High	98.3% (95% CI: 97.4%-98.9%)
Invalid Rate (after repeat testing)	Low, acceptable for clinical use	2.1% (95% CI: 1.5%, 3.1%)
Analytical Sensitivity (LOD) for Flu A	Lowest possible	Varies by strain (e.g., A/H1N1: 4.20 x 10^5 TCID50/mL, 4.59 x 10^6 Genome Equivalents/mL)
Analytical Sensitivity (LOD) for Flu B	Lowest possible	Varies by strain (e.g., B Victoria: 1.05 x 10^5 TCID50/mL, 2.29 x 10^6 Genome Equivalents/mL)
Analytical Specificity (Cross-Reactivity)	No cross-reactivity with common respiratory pathogens	No cross-reactivity with 53 tested microorganisms
Interference	No interference from common substances	No effect from 26 tested interfering substances
Carry-Over Contamination	No false positives	Minimal (one false positive for Flu B observed in one of the extensive carry-over studies)

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

Test Set Sample Size:
- Clinical Study: 1,270 viral transport media specimens were initially tested. After excluding ineligible specimens (3) and invalid results after repeat testing (27), a total of 1,243 specimens were used for performance analysis.
- Analytical Sensitivity (LOD): 20 replicates per virus concentration, for multiple strains.
- Analytical Reactivity: Triplicates initially, then additional 2-fold dilutions until a negative result was obtained.
- Analytical Specificity (Cross Reactivity): Not specified per microorganism, but 53 different microorganisms were tested.
- Interfering Substances: Not specified per substance, but 26 different substances were evaluated.
- Carry-Over Contamination: 15 rounds of alternating positive and negative swabs for direct swab testing, and 30 rounds for VTM samples.
Data Provenance: From a multi-site prospective clinical study conducted during the 2014-2015 flu season in the U.S. Analytical studies were conducted by the vendor.

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

The document does not specify the number of experts or their qualifications for establishing ground truth. The ground truth for the clinical study was established using an FDA-cleared influenza real-time Polymerase Chain Reaction (RT-PCR) test as the comparator method. Discrepant samples were re-tested on a different FDA-cleared influenza real-time RT-PCR assay by Alere Scarborough Inc.

4. Adjudication Method for the Test Set

The adjudication method for the clinical study was specified for discrepant samples:

An FDA-cleared influenza real-time RT-PCR test was used as the primary comparator.
All discrepant samples were tested on a different FDA-cleared influenza real-time RT-PCR assay at Alere Scarborough Inc. to confirm the influenza status.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance

No. This device is an in-vitro diagnostic assay (a molecular test) and not an imaging or AI-assisted diagnostic device requiring human reader interpretation. Therefore, a multi-reader multi-case (MRMC) comparative effectiveness study was not performed, and the concept of "human readers improve with AI" does not apply here.

6. If a Standalone (i.e. algorithm only without human-in-the loop performance) was done

Yes, the performance presented is for the standalone device (Alere™ i Influenza A & B assay on the Alere™ i Instrument) without human-in-the-loop performance influencing the result generation. The instrument automatically reports the results.

7. The type of ground truth used

Clinical Study: The ground truth was established by an FDA-cleared influenza real-time Polymerase Chain Reaction (RT-PCR) test, with additional confirmatory testing for discrepant results using a different FDA-cleared RT-PCR assay. This is a form of molecular diagnostic gold standard.
Analytical Studies (LOD, Reactivity, Cross-Reactivity, Interference): Ground truth was based on the known concentrations and identities of the viral strains, microorganisms, and substances used in the laboratory experiments.

8. The Sample Size for the Training Set

The document does not explicitly mention a "training set" in the context of device development or machine learning models, as this is a molecular diagnostic assay. The analytical and clinical studies described are for validation and performance assessment of the assay and instrument.

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

As no "training set" in the context of machine learning was described, this question is not applicable to the provided information. The device relies on specific nucleic acid amplification technology rather than machine learning models requiring large training datasets with established ground truth.

Summary

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Image /page/0/Picture/0 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo consists of a circular seal with the text "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" around the perimeter. Inside the circle is a stylized symbol that resembles a caduceus, with three figures in profile facing right. The symbol is black and the background is white.

DEPARTMENT OF HEALTH & HUMAN SERVICES

Public Health Service

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

July 28, 2015

Alere Scarborough, Inc. Angela Drysdale Vice President, Regulatory and Clinical Affairs - Infectious Disease 10 Southgate Road Scarborough, ME 04074

Re: K151464

Trade/Device Name: Alere™ i Influenza A&B Regulation Number: 21 CFR 866.3980 Regulation Name: Respiratory Viral Panel Multiplex Nucleic Acid Assay Regulatory Class: II Product Code: OCC, OZE, OOI Dated: June 02, 2015 Received: June 03, 2015

Dear Ms. Drysdale:

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

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

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); medical device reporting (reporting of

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medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

If you desire specific advice for your device on our labeling regulations (21 CFR Parts 801 and 809), please contact the Division of Small Manufacturers. International and Consumer Assistance at its toll-free number (800) 638 2041 or (301) 796-7100 or at its Internet address http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to

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

You may obtain other general information on your responsibilities under the Act from the Division of Small Manufacturers, International and Consumer Assistance at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.

Sincerely yours.

Tamara V. Feldblyum -S for

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

Enclosure

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Indications for Use

510(k) Number (if known) K151464

Device Name Alere™ i Influenza A & B

Indications for Use (Describe)

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.

Type of Use (Select one or both, as applicable)

☑ Prescription Use (Part 21 CFR 801 Subpart D)
☐ Over-The-Counter Use (21 CFR 801 Subpart C)

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510(K) SUMMARY

This summary of 510(k) safety and effectiveness information is being submitted in accordance with the requirements of SMDA 1990 and 21 CFR 807.92.

The assigned 510(k) number is: K151464

SUBMITTER

Alere Scarborough, Inc. 10 Southgate Road Scarborough, ME 04074 Establishment Registration Number: 1221359

CONTACT PERSON

Angela Drysdale (207) 730-5737 (Office) (207) 730-5767 (FAX) Angela.drysdale@alere.com (email)

DATE PREPARED

7/8/2015

TRADE NAME Alere™ i Influenza A & B

COMMON NAME Alere™ i flu, Alere™ i, Alere™ Influenza A & B

CLASSIFICATION NAME

Respiratory Viral Panel Multiplex Nucleic Acid Assay (per 21 CFR 866.3980) Instrumentation for Clinical Multiplex Test Systems (per 21 CFR 862.2570)

CLASSIFICATION Class II

PRODUCT CODE OCC, OZE, OOI

PANEL Microbiology (83)

PREDICATE DEVICE Alere™ i Influenza A & B Assay, K141520

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DEVICE DESCRIPTION

Sample Receiver - single use, disposable containing the elution buffer
. Test Base – single use, disposable comprising two sealed reaction tubes, each containing a lyophilized pellet
Transfer Cartridge – single use, disposable for transfer of the eluted sample to the Test Base, and
Alere™ i Instrument repeat use reader ●

INTENDED USE

The Alere™ i Influenza A & B assay performed on the Alere™ i Instrument is a rapid molecular in vitro diagnostic test utilizing an isothermal nucleic acid amplification technology for the qualitative detection and discrimination of influenza A and B viral RNA in direct nasal or nasopharyngeal swabs eluted in viral transport media from patients with signs and symptoms of respiratory infection. It is intended for use as an aid in the differential diagnosis of influenza A and B viral infections in conjunction with clinical and epidemiological risk factors. The assay is not intended to 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 2012-2013 and the 2014-2015 influenza seasons when influenza A/H3 and A/H1N1 pandemic were the predominant influenza A viruses in circulation. When other influenza A viruses are emerging, performance characteristics may vary.

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TECHNOLOGICAL CHARACTERISTICS

The purpose of this submission is to expand the Alere™ i Influenza A & B test Intended Use (hereby referred to as Expanded Claim Alere™ i Influenza A & B test) to include testing of swab samples that have been eluted in viral transport media. There have been no changes to the Alere™ i Influenza A & B test or principles of operation.

DEVICE COMPARISON

The expanded claim Alere™ i Influenza A & B test was compared to the legally marketed predicate device, the 510(k) cleared Alere™ i Influenza A & B test.

Parameter	Expanded ClaimAlere™ i Influenza A & B	Alere™ i Influenza A & B (K141520)
FDA Product Code	OCC,OZE, OOI	Same
Assay Target	Influenza A, Influenza B	Same
Intended Use	The Alere™ i Influenza A & B assayperformed on the Alere™ i Instrument isa rapid molecular in vitro diagnostic testutilizing an isothermal nucleic acidamplification technology for thequalitative detection and discriminationof influenza A and B viral RNA in nasalswabs and nasal or nasopharyngealswabs eluted in viral transport mediafrom patients with signs and symptomsof respiratory infection. It is intended foruse as an aid in the differential diagnosisof influenza A and B viral infections inhumans in conjunction with clinical andepidemiological risk factors. The assay isnot intended to detect the presence ofinfluenza C virus.Negative results do not precludeinfluenza virus infection and should notbe used as the sole basis for diagnosis,treatment or other patient managementdecisions.Performance characteristics for influenzaA were established during the 2012-2013 influenza season when influenzaA/H3 and A/H1N1 pandemic were thepredominant influenza A viruses incirculation. When other influenza Aviruses are emerging, performancecharacteristics may vary.If infection with a novel influenza A virusis suspected based on current clinicaland epidemiological screening criteriarecommended by public healthauthorities, specimens should becollected with appropriate infectioncontrol precautions for novel virulentInfluenza viruses and sent to state orlegal health de	The Alere™ i Influenza A & B assay performedon the Alere™ i Instrument is a rapidmolecular in vitro diagnostic test utilizing anisothermal nucleic acid amplificationtechnology for the qualitative detection anddiscrimination of influenza A and B viral RNAin nasal swab samples from patients withsigns and symptoms of respiratory infection.It is intended for use as an aid in thedifferential diagnosis of influenza A and Bviral infections in humans in conjunction withclinical and epidemiological risk factors. Theassay is not intended to detect the presence ofinfluenza C virus.Negative results do not preclude influenzavirus infection and should not be used as thesole basis for diagnosis, treatment or otherpatient management decisions.Performance characteristics for influenza Awere established during the 2012-2013influenza season when influenza A/H3 andA/H1N1 pandemic were the predominantinfluenza A viruses in circulation. When otherinfluenza A viruses are emerging,performance characteristics may vary.If infection with a novel influenza A virus issuspected based on current clinical andepidemiological screening criteriarecommended by public health authorities,specimens should be collected withappropriate infection control precautions fornovel virulent Influenza viruses and sent tostate or local health department for testing.Viral culture should not be attempted in thesecases unless a BSL 3+ facility is available toreceive and culture specimens.
Parameter	Expanded ClaimAlere™ i Influenza A & B	Alere™ i Influenza A & B (K141520)
	culture should not be attempted in thesecases unless a BSL 3+ facility is availableto receive and culture specimens.
Intended Environmentfor Use	CLIA waived for testing direct nasal swabsamples only. Professional use, in amedical laboratory or point-of-care.	CLIA Waived.Professional use, in a medical laboratory orpoint-of-care.
Instrumentation	Alere™ i Instrument	Same
Self-Contained System	Integrated PC, Software, and TouchScreen Display	Same
Automated Assay	Yes. Sample preparation, amplification,detection, and result interpretation.	Same
Assay Information
Sample Type	Nasal Swab and Nasal or NasopharyngealSwabs Eluted in Viral Transport Media	Nasal Swab
Influenza A Viral Target	PB2 segment	Same
Influenza B Viral Target	PA segment	Same
Technology	Isothermal nucleic acid amplification fordetecting the presence/absence of viralRNA in clinical specimens	Same
Detection Method	Assay uses different reporter dyes foreach target	Same
Internal Control	Yes	Same
Result Interpretation	Automated	Same
Assay Result	Qualitative	Same
Time to Result	< 15 minutes	Same

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SUBSTANTIAL EQUIVALENCE

Expansion of the Alere™ i Influenza A & B Intended Use did not result in significant changes to the Analytical Performance of the test.

PERFORMANCE SUMMARY

ANALYTICAL STUDIES

ANALYTICAL SENSITIVITY

Alere™ i Influenza A & B limit of detection (LOD) in natural nasal swab matrix was determined by evaluating different concentrations of 3 strains of influenza A and 2 strains of influenza B virus in Alere™ i Influenza A & B. Three strains of influenza A virus representing each of the three common currently or recently circulating influenza A subtypes (i.e., A/H1N1, A/H3N2 seasonal, and A/H1N1 pandemic (pdm)) and two strains of influenza B virus representing each of the two influenza B genetic lineages (i.e., Victoria and Yamagata) were included in this study.

Presumed negative natural nasal swab specimens were eluted in UTM. Swab elutes were combined and mixed thoroughly to create a clinical matrix pool to be used as the diluent. Each influenza virus strain was diluted in this natural nasal swab matrix pool to generate virus dilutions for testing. The vender provided virus strains were re-titered and the concentrations (in TCID50/mL) were determined by standard virologic method. The concentration for each dilution (in genome equivalents/mL) was also assessed using laboratory developed and validated influenza A and influenza B quantitative real-time PCR assays.

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Contrived nasal swab samples were prepared by coating 10 microliters of each virus dilution onto the swab. The contrived swab samples were tested without further elution in viral transport media according to the test procedure for Direct Nasal Swab.

An additional LOD study was conducted with contrived swab samples eluted into VTM and tested according to the test procedure for Nasal or Nasopharyngeal Swab Eluted in Viral Transport Media.

The LOD for each influenza strain tested was determined as the lowest virus concentration that was detected ≥ 95% of the time (i.e., concentration at which at least 19 out of 20 replicates tested positive).

The confirmed LODs in natural nasal swab matrix for both direct swab and swab eluted in VTM for each influenza strain tested are presented in the tables below:

Limit of Detection (LOD) Study Results – Natural Nasal Swab Matrix (Direct Swab Testing)

Influenza Strain	Influenza ASubtype orInfluenza BGenetic Lineage	LOD(TCID50/mL)	LOD(TCID50/Swab)*	LOD (GenomeEquivalents/mL)	LOD (GenomeEquivalents/Swab)*
A/Puerto Rico/8/34	A/H1N1	$1.88 x 10^5$	$1.88 x 10^3$	$4.22 x 10^6$	$4.22 x 10^4$
A/Perth/16/2009	A/H3N2	$8.60 x 10^2$	$8.60 x 10^0$	$7.91 x 10^4$	$7.91 x 10^2$
A/California/7/2009	A/2009 H1N1 pdm	$1.25 x 10^4$	$1.25 x 10^2$	$5.20 x 10^6$	$5.20 x 10^4$
B/Malaysia/2506/2004	B Victoria lineage	$1.90 x 10^3$	$1.90 x 10^1$	$7.24 x 10^4$	$7.24 x 10^2$
B/Bangladesh/3333/2007	B Yamagata lineage	$5.55 x 10^2$	$5.55 x 10^0$	$7.36 x 10^4$	$7.36 x 10^2$

*Note: 10 ul of each virus dilution was coated onto a swab

Limit of Detection (LOD) Study Results - Natural Nasal Swab Matrix (Swab Eluted in VTM Testing)

Influenza Strain	Influenza ASubtype orInfluenza BGenetic Lineage	LOD(TCID50/mL)	LOD(TCID50/Swab)*	LOD (GenomeEquivalents/mL)	LOD (GenomeEquivalents/Swab)*
A/Puerto Rico/8/34	A/H1N1	$4.20 \times 10^5$	$4.20 \times 10^3$	$4.59 \times 10^6$	$4.59 \times 10^4$
A/Perth/16/2009	A/H3N2	$9.82 \times 10^3$	$9.82 \times 10^1$	$1.25 \times 10^6$	$1.25 \times 10^4$
A/California/7/2009	A/2009 H1N1 pdm	$5.20 \times 10^5$	$5.20 \times 10^3$	$7.77 \times 10^6$	$7.77 \times 10^4$
B/Malaysia/2506/2004	B Victoria lineage	$1.05 \times 10^5$	$1.05 \times 10^3$	$2.29 \times 10^6$	$2.29 \times 10^4$
B/Bangladesh/3333/2007	B Yamagata lineage	$1.34 \times 10^4$	$1.34 \times 10^2$	$1.98 \times 10^6$	$1.98 \times 10^4$

Note: 10 ul of each virus dilution was coated onto a swab; each contrived swab was further diluted into 3 mL of UTM

REACTIVITY TESTING

An analytical reactivity (inclusivity) study was performed to determine whether the Alere™ i Influenza A & B assay is able to detect a variety of influenza A and B strains that represent temporal and geographic diversity.

Vender provided stocks of influenza A and B strains were diluted in UTM to generate virus dilutions for testing. The concentration (in TCIDso/mL, or EID50/mL) for each strain was determined by standard virologic method. The concentration for each dilution (in genome equivalents/mL) was also assessed using laboratory developed and validated influenza A and influenza B quantitative real-time PCR assays.

Contrived swab samples were prepared by coating 10 microliters of virus dilution onto each swab. The contrived swab samples were tested without further elution in viral transport media according to the test procedure for Direct Nasal Swab.

The starting dilution concentration selected for testing in this study was higher than the established LODs in the Limit of Detection study. Each starting dilution per virus strain was tested in triplicates initially. If the initial testing concentration tested positive for all three replicates, the strain was further diluted 10-fold and tested in triplicates until at least one out three replicates generated a negative result. When a negative result was obtained, additional 2-fold dilutions were tested, starting from the highest dilution that produced 100% (3/3) positive results. A concentration level was considered "reactive/positive" in this study for all but one strain tested (i.e., B/Texas/06/2011 – see footnote "c" under the table below) if all three replicates generated a positive result for the expected influenza virus.

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The Alere™ i Influenza A & B assay detected all strains tested at the concentrations indicated in the table below:

Analytical Reactivity Study Results

Influenza Strain	Influenza ASubtype orInfluenza BGenetic Lineage	Test Concentration (in TCID50 or Genome Equivalents,unless indicated otherwise)				Flu AResult(n=3,unlessindicateotherwise)	Flu BResult(n=3,unlessindicateotherwise)
		TCID50/mL	TCID50/Swab*	GenomeEquivalents/mL	GenomeEquivalents/Swab*
A/New Caledonia/20/1999 a	A/H1N1	$9.19 x 10^5$	$9.19 x 10^3$	$4.09 x 10^6$	$4.09 x 10^4$	+	-
A/New Jersey/8/76 a	A/H1N1	$3.41 x 10^1$	$3.41 x 10^{-1}$	$1.52 x 10^5$	$1.52 x 10^3$	+	-
A/Brisbane/59/2007 a	A/H1N1	$2.11 x 10^4$	$2.11 x 10^2$	$3.39 x 10^5$	$3.39 x 10^3$	+	-
A/WSN/33 a	A/H1N1	$2.11 x 10^2$	$2.11 x 10^0$	$2.43 x 10^5$	$2.43 x 10^3$	+	-
A/Port Chalmers/1/73	A/H3N2	$4.22 x 10^4$	$4.22 x 10^2$	$1.31 x 10^6$	$1.31 x 10^4$	+	-
A/Hong Kong/8/68	A/H3N2	$7.03 x 10^0$	$7.03 x 10^{-2}$	$2.70 x 10^5$	$2.70 x 10^3$	+	-
A/Aichi/2/68	A/H3N2	$2.08 x 10^5$	$2.08 x 10^3$	$7.47 x 10^6$	$7.47 x 10^4$	+	-
A/Victoria/3/75	A/H3N2	$3.68 x 10^5$	$3.68 x 10^3$	$3.39 x 10^6$	$3.39 x 10^4$	+	-
A/Wisconsin/67/2005	A/H3N2	$6.81 x 10^4$	$6.81 x 10^2$	$2.57 x 10^6$	$2.57 x 10^4$	+	-
A/Brisbane/10/2007	A/H3N2	$3.16 x 10^2$	$3.16 x 10^0$	$3.37 x 10^5$	$3.37 x 10^3$	+	-
A/Texas/50/2012	A/H3N2	$2.5 x 10^0$	$2.50 x 10^{-2}$	$6.35 x 10^3$	$6.35 x 10^1$	+	-
A/Victoria/361/2011	A/H3N2	$1.56 x 10^1$	$1.56 x 10^{-1}$	$3.53 x 10^5$	$3.53 x 10^3$	+	-
A/California/4/2009	A/H1N1 (pdm)	$1.47 x 10^4$	$1.47 x 10^2$	$1.07 x 10^6$	$1.07 x 10^4$	+	-
A/Maryland/04/2011	A/H1N1 (pdm)	$7.88 x 10^4$	$7.88 x 10^2$	$3.81 x 10^6$	$3.81 x 10^4$	+	-
A/New York/18/2009	A/H1N1 (pdm)	$1.25 x 10^2$	$1.25 x 10^0$	$9.16 x 10^5$	$9.16 x 10^3$	+	-
A/Anhui/1/2013(Inactivated)a	A/H7N9(Detected in Chinain 2013)	$4.00 x 10^6$EID50/mL	$4.00 x 10^4$EID50/Swab	$1.72 x 10^6$	$1.72 x 10^4$	+	-
A/Indiana/10/2011ª	A/H3N2v	$2.00 x 10^8$EID50/mL	$2.00 x 10^6$EID50/Swab	$5.94 x 10^4$	$5.94 x 10^2$	+	-
B/Lee/40	Victoria Lineage	$5.00 x 10^1$CEID50/mL	$5.00 x 10^{-1}$CEID50/Swab	$5.40 x 10^4$	$5.40 x 10^2$	-	+
B/Victoria/504/2000	Victoria Lineage	$1.19 x 10^3$	$1.19 x 10^1$	$6.24 x 10^4$	$6.24 x 10^2$	-	+
B/Nevada/03/2011	Victoria Lineage	$1.75 x 10^3$	$1.75 x 10^1$	$8.29 x 10^4$	$8.29 x 10^2$	-	+
B/Montana/05/2012	Victoria Lineage	$9.00 x 10^1$	$9.00 x 10^{-1}$	$2.55 x 10^4$	$2.55 x 10^2$	-	+
B/Maryland/1/59	Yamagata Lineage	$8.51 x 10^2$	$8.51 x 10^0$	$1.13 x 10^5$	$1.13 x 10^3$	-	+
B/Russia/69b	Yamagata Lineage	$4.44 x 10^1$	$4.44 x 10^{-1}$	$2.96 x 10^6$	$2.96 x 10^4$	-	+
B/Wisconsin/01/2010c	Yamagata Lineage	$3.68 x 10^4$	$3.68 x 10^2$	$1.16 x 10^6$	$1.16 x 10^4$	-	+
B/Massachusetts/2/2012	Yamagata Lineage	$6.25 x 10^1$	$6.25 x 10^{-1}$	$2.28 x 10^5$	$2.28 x 10^3$	-	+
B/Texas/06/2011c	Yamagata Lineage	$2.89 x 10^5$	$6.25 x 10^3$	$2.00 x 10^6$	$2.00 x 10^4$	-	+

*Note: 10 ul of each virus dilution was coated onto a swab

4 Although this test has been shown to detect A/H1N9 (detected in China in 2013) and A/H3N2v viruses cultured from positive human respiratory specimens, the performance characteristics of this device with clinical speciment that are positive for the A/H1N1 (pre-2009 pandemic), A/H7N9 (detected in China in 2013) and A/H3N2v viruses have not been established.

4 Influenza B/Russia/69 lowest level in which 3/3 replicates were positive is approximately 40 to 150 x the LOD (as comparing to the Genome Equivalents/Swab values generated in the LOD with simulated clinical matrix study testing B/Malaysia/2008, respectively). A polymorphism within segment PA of the Influenza B genome was identified at a position which is 4 nucleotides from the 3 -end of template 2. This G to A polymorphism results in a G/C (product/template) match to an A/C (product/template) mismatch is determined to be moderately destabilizing, and coupled to its position only 4 nucleotides from the 2 recognition region, its impact on annealing is potentially great. The frequency of this is determined to be very low. In analyzing the strains present in the NCBI Influenza Virus Resource database from 2/2005 to 3/2014 (N=986), no strains contained this polymorphism, suggesting that it has not been circulating for an extended period of time.

c Influenza B/Wisconsin/01/2010 lowest level in which 3/3 replicates were positive is approximately 15 to 60 x the LOD, and Influenza B/Texas/06/2011 lowest level in which at least 1/3 replicates were positive is approximately 25 to 100 x the LOD (as comparing to the Genome Equivalents/Swab values generated in the LOD with simulated clinical matrix study testing B/Malaysia / 60/2008, respectively). A single G to A polymorphism within segment PA of the Influenza B genome was identified at a position which is 5 not the 3end of the molecular beacon annealing region in both strains. The G to A polymorphism results in a C/A mismatch between the molecular beacon and product 1. The C./A mismatch is determined to be moderately destabilizing that can significantly reduce assay sensitivity. An assessment of what impact this polymorphism would have on the melting temperature (Tm) of the molecular beacon/product 1 annealing was performed and the results showed a Tm drop from 62.3°C to 55.6°C, just below the assay running temperature. This suggests that annealing would occur, but at a greatly reduced level, with a concomitant loss of assay sensitivity. The frequency of this G to A polymorphism is approximately 5% within the NCBI Influenza Virus Resource database covering the time from 2/2005 through 3/2014.

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Transport Media, and demonstrated equivalent analytical reactivity performance to that of testing direct swab samples.

Analytical Specificity (Cross Reactivity)

To determine the analytical specificity of Alere™ i Influenza A & B, 53 commensal and pathogenic microorganisms (37 bacteria, 15 viruses and 1 yeast) that may be present in the nasal cavity or nasopharynx were tested. All of the following microorganisms were negative when tested at concentrations ranging from 10° to 10° cells/mL, CFU/mL or IFU/mL (bacteria), 104 to 10° TCIDs0/mL (viruses), and 10° cells/mL (yeast).

Bacteria	Viruses	Yeast
Acinetobacter calcoaceticus	Adenovirus type 1	Candida albicans
Bacteroides fragilis	Adenovirus type 7
Bordetella pertussis	Human Coronavirus OC43
Chlamydia pneumoniae	Human Coronavirus 229E
Corynebacterium diphtheriae	Enterovirus/Coxsackievirus B4
Enterococcus faecalis	Human Cytomegalovirus (CMV) (Herpes V)
Escherichia coli	Epstein Barr Virus
Gardnerella vaginalis	Human metapneumovirus
Haemophilus influenzae	Measles (Edmonston)
Klebsiella pneumoniae	Mumps (Enders)
Lactobacillus casei	Parainfluenza 1
Lactobacillus plantarum	Parainfluenza 2
Legionella pneumophila	Parainfluenza 3
Listeria monocytogenes	Respiratory Syncytial Virus type B
Moraxella/Branhamella catarrhalis	Rhinovirus type 1A
Mycobacterium avium
Mycobacterium intracellulare
Mycobacterium tuberculosis
Mycoplasma pneumoniae
Neisseria gonorrhoeae
Neisseria meningitidis
Neisseria sicca
Neisseria subflava
Proteus vulgaris
Pseudomonas aeruginosa
Serratia marcescens
Staphylococcus aureus
Staphylococcus epidermidis
Streptococcus, Group A
Streptococcus, Group B
Streptococcus, Group C
Streptococcus, Group F
Streptococcus, Group G
Streptococcus mutans
Streptococcus pneumoniae
Streptococcus salivarius

Streptococcus sanguinis

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Interfering Substances

The following substances, naturally present in respiratory specimens or that may be artificially introduced into the nasal cavity or nasopharynx, were evaluated with Alere™ i Influenza A & B at the concentrations listed below and were found not to affect test performance.

Substance	Concentration
Mucin	20 µg/mL
Whole Blood	50 µl/mL
Sinus Buster Nasal Spray	200 µl/mL
NeoSynephrine Cold & Sinus Extra Strength Spray	200 µl/mL
Zicam Extreme Congestion Relief	200 µl/mL
Afrin PumpMist Original	200 µl/mL
4-acetamidophenol	200 µg/mL
Acetylsalicylic acid	650 µg/mL
Albuterol	400 ng/mL
Chlopheniramine	145 ng/mL
Dexamethasone	0.80 mg/mL
Dextromethorphan	1 µl/mL
Diphenhydramine	5 µg/mL
Doxylamine Succinate	236 ng/mL
Ephedrine	237 ng/mL
Flunisolide	6.8 ng/mL
Guaiacol glycerol ether	3.5 ng/mL
Mupirocin	12 mg/mL
Oxymetazoline	0.6 mg/mL
Phenylephrine	12 mg/mL
Rebetol	4.5 µg/mL
Relenza	282 ng/mL
Rimatadine	282 ng/mL
Tamiflu	1.1 µg/mL
Tobryamycin	2.43 mg/mL
Triamcinolone	40 µg/mL

Inhibition by other Microorganisms

Alere™ i Influenza A & B test performance in the presence of non-influenza respiratory pathogens was evaluated. Vendor provided stocks of influenza A and B strains were diluted in UTM to approximately 2 to 3 times the limit of detection. Contrived influenza A and B positive swab specimens were prepared by coating 10 microliters of virus dilution onto each swab. The following panel of non-influenza viruses were tested at the concentration provided in the table below and was found not to affect test performance.

Virus Panel	Concentration(TCID50/ml)
Adenovirus Type 1	1.58 x 107
Rhinovirus Type 1A	1.58 x 107
Respiratory Syncytial Virus, Type B, Strain18537	8.89 x 105

In an additional study, contrived influenza A and B positive swab specimens were also eluted into UTM and tested according the test procedure for Nasal or Nasopharyngeal Swab Eluted in Viral Transport Media using

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the same panel of non-influenza respiratory viruses as described in the table above. None of the non-influenza respiratory viruses tested was found to affect test performance at the concentrations evaluated.

Inhibition by High Levels of Influenza A and B

Alere™ i Influenza A & B test performance in the presence of influenza A and B was evaluated. Vendor provided stocks of influenza A and B strains were diluted in UTM to approximately 2 to 3 times the limit of detection. Contrived influenza A and B positive swab specimens were prepared by coating 10 microliters of virus dilution onto each swab. To create the co-infection swabs, diluted influenza A (at a concentration approximately 5 times the LOD) was added to the near LOD Flu B swab. Likewise, diluted influenza B (at a concentration approximately 40 times the LOD) was added to the near LOD Flu A swab. No impact on test performance was observed.

Alere™ i Influenza A & B test performance in the presence of influenza A and B was also evaluated in an additional study following the test procedure for Nasopharyngeal Swab Eluted in Viral Transport Media. No impact on test performance was observed at the concentrations tested.

Carry-Over Contamination

An analytical carry-over study was performed to demonstrate that when recommended laboratory practices are followed, there is little risk of false positive results caused by carryover or cross-contamination in the Alere™ i Influenza A & B test. Vendor provided stocks of influenza A and B strains were diluted in UTM to a minimum of 106 TCID50/ml. Contrived influenza A and B positive swab specimens were prepared by coating 10 microliters of virus dilution onto each swab. Testing of the contrived positive swabs was alternated with testing of a negative swab sample for a total of 15 rounds. There were no false positive results obtained.

An additional analytical carry-over study was performed testing contrived positive VTM samples alternated with negative VTM samples following the test procedure for Nasopharyngeal Swab Eluted in Viral Transport Media for a total of 30 rounds. No false positive results were observed in this study except for one Flu B false positive result.

An additional analytical reactivity study was also performed testing the same set of influenza A and B strains as described in the table above following the test procedure for Nasopharyngeal Swab Eluted in Viral Transport Media, and demonstrated equivalent analytical reactivity performance to that of testing direct swab samples.

CLINICAL STUDY

Nasal or Nasopharyngeal Swabs Eluted in Viral Transport Media

Clinical performance characteristics of Alere™ i Influenza A & B were evaluated in a multi-site prospective study during the 2014-2015 flu season in the U.S. A total of three investigational sites across the U.S. participated in the study. To be enrolled in the study, patients had to be presenting at the participating study centers with flu-like symptoms. Nasal or nasopharyngeal swab specimens were collected from patients with flu-like symptoms and were placed in viral transport media. The samples were processed and tested using the Alere™ i Influenza A & B assay according to the test procedure for Nasal or Nasopharyngeal Swab Eluted in Viral Transport Media. An FDA-cleared influenza real-time Polymerase Chain Reaction (RT-PCR) test was utilized as the comparator method for this study. All discrepant samples were tested on a different FDAcleared influenza real-time RT-PCR assay at Alere Scarborough Inc. to confirm influenza status. External control testing, using Alere™ i Influenza A & B Positive and Negative Controls, was performed prior to sample testing each day and on each Alere™ i instrument for the dinical study. A total of 1,273 nasal or nasopharyngeal swabs eluted in viral transport media were enrolled in this study. Of those, 3 specimens did not meet eligibility criteria. A total of 1,270 viral transport media specimens were tested with the Alere™ i Influenza A & B assay. Patient age and gender distribution for all included specimens in this study is presented in the table below.

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Age Group	Female	Male
<1 year	92	84
1 to 5 years	258	282
6 to 10 years	113	131
11 to 15 years	63	56
16 to 21 years	16	20
>21 to 60 years	52	37
>60 years	42	24
Total	636	634

Age and Gender Distribution - Nasal or Nasopharyngeal Swabs Eluted in Viral Transport Media Study

Of the 1,270 specimens, Alere™ i Influenza A & B generated invalid results for 27 specimens after repeat testing per the product instructions, resulting in a total of 1,243 specimens for performance analysis.

Compared to the comparator method, the performance of Alere™ i Influenza A & B for influenza B are presented in the two tables below.

Nasal or Nasopharyngeal Swabs Eluted in Viral Transport Media - Performance Obtained for Influenza A with Alere™ i Influenza A & B against the Comparator Method

Alere™ iInfluenza A & B- Flu A		Comparator Method
	Positive	Negative	Total
Positive	221	35a	256
Negative	5	982	987
Total	226	1017	1243
Sensitivity: 221/226	97.8%	(95%CI: 94.9%-99.1%)
Specificity: 982/1017	96.6%	(95%CI: 95.3%-97.5%)

a Flu A nucleic acid was detected in 31/35 False Positive specimens using an alternative FDA-cleared molecular test

Nasal or Nasopharyngeal Swabs Eluted in Viral Transport Media - Performance Obtained for Influenza B with Alere™ i Influenza A & B against the Comparator Method

Alere™ iInfluenza A & B- Flu B	Comparator Method
	Positive	Negative	Total
Positive	92	19a	111
Negative	7	1125	1132
Total	99	1144	1243
Sensitivity: 92/99	92.9%	(95%CI: 86.1%-96.5%)
Specificity: 1125/1141	98.3%	(95%CI: 97.4%-98.9%)

ª Flu B nucleic acid was detected in 3/19 False Positive specimens using an alternative FDA-cleared molecular test

Performance of Alere™ i Influenza A & B for the detection of influenza A and influenza B versus the comparator method in this study is presented in the table below stratified by patient age.

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Nasal or Nasopharyngeal Swabs Eluted in Viral Transport Media - Performance Obtained for Influenza A and Influenza B with Alere™ i Influenza A & B in Comparison to the Comparator Method – Stratified by Patient Age

	≤ 5 Years of Age(n = 706)		6 - ≤ 21 Years of Age(n = 388)		≥ 22 Years of Age(n = 149)
InfluenzaType	Sensitivity95% CI	Specificity95% CI	Sensitivity95% CI	Specificity95% CI	Sensitivity95% CI	Specificity95% CI
Flu A	99.1%(105/106)94.8%, 99.8%	98.2%(589/600)96.7%, 99.0%	97.0%(98/101)91.6%, 99.0%	95.8%(275/287)92.8%, 97.6%	94.7%(18/19)75.4%, 99.1%	90.8%(118/130)84.6%, 94.6%
Flu B	100%(39/39)91.0%, 100%	98.2%(655/667)96.9%, 99.0%	94.2%(49/52)84.4%, 98.0%	98.5%(331/336)96.6%, 99.4%	50%(4/8)21.5%, 78.5%	98.6%(139/141)95.0%, 99.6%

During this prospective clinical study, the initial invalid rate (before repeat testing per the product instructions) was 4.3% (54/1270) (95% Cl: 3.3% to 5.5%). After repeat testing per the product instructions, the invalid rate was 2.1% (27/1270) (95% CI: 1.5%, 3.1%).

Regulation Number and Section

§ 866.3980 Respiratory viral panel multiplex nucleic acid assay.

(a)
Identification. A respiratory viral panel multiplex nucleic acid assay is a qualitative in vitro diagnostic device intended to simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or viral culture. The detection and identification of a specific viral nucleic acid from individuals exhibiting signs and symptoms of respiratory infection aids in the diagnosis of respiratory viral infection when used in conjunction with other clinical and laboratory findings. The device is intended for detection and identification of a combination of the following viruses:(1) Influenza A and Influenza B;
(2) Influenza A subtype H1 and Influenza A subtype H3;
(3) Respiratory Syncytial Virus subtype A and Respiratory Syncytial Virus subtype B;
(4) Parainfluenza 1, Parainfluenza 2, and Parainfluenza 3 virus;
(5) Human Metapneumovirus;
(6) Rhinovirus; and
(7) Adenovirus.
(b)
Classification. Class II (special controls). The special controls are:(1) FDA's guidance document entitled “Class II Special Controls Guidance Document: Respiratory Viral Panel Multiplex Nucleic Acid Assay;”
(2) For a device that detects and identifies Human Metapneumovirus, FDA's guidance document entitled “Class II Special Controls Guidance Document: Testing for Human Metapneumovirus (hMPV) Using Nucleic Acid Assays;” and
(3) For a device that detects and differentiates Influenza A subtype H1 and subtype H3, FDA's guidance document entitled “Class II Special Controls Guidance Document: Testing for Detection and Differentiation of Influenza A Virus Subtypes Using Multiplex Nucleic Acid Assays.” See § 866.1(e) for the availability of these guidance documents.