Predicate Devices

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The description focuses on PCR-based detection and analysis of melting temperatures (Tm) for target identification, which are standard molecular diagnostic techniques and do not indicate the use of AI/ML. There is no mention of AI, ML, or related terms in the document.

Therapeutic

No
Explanation: This device is an in vitro diagnostic test designed to detect and differentiate viral nucleic acids to aid in diagnosis, not to provide therapy or treatment.

Diagnostic

Yes

The "Intended Use" section explicitly states that "The ARIES® Flu A/B & RSV Assay is a polymerase chain reaction (PCR) based qualitative in vitro diagnostic test." It also indicates its purpose is for "direct detection and differentiation of influenza A virus, influenza B virus, and respiratory syncytial virus (RSV) nucleic acid" and "is intended for use as an aid in the differential diagnosis."

SaMD (Software as a Medical Device)

No

The device is described as a "qualitative in vitro diagnostic test system that will consist of an ARIES® System with its included software, an assay-specific cassette, and an assay-specific protocol file." This explicitly includes hardware components (ARIES® System and assay-specific cassette) in addition to software.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

Here's why:

Intended Use: The "Intended Use / Indications for Use" section explicitly states that the ARIES® Flu A/B & RSV Assay is a "qualitative in vitro diagnostic test".
Device Description: The "Device Description" section also refers to it as a "qualitative in vitro diagnostic test system".
Function: The device is designed to detect and differentiate specific viral nucleic acids (Influenza A, Influenza B, and RSV) in human specimens (nasopharyngeal swabs) to aid in diagnosis. This is a core function of an in vitro diagnostic device.
Specimen Type: It uses biological specimens (nasopharyngeal swabs) collected from the human body.
Purpose: The results are intended to be used in conjunction with clinical and laboratory findings to aid in the differential diagnosis of respiratory viral infections.

All of these points align with the definition of an In Vitro Diagnostic device.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The ARIES® Flu A/B & RSV Assay is a polymerase chain reaction (PCR) based qualitative in vitro diagnostic test for the direct detection and differentiation of influenza A virus, influenza B virus, and respiratory syncytial virus (RSV) nucleic acid in nasopharyngeal swab (NPS) specimens from patients with signs and symptoms of respiratory tract infection in conjunction with clinical and laboratory findings. The test is intended for use as an aid in the differential diagnosis of Influenza A, Influenza B, and RSV in humans and is not intended to detect Influenza C.

Negative results do not preclude influenza virus or RSV infection and should not be used as the sole basis for diagnosis, treatment or other management decisions. Conversely, positive results do not rule-out bacterial infection or co-infection with other viruses. The agent detected may not be the definite cause of disease. The use of additional laboratory testing (e.g. bacterial culture, immunofluorescence, X-ray findings) and clinical presentation must be taken into consideration in order to obtain the final diagnosis of respiratory viral infection.

Performance characteristics for influenza A were established during the 2015-2016 influenza seasons when influenza A/H3N2 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 departments for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.

The ARIES® Flu A/B & RSV Assay is indicated for use with the ARIES® Systems.

Product codes (comma separated list FDA assigned to the subject device)

OCC, OOI, OZE

Device Description

The ARIES® Flu A/B & RSV Assay is a polymerase chain reaction (PCR)-based qualitative in vitro diagnostic test system that will consist of an ARIES® System with its included software, an assay-specific cassette, and an assay-specific protocol file. The ARIES® Flu A/B & RSV Assay cassette is a disposable, single-use cassette containing nucleic acid purification reagents, internal sample process control (SPC), and an assay-specific master mix capable of performing the designated assay on one sample. The ARIES® Flu A/B & RSV Assay cassette directly detects and differentiates influenza A virus, influenza B virus, and respiratory syncytial virus (RSV) nucleic acid in nasopharyngeal swabs (NPS) specimens from patients with signs and symptoms of respiratory tract infection in conjunction with clinical and laboratory findings. Specifically, the ARIES® Flu A/B & RSV Assay cassette detects the matrix protein genes of influenza A and influenza B viruses, and the fusion gene of RSV and a RNA Sample Processing Control.

The specimen is lysed and nucleic acid is extracted using an ARIES® System. An extractable sample processing control (SPC) target is present in the ARIES® Flu A/B & RSV assay cassette and is processed with the specimen. The SPC controls for specimen lysis, for recovery of extracted nucleic acid, for inhibitory substances and for PCR reagent and instrument integrity. The Ct value of the SPC is designed to verify proper specimen lysis and nucleic acid extraction, to identify PCR inhibition, if any, and verify proper function of the extraction system and real-time instrument. The Tm value of the SPC is used as a reference for determining the target Tm.

The extracted nucleic acid and SPC are transferred via magnetic beads through the cassette to the ARIES® Flu A/B & RSV Assay lyophilized PCR reagents in the PCR tube that contain primer pairs specific to influenza A, influenza B, RSV, and the SPC sequence. The specific primer pairs are labeled with distinct fluorophore labels. PCR amplification is performed and assay fluorescence is monitored on an ARIES® System. Incorporation of the quencherlabeled nucleotide causes a decrease in assay fluorescence. Following amplification, the reaction is slowly heated and fluorescence is monitored. The strands of the amplification products will separate at a specific melting temperature (Tm) that is determined by an increase in fluorescence as the strands are separated. The instrument fluorescence output is analyzed and test results are determined using the ARIES® Flu A/B & RSV Assay protocol file. A printed results report is generated.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

nasopharyngeal swab (NPS) specimens

Indicated Patient Age Range

Not Found

Intended User / Care Setting

Not Found

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

A total of 2504 nasopharyngeal swab specimens from subjects suspected of having respiratory tract infection were collected in the prospective study. Twenty-five (25) specimens were excluded based on inclusion criteria violation or protocol deviation leaving a total of 2479 eligible unique specimens for subsequent data analysis. Of these, 1017 were collected during the 2014/2015 Flu season while the remaining 1462 specimens were enrolled during the 2015/2016 Flu season.

All 2479 eligible clinical specimens were tested by an FDA-cleared molecular comparator and the ARIES® Flu A/B & RSV Assav. The comparator assay was performed in accordance with manufacturer's instructions at a centralized testing facility. Clinical runs and re-runs using ARIES® Flu A/B & RSV Assay were carried out by trained operators at the testing sites on clinical specimens that were either stored frozen at -65°C to -80°C (N=1316; 53.1%) or kept refrigerated at 2°C to 8°C (N=1163; 46.9%) prior to testing.

Out of the 2479 clinical specimens included in the prospective analysis, 2458 (2458/2479; 99.2%) generated valid results with ARIES® Flu A/B & RSV Assay during initial testing. There were 21 specimens (21/2479, 0.8%) that were re-tested with ARIES® Flu A/B & RSV Assay because they yielded initial invalid results. All 21 specimens in question generated valid ARIES results upon repeat testing.

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

Clinical Performance:
The clinical performance of the ARIES® Flu A/B & RSV Assay was evaluated using leftover, de-identified, nasopharyngeal swab (NPS) specimens prospectively collected from pediatric or adult patients suspected of having respiratory tract infection during the 2014/2015 and 2015/2016 flu seasons.

Sample size: N=2479 eligible unique specimens.

Results:
Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) for Influenza A, Influenza B and RSV are summarized in the tables below.

Influenza A (N=2479):

Positive Percent Agreement: 95.8% (95% CI: 93.0% - 97.8%)
Negative Percent Agreement: 98.4% (95% CI: 97.8% - 98.9%)

Influenza B (N=2479):

Positive Percent Agreement: 93.8% (95% CI: 82.8% - 98.7%)
Negative Percent Agreement: 99.4% (95% CI: 99.0% - 99.7%)

RSV (N=2479):

Positive Percent Agreement: 97.1% (95% CI: 94.4% - 98.7%)
Negative Percent Agreement: 98.4% (95% CI: 97.7% - 98.9%)

Supplemented Influenza B Study:
Due to low prevalence, 40 banked Influenza B positive specimens and 40 negative specimens were studied separately.

ARIES® Flu A/B & RSV Assay accurately detected all 40 Influenza B positive specimens tested (100%; 95% confidence interval, 91.2% - 100%).

Conclusion: The diagnostic accuracy of the ARIES® Flu A/B & RSV Assay is acceptable for the detection of Influenza A, Influenza B and RSV in nasopharyngeal swabs collected from patients suspected of respiratory tract infection (RTI).

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

Not Found. (Positive Percent Agreement and Negative Percent Agreement provided as key metrics)

Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.

K120413

Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).

Not Found

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.

Summary

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

August 2, 2016

Luminex Corporation Wendy Ricker Senior Regulatory Affairs Associate 12212 Technology Blvd. Austin TX 78727

Re: K161220 Trade/Device Name: ARIES® Flu A/B & RSV Assay Regulation Number: 21 CFR 866.3980 Regulation Name: Respiratory Viral Panel Multiplex Nucleic Acid Assay Regulatory Class: II Product Code: OCC, OOI, OZE Dated: April 29, 2016 Received: April 29, 2016

Dear Ms. Ricker:

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

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

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (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.

1

If you desire specific advice for your device on our labeling regulations (21 CFR Parts 801 and 809), please contact the Division of Industry and Consumer Education at its toll-free number (800) 638 2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/Resourcesfor You/Industry/default.htm. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to

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

You may obtain other general information on your responsibilities under the Act from the Division of Industry and Consumer Education at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.

Sincerely yours,

Tamara V. Feldblyum -S for

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

Enclosure

2

Indications for Use

510(k) Number (if known) K161220

Device Name

ARIES® Flu A/B & RSV Assay

Indications for Use (Describe)

The ARIES® Flu A/B & RSV Assay is a polymerase chain reaction (PCR) based qualitative in vitro diagnostic test for the direct detection and differentiation of influenza A virus, influenza B virus, and respiratory syncytial virus (RSV) nucleic acid in nasopharyngeal swab (NPS) specimens from patients with signs and symptoms of respiratory tract infection in conjunction with clinical and laboratory findings. The test is intended for use as an aid in the differential diagnosis of Influenza A, Influenza B, and RSV in humans and is not intended to detect Influenza C.

Neqative results do not preclude influenza virus or RSV infection and should not be used as the sole basis for diagnosis, treatment or other management decisions. Conversely, positive results do not rule-out bacterial infection or co-infection with other viruses. The agent detected may not be the definite cause of disease. The use of additional laboratory testing (e.g. bacterial culture, immunofluorescence, X-ray findings) and clinical presentation must be taken into consideration in order to obtain the final diagnosis of respiratory viral infection.

Performance characteristics for influenza A were established during the 2015-2016 influenza seasons when influenza A/H3N2 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 departments for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.

The ARIES® Flu A/B & RSV Assay is indicated for use with the ARIES® Systems.

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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11.0 Executive Summary 510(k)

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

A. 510(k) Number:

K161220

B. Purpose for Submission:

Traditional 510(k), New Device

C. Measurand:

Targets RNA sequences for the conserved regions of the matrix protein genes of influenza A and influenza B viruses, and the fusion gene of Respiratory Syncytial Virus (RSV).

D. Type of Test:

Qualitative Real Time Polymerase Chain Reaction (PCR)

E. Applicant:

Luminex Corporation

F. Proprietary and Established Names:

ARIES® Flu A/B & RSV Assay

G. Regulatory Information:

| Product

Code	Classification	Regulation Section
OCC	II	21 CFR 866.3980—Respiratory viral panel
multiplex nucleic acid assay	Microbiology
(83)

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H. Intended Use:

1. Intended use(s):

The ARIES® Flu A/B & RSV Assay is a polymerase chain reaction (PCR) based qualitative in vitro diagnostic test for the direct detection and differentiation of influenza A virus, influenza B virus, and respiratory syncytial virus (RSV) nucleic acid in nasopharyngeal swabs (NPS) specimens from patients with signs and symptoms of respiratory tract infection in conjunction with clinical and laboratory findings. The test is intended for use as an aid in the differential diagnosis of Influenza B, and RSV in humans and is not intended to detect Influenza C.

Negative results do not preclude influenza virus or RSV infection and should not be used as the sole basis for diagnosis, treatment or other management decisions. Conversely, positive results do not rule-out bacterial infection with other viruses.

The agent detected may not be the definite cause of disease. The use of additional laboratory testing (e.g. bacterial culture, immunofluorescence, X-ray findings) and clinical presentation must be taken into consideration in order to obtain the final diagnosis of respiratory viral infection.

Performance characteristics for influenza A were established during the 2014-2015 and the 2015-2016 influenza seasons when influenza A/H3N2 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 departments for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.

The ARIES® Flu A/B & RSV Assay is indicated for use with the ARIES® Systems.

1. Indication(s) for use:
  Same as intended use.
1. Special conditions for use statement(s):
  For prescription use only.

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4. Special instrument requirements:

For use with the ARIES® System or ARIES® M1 System.

l. Device Description:

The ARIES® Flu A/B & RSV Assay is a polymerase chain reaction (PCR)-based qualitative in vitro diagnostic test system that will consist of an ARIES® System with its included software, an assay-specific cassette, and an assay-specific protocol file. The ARIES® Flu A/B & RSV Assay cassette is a disposable, single-use cassette containing nucleic acid purification reagents, internal sample process control (SPC), and an assay-specific master mix capable of performing the designated assay on one sample. The ARIES® Flu A/B & RSV Assay cassette directly detects and differentiates influenza A virus, influenza B virus, and respiratory syncytial virus (RSV) nucleic acid in nasopharyngeal swabs (NPS) specimens from patients with signs and symptoms of respiratory tract infection in conjunction with clinical and laboratory findings. Specifically, the ARIES® Flu A/B & RSV Assay cassette detects the matrix protein genes of influenza A and influenza B viruses, and the fusion gene of RSV and a RNA Sample Processing Control.

The specimen is lysed and nucleic acid is extracted using an ARIES® System. An extractable sample processing control (SPC) target is present in the ARIES® Flu A/B & RSV assay cassette and is processed with the specimen. The SPC controls for specimen lysis, for recovery of extracted nucleic acid, for inhibitory substances and for PCR reagent and instrument integrity. The Ct value of the SPC is designed to verify proper specimen lysis and nucleic acid extraction, to identify PCR inhibition, if any, and verify proper function of the extraction system and real-time instrument. The Tm value of the SPC is used as a reference for determining the target Tm.

The extracted nucleic acid and SPC are transferred via magnetic beads through the cassette to the ARIES® Flu A/B & RSV Assay lyophilized PCR reagents in the PCR tube that contain primer pairs specific to influenza A, influenza B, RSV, and the SPC sequence. The specific primer pairs are labeled with distinct fluorophore labels. PCR amplification is performed and assay fluorescence is monitored on an ARIES® System. Incorporation of the quencherlabeled nucleotide causes a decrease in assay fluorescence. Following amplification, the reaction is slowly heated and fluorescence is monitored. The strands of the amplification products will separate at a specific melting temperature (Tm) that is determined by an increase in fluorescence as the strands are separated. The instrument fluorescence output is analyzed and test results are determined using the ARIES® Flu A/B & RSV Assay protocol file. A printed results report is generated.

Substantial Equivalence Information: J.

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1. Predicate device name(s):

Simplexa Flu A/B & RSV Direct, Simplexa Flu A/B & RSV Positive Control Pack (manufactured by Focus Diagnostics, Inc.)

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

K120413

3. Comparison with predicate:

The following table compares the ARIES® Flu A/B & RSV Assay to Focus' Simplexa Flu A/B & RSV Direct, Simplexa Flu A/B & RSV Positive Control Pack (K120413). Table 11.1 shows similarities between the new device and the predicate, while Table 11.2 shows the differences.

Similarities
Attribute	New Device	Predicate Device (K120413)
Intended Use	The ARIES® Flu A/B & RSV Assay is a
polymerase chain reaction (PCR) based
qualitative in vitro diagnostic test for the
direct detection and differentiation of
influenza A virus, influenza B virus, and
respiratory syncytial virus (RSV) nucleic
acid in nasopharyngeal swabs (NPS)
specimens from patients with signs and
symptoms of respiratory tract infection
in conjunction with clinical and
laboratory findings. The test is intended
for use as an aid in the differential
diagnosis of Influenza A, Influenza B, and
RSV in humans and is not intended to
detect Influenza C.
Negative results do not preclude
influenza virus or RSV infection and
should not be used as the sole basis for
diagnosis, treatment or other
management decisions. Conversely,
positive results do not rule-out bacterial
infection or co-infection with other
viruses.
The agent detected may not be the
definite cause of disease. The use of
additional laboratory testing (e.g.
bacterial culture immunofluorescence	The Focus Diagnostics Simplexa™ Flu A/B
& RSV Direct assay is intended for use on
the 3M Integrated Cycler instrument for
the in vitro qualitative detection and
differentiation of influenza A virus,
influenza B virus, and respiratory
syncytial virus (RSV) RNA in
nasopharyngeal swabs (NPS) from human
patients with signs and symptoms of
respiratory tract infection in conjunction
with clinical and epidemiological risk
factors. This test is intended for use as an
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 virus or RSV infection and
should not be used as the sole basis for
treatment or other patient management
decisions.
Performance characteristics for influenza
A were established with clinical
specimens collected during the
2010/2011 influenza season when 2009

Table 11.1: Similarities between New Device and Predicate

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Assay Targets
	X-ray findings) and clinical presentation must be taken into consideration in order to obtain the final diagnosis of respiratory viral infection.
Performance characteristics for influenza A were established during the 2014-2015 and the 2015-2016 influenza seasons when influenza A/H3N2 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 departments for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.
The ARIES® Flu A/B & RSV Assay is indicated for use with the ARIES® Systems.	H1N1 influenza and H3N2 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.
Assay Targets	RNA from Influenza A, Influenza B, and RSV	RNA from Influenza A, Influenza B, and RSV
Influenza A Target	Matrix gene	Matrix gene
Influenza B Target	Matrix gene	Matrix gene
Sample type	Nasopharyngeal swabs (NPS)	Nasopharyngeal swabs (NPS)
Assay format	Real-time PCR	Real-time PCR
Assay results	Qualitative	Qualitative

Table 11.2: Differences between New Device and Predicate

Differences
Attribute	New Device	Predicate Device (K120413)
Extraction Method	Automated by an ARIES® System	No extraction
RSV Target	Fusion gene	M gene
Controls	Sample processing control	RNA Internal Control, Positive Controls
Sold Separately
Instrument	ARIES® System or ARIES® M1 System	3M™ Integrated Cycler

K. Standards/Guidance Documents Referenced:

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1. Guidance for Industry and FDA Staff, Class II Special Controls Guidance Document: Respiratory Viral Panel Multiplex Nucleic Acid Assay, October 9, 2009
1. Guidance for Industry and FDA Staff Establishing the Performance Characteristics of In Vitro Diagnostic Devices for the Detection or Detection and Differentiation of Influenza Viruses, July 15, 2011
1. Guidance for Industry and FDA Staff, In Vitro Diagnostic Devices to Detect Influenza A Viruses: Labeling and Regulatory Path, May 1, 2007.

L. Test Principle:

The ARIES® Flu A/B & RSV Assay chemistry is based on an expanded genetic alphabet technology, consisting of synthetic DNA base pair 2'-deoxy-5-methyl-isocytidine (iC): 2'deoxyisoguanosine (iG). The isobases (iC and iG) pair specifically with each other and not with natural nucleotides. In addition, isobases are efficiently incorporated during PCR. During PCR amplification, a quencher-modified iGTP is incorporated by the polymerase opposite an iC and a fluorophore reporter attached to a PCR primer. If the target is present and is amplified, assay fluorescence decreases with every cycle as amplification product accumulates. The decrease in assay fluorescence is monitored in real time using an ARIES® System. Following PCR, the amplification products are thermally denatured and assay fluorescence is monitored. The strands of the amplification products are separated and assay fluorescence increases, thus enabling determination of the melting temperature (Tm) of the amplicon.

M. Performance Characteristics:

1. Analytical performance:

a. Precision/Reproducibility:

Precision

Within Laboratory Precision of the ARIES® Flu A/B & RSV Assay was evaluated by two operators performing testing with four ARIES "Systems using three lots of ARIES® Flu A/B & RSV Assay cassettes. Testing was performed on 12 nonconsecutive days and included a total of 675 replicates of a representative reproducibility panel. The reproducibility panel contained influenza A, influenza B, RSV-A, and RSV-B and representative viral cultures were diluted to three concentrations: moderate positive (10x LoD), low positive (3x LoD), and high negative (0.125x LoD for influenza A, and 0.25x LoD for influenza B, RSV-A and RSV-B). All dilutions were prepared in a simulated nasal matrix (SNM); negative samples consisted of only SNM. The overall

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invalid percentage for this study was 1.3% (9/675). The results of the reproducibility study are summarized in Table 11-3.

| Strain | Target Concentration | Expected Positivity | Positivity | 95%
Confidence
Interval |
|-------------------------------|----------------------|---------------------|-----------------|-------------------------------|
| Influenza A/Hong
Kong/8/68 | Moderate Positive | 100% | 100%
(48/48) | 93% - 100% |
| | Low Positive | Approximately 95% | 100%
(48/48) | 93% - 100% |
| | High Negative | 20% - 80% | 79%
(38/48) | 65% - 90% |
| Influenza
B/Florida/04/06 | Moderate Positive | 100% | 100%
(48/48) | 93% - 100% |
| | Low Positive | Approximately 95% | 100%
(48/48) | 93% - 100% |
| | High Negative | 20% – 80% | 81%
(39/48)a | 67% - 91% |
| RSV A2 | Moderate Positive | 100% | 100%
(48/48) | 93% - 100% |
| | Low Positive | Approximately 95% | 96%
(46/48) | 86% - 99% |
| | High Negative | 20% - 80% | 48%
(23/48) | 33% - 63% |
| RSV B WV/14617/85 | Moderate Positive | 100% | 100%
(48/48) | 93% - 100% |
| | Low Positive | Approximately 95% | 100%
(48/48) | 93% - 100% |
| | High Negative | 20% – 80% | 90%
(43/48)a | 77% - 97% |
| Flu A/B & RSV
Negative | Negative | 0% | 2.1%
(2/96)b | 0% - 7% |

Table 11-3: ARIES® Flu A/B & RSV Assav Within Laboratory Precision Determination Results
------------------------------------------------------------------------------------------	--	--

° RSV B WV/14617/45 and influenza B/Florida/04/06 High Negative samples generated positivity that expected positivity results of 20-80%.

' Two Flu A/B & RSV Negative replicates tested by different test dates, on different instruments, with different cassette lots generated late Ct false influenza B Positive results. Overal percentage of negative results for all three assay targets is 98%.

Reproducibility

Reproducibility of the ARIES® Flu A/B & RSV Assay was evaluated by testing one lot of ARIES® Flu A/B & RSV Assay Cassettes on two ARIES "Systems by two operators at each of three clinical laboratory sites on five non-consecutive days. A reproducibility panel was prepared containing a moderate positive (10x LoD), low positive (3x LoD), and high negative (0.2x LoD) independently for influenza A, influenza B, RSV-A, and RSV-B, as well as a negative sample. The reproducibility panels were created by an independent operator and blinded to the testing sites. Each panel member was

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tested in triplicate by each operator on each day of testing. The results of the reproducibility study are shown in Tables 11-4 and 11-5.

Table 11-4: ARIES c Flu A/B & RSV Assay Site-to-Site Reproducibility Results
		Site 1		Site 2		Site 3
		Positivity		Positivity		Positivity
	High Negative	27/30	90.0%	30/30	100.0%	26/30	86.7%
Influenza A	Low Positive	30/30	100.0%	30/30	100.0%	30/30	100.0%
	Moderate Positive	30/30	100.0%	30/30	100.0%	30/30	100.0%
	Negative	1/30	3.3%	0/30	0.0%	0/30	0.0%
	High Negative	9/30	30.0%	7/30	23.3%	15/30	50.0%
Influenza B	Low Positive	29/30	96.7%	30/30	100.0%	30/30	100.0%
	Moderate Positive	30/30	100.0%	30/30	100.0%	30/30	100.0%
	Negative	0/30	0.0%	0/30	0.0%	0/30	0.0%
	High Negative	39/60	65.0%	50/60	83.3%	43/60	71.7%
RSVc	Low Positive	60/60	100.0%	60/60	100.0%	60/60	100.0%
	Moderate Positive	60/60	100.0%	60/60	100.0%	59/60	98.3%
	Negative	0/30	0.0%	0/30	0.0%	0/30	0.0%

Table 11-4: ARIES® Flu A/B & RSV Assay Site-to-Site Reproducibility Results 30
--------------------------------------------------------------------------------	--	--	--	--	--

a An overall invalid rate of 0.9% (11/1260) was observed in the target replicates.

° The expected result for: a moderate positive, low positive target was approximately 95% positive, high negative was 20 – 80% positive, and negative was 0% positive.

^ RSV-A and RSV-B are not differentiated by the ARES Flu A/B & RSV Assay and, therefore, are combined and represented as RSV only.

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		Positivity		95% C.I. lower limit	upper limit
	High Negative	83/90	92.2%	84.6%	96.8%
Influenza A	Low Positive	90/90	100.0%	95.9%	100.0%
	Moderate Positive	90/90	100.0%	95.9%	100.0%
	Negative	1/90	1.1%	1.1%	6.0%
	High Negative	31/90	34.4%	24.7%	45.2%
Influenza B	Low Positive	89/90	98.9%	94.0%	100.0%
	Moderate Positive	90/90	100.0%	95.9%	100.0%
	Negative	0/90	0.0%	0.0%	4.0%
RSVc	High Negative	132/180	73.3%	66.2%	79.6%
	Low Positive	180/180	100.0%	95.9%	100.0%
	Moderate Positive	179/180	99.4%	96.9%	100.0%
	Negative	0/90	0.0%	0.0%	4.0%

Table 11-5: Reproducibility Panel Total Results 3.6

a An overall invalid rate of 0.9% (11/1260) was observed in the target replicates.

ٌ The expected result for: a moderate positive, low positive target was approximately 95% positive, high negative was 20 – 80% positive, and negative was 0% positive.

် SV-A and RSV-B are not differentiated by the ARIES Flu A/B & RSV Assay and, therefore, are combined and represented as RSV only.

b. Linearity/assay reportable range:

Not applicable. The ARIES® Flu A/B & RSV Assay is a qualitative assay.

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

Specimen Stability (Fresh vs. Frozen)

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Fresh vs. frozen specimen stability was determined with contrived fresh and frozen specimens stored at either 2-8°C (fresh) or -65 to -95°C (frozen) and tested with the ARIES® Flu A/B & RSV Assay. This was assessed by testing 60 replicates for a single target pathogen (influenza A, influenza B, or RSV) prepared at 3 test concentrations (30 low positive, 15 moderate positive, and 15 high positive) across 9 different time points extending out to 12 months. In addition to the 60 replicates for each target organism, 6 replicates of an influenza A/B and RSV negative specimen in simulated nasal matrix (SNM) were tested for each time point extending out to 12 months. Data up to 3 months was collected with all targets yielding the expected result. Overall, high positive specimens were 100% positive, moderate positive specimens were 100% positive, and low positive specimens were positive approximately 95% of the time for all three evaluated targets. Negative specimens were negative 100% of the time. Influenza A, influenza B, and RSV specimens are stable for up to 7 days when stored at 2-8°C and 3 months when stored at -65 to -95°C.

Shelf-Life Stability

Real time stability study was performed to evaluate the shelf life of ARIES® Flu A/B & RSV Assay cassettes. Stability was established by testing 6 replicates of ARIES® Flu A/B & RSV Assay Extractable Control, 100X LOD blend (PN 10188) and negative targets (UTM) on three different lots of ARIES® Flu A/B & RSV Assay cassettes stored at 2 different temperatures: 4°C (2 = 8°C) and room temperature (15 = 30°C) at 10 different time points extending out to 19 months. Acceptance criteria for stability at each time point and temperature was established as 100% positivity for all influenza A/B and RSV replicates, and 100% negativity for all negative replicates. Data collected up to 5 months gave expected results indicating stability of the of ARIES® Flu A/B & RSV Assay cassettes up to 5 months.

Open Box Stability

An Open Box Stability study was performed in order to evaluate performance of ARIES® Flu A/B & RSV Assay Cassettes after they were removed from their individual pouches. Cassettes were removed from their pouches and placed on a laboratory bench where they were exposed to ambient temperatures, humidity, and light for up to ten (10) hours. Data was collected for contrived influenza A, influenza B, and RSV positive and influenza A, influenza B, and RSV negative (Copan Universal Transport Media) samples at five time points throughout a ten (10) hour duration. Three lots of cassettes were used to assess Open Box Stability. At the end of 10 hours, all three lots of cassettes produced expected results showing that ARIES® Flu A/B and RSV Assay Cassettes are stable in ambient laboratory conditions for up to ten (10) hours after they have been removed from the storage pouch.

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Controls:

Process Control

Each ARIES® assay cassette contains a Sample Process Control (SPC), which is processed with the sample and analyzed during the amplification reaction. The SPC verifies sample lysis, nucleic acid extraction, and proper reagent, cassette, ARIES® System, and assay protocol performance. The SPC has a known melting temperature (Tm) range and Ct range. Each time an assay is run, the system measures the temperature and fluorescence intensity of the SPC control to ensure the thermal and optical subsystems have remained in calibration.

External Controls

External controls should be tested according to guidelines or requirements of local, provincial and/or federal regulations or accreditation organizations. For example, reference influenza A, influenza B & RSV strains or well characterized influenza A, influenza B, and RSV clinical isolates may be used as positive controls. The ARIES® Flu A/B & RSV Assay Cassette Kit does not include external positive and negative controls.

d. Detection Limit:

Limit of Detection (LoD) was established for the ARIES® Flu A/B & RSV Assay using three influenza A, two influenza B, and two respiratory syncytial viral strains (1 RSV-A, and 1 RSV-B) diluted in a simulated nasal matrix (SNM) containing negative pooled human clinical matrix and Universal Transport media (UTM). The LoD for each viral strain was determined as the lowest concentration (TCIDso/mL) that had a positivity rate of ≥ 95%. Serial dilutions of each quantified viral strain in SNM were initially tested in a range finding study where the preliminary LoD TCIDso/mL concentrations were determined. The preliminary LoD concentrations were then confirmed by testing twenty (20) replicates of each strain. The final LoD concentrations for the seven viral strains are shown in Table 11-6.

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| Assay Target | Strain | Concentration
(TCID50/mL or CEID50/mL) | Positivity | 95% Confidence
Interval (Lower Limit /
Upper Limit) |
|--------------|---------------------------------|-------------------------------------------|------------|-----------------------------------------------------------|
| Influenza A | PR/8/34 | 1 x 10 $$ | 100% | 83.2% - 100% |
| | Hong Kong/8/68 | 1 x 10 $$ | 100% | 83.2% - 100% |
| | Mexico/4108/2009
(H1N1)pdm09 | 1 x 10 $$ | 95% | 75.1% - 99.9% |
| Influenza B | Florida/04/06 | 1 x 10 $$ | 100% | 83.2% - 100% |
| | Malaysia/2506/04 | 1 x 10 $$ | 100% | 83.2% - 100% |
| RSV | A2 (VR-1540) | 1 x 10 $$ | 95% | 75.1% - 99.9% |
| | WV/14617/85 (VR-1400) | 1 x 10 $$ | 100% | 83.2% - 100% |

Table 11-6: Limit of Detection of the ARIES® Flu A/B & RSV Assay

e. Analytical Reactivity (Inclusivity)

The analytical reactivity/inclusivity of the ARIES® Flu A/B and RSV Assay was evaluated against twenty-four influenza A strains, seven influenza B strains, and three respiratory syncytial virus (RSV) strains, which differ from those strains included in the Limit of Detection (LoD) study. Each strain was diluted in simulated nasal matrix (SNM) to a concentration near the LoD (1x10+ TCID50/mL or CEID50/mL) and tested in triplicate; SNM consists of a 1:1 mixture of Universal Transport Medium (UTM) and 100% negative clinical specimen pool. For five strains, additional testing at 1x10 TCID50/mL or CEID50/mL was required to achieve 100% positivity. For two strains, additional testing at 1x103 TCID50/mL or CEID50/mL was required to achieve 100% positivity. The study results are shown in Table 11-7.

Table 11-7: ARIES® Flu A/B & RSV Assay Analytical Reactivity/Inclusivity Results

| Inclusivity Strains | 100% Positivity
Concentration | Result |
|--------------------------------------------|----------------------------------|----------------------|
| Influenza A /Perth/16/2009 (H3N2)-like | $1x10^1$ | Influenza A Detected |
| Influenza A/Brisbane/10/07 H3 | $1x10^1$ | Influenza A Detected |
| Influenza A/Brisbane/59/07 H1 | $1x10^1$ | Influenza A Detected |
| Influenza A/Port Chalmers/1/73 H3N2 | $1x10^1$ | Influenza A Detected |
| Influenza A/Solomon Island/03/06 H1 | $1x10^1$ | Influenza A Detected |
| Influenza A/Swine H1N1/Iowa/15/1930 | $1x10^1$ | Influenza A Detected |
| Influenza A/Taiwan/42/06 H1N1 | $1x10^1$ | Influenza A Detected |
| Influenza A/Wisconsin/67/05 H3 | $1x10^1$ | Influenza A Detected |
| Influenza A/California/7/2009-like (pH1N1) | $1x10^1$ | Influenza A Detected |

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Influenza A/Hong Kong/33982/2009 H9N2 x PR8-	1x101	Influenza A Detected
Influenza A/Indiana/08/2011 (H3N2)v	1x101	Influenza A Detected
Influenza A/Texas/50/2012 H3N2	1x101	Influenza A Detected
Influenza A/WS/33 H1N1	1x101	Influenza A Detected
Influenza A/New Caledonia/20/99 H1N1	1x102	Influenza A Detected
Influenza A/Swine H1N1/USA/1976/1931	1x102	Influenza A Detected
Influenza A/California/07/2009 NYMC x-179A	1x102	Influenza A Detected
Influenza A/Victoria/361/2011-like (H3N2)	1x102	Influenza A Detected
Influenza A/Minnesota/11/2010 (H3N2)v	1x103	Influenza A Detected
Influenza A/Ohio/02/2012 (H3N2)	1x103	Influenza A Detected
A/Anhui/01/2005 (H5N1)a	1x101	Influenza A Detected
A/Anhui/1/2013 (H7N9)a	1x101	Influenza A Detected
A/Egypt/321/2007 (H5N1)a	1x101	Influenza A Detected
A/Shanghai/1/2013 (H7N9)a	1x101	Influenza A Detected
A/Vietnam/1194/2004 (H5N1)a	1x101	Influenza A Detected
Influenza B/Massachusetts/2/2012-like	1x101	Influenza B Detected
Influenza B/Wisconsin/1/2010-like	1x101	Influenza B Detected
Influenza B/Florida/02/2006 (Victoria)	1x101	Influenza B Detected
Influenza B/Lee/40	1x101	Influenza B Detected
Influenza B/Panama/45/90 (Yamagata)	1x101	Influenza B Detected
Influenza B/Brisbane/60/2008	1x101	Influenza B Detected
Influenza B/Florida/07/04 (Yamagata)	1x102	Influenza B Detected
RSV A/Long	1x101	RSV Detected
RSV B/9320	1x101	RSV Detected
RSV B/Wash/18537/62	1x101	RSV Detected

a BPL-inactivated viral culture fluid provided from IRR with no unit/titer information. Dilution factors based on prior preliminary LoD testing of these strains with the ARIES® Flu A/B & RSV assay to approximate limit of detection.

Analytical specificity: f.

Interfering Substances:

The potential inhibitory effect of non-microbial substances expected to be found in nasopharyngeal swab specimens was evaluated by the testing with the ARIES® FLU A/B & RSV Assay. Three (3) replicates each of influenza A, influenza B, RSV-A, and RSV-B were tested at concentrations near the assay LoD with a clinically relevant

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concentration of each potentially interfering substance spiked into the contrived sample; additionally, negative Copan Universal Transport Medium (UTM) was spiked with the same concentration of each substance and tested for assay interference.

The results of the study demonstrate that all influenza A, influenza B, and RSV samples were 100% positive in the presence of a non-microbial substance at concentrations shown below; all negative samples containing only the non-microbial substance were 100% negative, with the exception of FluMist®.

Positive influenza results obtained in a patient who received FluMist prior to sample collection may be due to detection of the live attenuated influenza vaccine virus and may mask a true positive result caused by an influenza infection; additionally, FluMist may interfere with RSV detection due to high concentration of vaccine virus nucleic acid, causing a possible RSV false negative result. This statement has been added to the Analytical Performance section of the ARIES® FLU A/B & RSV Product Insert.

Interfering Substance	Test Concentration
Benzocaine	2.5% w/v
Budesonide	25 mg/mL
Dexamethasone	3 mg/mL
FluMist°	0.5% v/v
Flunisolide	55 mg/mL
Menthol	1.7 mg/mL
Mometasone	2.5 mg/mL
Phenylephrine	0.5% w/v
Afrin° (Oxymetazoline)	15% v/v
Tobramycin	4 µG/mL
Mupirocin	6.6 mg/mL
Beconase AQ° (Beclomethasone)	5% v/v
Flonase° (Fluticasone)	5% v/v
Zanamivir	3.3 mg/mL
Tamiflu°	1 μΜ
Triamcinolone	5.5 mg/mL
Sodium chloride	0.65% v/v
Human Whole Blood	2% v/v
Mucin Protein	60 μG/mL
ZICAM° (Galphimia glauca, Histaminum hydrochloricum, Luffa operculata, Sulfur)	5% v/v

Table 11-8: Interfering Substances Tested

Cross-Reactivity:

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The analytical specificity for the ARIES® Flu A/B & RSV Assay was evaluated by testing the potential cross-reactivity of 32 microorganisms listed in Table 11-9. The microorganisms tested consisted of 14 viral and 18 bacterial strains representing common respiratory pathogens, or those potentially encountered in the human nasopharynx region. The potential cross reactive organisms were spiked into simulated nasal matrix (SNM) that was negative for influenza A, influenza B, and RSV and tested with the ARIES® Flu A/B & RSV Assay in triplicate. Bacterial organisms were tested at concentrations ≥10° CFU/mL and viral organisms at ≥10° TCID50/mL, or the highest available concentration for both types of potential cross-reactive microorganism. All replicates of 31 microorganisms that were evaluated yielded negative results following initial testing. One replicate of three tested for Parainfluenza type 1 generated a late Ct indicating a weak influenza B positive result (Ct = 40) during the initial testing. An additional five replicates of the same Parainfluenza type 1 strain was tested with all 5 replicates yielding negative target results. Parainfluenza type 1 was considered to be non-reactive with the ARIES® Flu A/B & RSV Assay after the repeat testing.

Microorganism
Adenovirus type 1	Haemophilus influenzae	Parainfluenza type 1
Adenovirus 7a	Mycoplasma pneumonia M129	Parainfluenza type 2
Bordetella pertussis (A639)	Mycobacterium tuberculosis	Parainfluenza type 3
Chlamydia pneumoniae	Lactobacillus plantarum (17-5)	Pseudomonas aeruginosa
Coronavirus 229E	Legionella longbeachae	Rhinovirus type 1A
Coronavirus OC43	Measles	Staphylococcus aureus (COL)
Corynebacterium diphtheriae	Metapneumovirus	Staphylococcus epidermidis
Cytomegalovirus (CMV)	Moraxella catarrhalis Ne 11	Streptococcus pneumoniae
Enterovirus 71	Mumps	Streptococcus pyogenes
Epstein Barr virus	Neisseria elongata	Streptococcus salivarius
Escherichia coli O157	Neisseria meningitidis

Table 11-9: Microorganism Information

Microbial Interference:

Microbial interference for the ARIES® Flu A/B & RSV Assay was assessed with the 32 potential cross reactive microorganisms evaluated in the Cross-Reactivity Study, and identified in Table 11-9 above. Bacteria were tested at ≥10° CFU/mL or the highest available concentration, and viruses were tested at ≥10° TCID50/mL or the highest available concentration. The potential interfering organisms were spiked into simulated nasal matrix (SNM) containing either representative strains of influenza A, influenza B, RSV A, or RSV B near the LoD concentration. All target strain + cross reacting organism samples were tested in triplicate (n=3) on the ARIES® System.

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Influenza A was correctly detected in all replicates. Influenza B was correctly detected in the presence of 29 cross reacting organisms, with 3 cross reacting organisms (Parainfluenza type 2, Coronavirus OC43, Corynebacterium diphtheriae) requiring additional testing of 5 replicates. For all repeated cross reacting organism + influenza B combinations, all 5 replicates correctly detected influenza B. Parainfluenza type 2, Coronavirus OC43, and Corynebacterium diphtheriae are considered non-reactive since 7 of 8 tested replicates generated correct results (initial testing of 3 replicates resulted in 2 out of 3 positive, and the repeat testing of 5 replicates resulted in 5 out of 5 positive results, denoted as 7 of 8).

RSV A was correctly detected in the presence of 28 cross reacting organisms, with 4 cross reacting organisms (Bordetella pertussis, Cytomegalovirus, Parainfluenza type 2, Epstein Barr virus) requiring additional testing of 5 replicates. For all repeated cross reacting organism + RSV A combinations, all 5 replicates correctly detected RSV A. Bordetella pertussis, Cytomegalovirus, Parainfluenza type 2, and Epstein Barr virus are considered non-reactive since 7 of 8 tested replicates generated correct results. RSV B was correctly detected in the presence of 28 cross reacting organisms, with 4 cross reacting organisms (Parainfluenza type 2, Rhinovirus type 1A, Enterovirus 71, Neisseria meningtidis) requiring additional testing of 5 replicates. For all repeated cross reacting organism + RSV B combinations excluding Enterovirus 71, all 5 replicates correctly detected RSV A. Parainfluenza type 2, Rhinovirus type 1A, and Neisseria meninqtidis are considered non-reactive since 7 of 8 tested replicates generated correct results. RSV B had an overall positivity of 6/8 when tested in the presence of Enterovirus 71. Additional testing of RSV B + Enterovirus 71 produced a final positivity of 3/3 using freshly prepared target material. Based on the final test result of 100% positivity (3/3) for RSV B + Enterovirus 71, Enterovirus 71 is considered non-reactive.

Carry-Over/Cross-Contamination:

Carry-over and Cross Contamination for the ARIES® Flu A/B and RSV Assay were evaluated by testing thirty (30) high concentration influenza A positive samples in series alternating with thirty (30) influenza A negative samples (UTM). The high positive samples were run adjacent to negative samples across ten (10) consecutive runs on one ARIES® System. No carry-over or cross contamination was observed, and the overall percent agreement was 100% for positive and negative samples.

Competitive Interference/Co-infection:

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A study was designed to evaluate the ability of the ARIES® Flu A/B and RSV assay to detect influenza A, influenza B, RSV A, and RSV B in the presence of a co-infection. Competitive interference can occur when one analyte is near the LoD and an additional analyte is present at high concentration. Analytes were tested at high (> 1x10* TCID50/mL) and low concentrations (1.5X LoD) using 3 replicates in various combinations, excluding RSV A + RSV B due to the assay's inability to distinguish between RSV subtypes. Low concentration analytes were detected for 3 of the 4 assay targets with 100% positivity in all high concentration combinations. RSV A in presence of high concentration influenza A required a 3X LoD dilution to achieve 100% positivity (Table 11-10).

Table 11-10: Co-infection Results Summary

Low Target Analyte	High Target Analyte	Condition	Percent
A/Hong Kong/8/68	B/Florida/04/06	Influenza A + Influenza B	100%
A/Hong Kong/8/68	RSV A2	Influenza A + RSV A	100%
A/Hong Kong/8/68	RSV B WV/14617/85	Influenza A + RSV B	100%
B/Florida/04/06	A/Hong Kong/8/68	Influenza B + Influenza A	100%
B/Florida/04/06	RSV A2	Influenza B + RSV A	100%
B/Florida/04/06	RSV B WV/14617/85	Influenza B + RSV B	100%
RSV A2	B/Florida/04/06	RSV A + Influenza B	100%
RSV A2 (3X LoD)	A/Hong Kong/8/68	RSV A + Influenza A	100%
RSV B WV/14617/85	A/Hong Kong/8/68	RSV B + Influenza A	100%
RSV B WV/14617/85	B/Florida/04/06	RSV B + Influenza B	100%

g. Assay cut-off:
Not applicable.
1. Comparison Studies:
- Method comparison with predicate device: a.

Not applicable.

b. Matrix comparison:
A matrix equivalency study was performed to establish equivalency of a simulated matrix (50:50 dilution of pooled negative clinical specimens in Copan Universal Transport Media (UTM)) with a natural matrix (pooled negative clinical specimens)

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when used in the ARIES® Flu A/B and RSV Assay. Study samples were prepared as a 4 point, 5-fold dilution series of representative Influenza A, Influenza B, and Respiratory Syncytial Virus (RSV) viral strain targets in both the simulated matrix and the pooled negative clinical matrix with the lowest dilution point targeting 3-5x the determined limit of detection. Each concentration level for each viral target strain was tested in 6 replicates for each matrix type for a total of 48 tests per viral strain. Additionally, 6 replicates of the simulated matrix and 6 replicates of the pooled negative clinical matrix were tested without the introduction of viral strain material. Equivalency was demonstrated based on the compared mean target Ct and SPC Ct values falling within 1 log (3.3 cycles) across the matrix types, and the accuracy of the clinical call in the simulated matrix compared with that of the natural matrix. The results of the study are shown in Tables 11-11 to 11-14. These results demonstrate the analytical performance of the ARIES® Flu A/B and RSV Assay is equivalent between the simulated matrix and the natural matrix.

Matrix type	Dilution 1			Dilution 2			Dilution 3			Dilution 4
	Mean Ct ± SD (cycles)	Positivity	Delta Ct	Mean Ct ± SD (cycles)	Positivity	Delta Ct	Mean Ct ± SD (cycles)	Positivity	Delta Ct	Mean Ct ± SD (cycles)	Positivity	Delta Ct
Simulated matrix	26.2 ± 0.7	100%		28.4 ± 0.4	100%		32.1 ± 0.8	100%		33.8 ± 1.3	100%
Negative Clinical Specimen matrix	26.3 ± 1.1	100%	-0.1	29.4 ± 1.0	100%	-1.0	30.8 ± 0.4	100%	1.3	34.0 ± 1.0	83%a	-0.2

a Positivity is within the 95% Cl of expected results of 35.9% - 99.6%

Table 11-12: ARIES® Flu A/B and RSV Assay Matrix Equivalency Results for Flu B

	Influenza B/Florida/04/06
Matrix type	Dilution 1	Dilution 2	Dilution 3	Dilution 4
	Mean Ct ± SD (cycles)
Positivity
Delta Ct	Mean Ct ± SD (cycles)
Positivity
Delta Ct	Mean Ct ± SD (cycles)
Positivity
Delta Ct	Mean Ct ± SD (cycles)
Positivity
Delta Ct

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Simulated matrix	$28.0 \pm 0.5$	100%	0.1	$30.2 \pm 0.7$	100%	-1.2	$33.0 \pm 0.6$	100%	0.2	$35.4 \pm 0.8$	100%	1.0
Negative Clinical
Specimen matrix	$28.1 \pm 0.3$	100%		$31.4 \pm 0.7$	100%		$32.9 \pm 0.8$	100%		$34.4 \pm 0.9$	100%

Table 11-13: ARIES® Flu A/B and RSV Assay Matrix Equivalency Results for RSV

| Matrix

type	RSV A2
	Dilution 1	Dilution 2	Dilution 3	Dilution 4
	Mean Ct ±
SD (cycles)	Positivity	Delta Ct	Mean Ct ±
SD (cycles)	Positivity	Delta Ct	Mean Ct ±
SD (cycles)	Positivity	Delta Ct	Mean Ct ±
SD (cycles)	Positivity	Delta Ct
Simulated
matrix	30.9 ±
0.9	100%		32.5 ±
0.6	100%		34.9 ±
0.7	100%		35.3 ±
1.4	100%
Negative
Clinical
Specimen
matrix	30.8 ±
0.6	100%	0.2	33.1 ±
0.6	100%	-0.6	35.6 ±
1.0	100%	-0.7	35.6 ±
0.8	100%	-0.4

Table 11-14: ARIES® Flu A/B and RSV Assay Matrix Equivalency Results for SPC

Matrix type	SPC
	Mean Ct ± SD (cycles)	Positivity	Delta Ct
Simulated matrix	27.6 ± 0.7	100%
Negative Clinical
Specimen matrix	27.6 ± 0.8	100%	0.0

Swab comparison: C.

A Nasopharyngeal Swab Equivalency study was performed to evaluate the reproducibility of the ARIES® Flu A/B & RSV Assay using three representative nasopharyngeal swab (NPS) types (flocked, rayon, and polyester) and two different swab sizes (minitip and regular). All swab types and swab sizes were evaluated with influenza A, influenza B, and RSV viral cultures prepared as a three point five-fold dilution series, transferred via select swab type and size to universal transport media (UTM), and tested with the ARIES® Flu A/B & RSV assay. In addition to the viral

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cultures, a negative sample consisting of swab transferred UTM was also evaluated. Overall, all swab types and sizes performed as expected generating 100% positivity for all evaluated target concentrations and 100% negativity for negative replicates (Table 11-15).

| Assay Target Concentration
(TCID50/mL) | Swab Type | Regular Size |------------------------------------|-------- | | 1x104.5 | A/Hong Kong/8/68
LoD = 1x102.40 | 1x103.8 | | 1x103.1 | | 1x104.5 | | 1x103.8 | | 1x103.1 | | 1x104.5 | | 1x103.8 | | 1x103.1 | | 1x102.4 | B/Florida/04/06
LoD = 1x100.30 | 1x101.7 | | 1x101.0 | | 1x102.4 | | 1x101.7 | | 1x101.0 | | 1x101.5 | RSV A2
LoD = 1x10-0.57 | 1x100.8 | | 1x100.1 | | 1x101.5 | | 1x100.8 | | 1x100.1 | | 1x101.5 | | 1x100.8 | | 1x100.1 | Final
Positivity | Minitip Size Positivity |
-------------------------------|-----------|-------------------------|-------------------------|
| Flocked | 100% | 100% |
| | 100% | 100% |
| | 100% | 100% |
| Polyester | 100% | 100% |
| | 100% | 100% |
| | 100% | 100% |
| Rayon | 100% | 100% |
| | 100% | 100% |
| | 100% | 100% |
| Flocked | 100% | 100% |
| | 100% | 100% |
| | 100% | 100% |
| Polyester | 100% | 100% |
| | 100% | 100% |
| | 100% | 100% |
| Flocked | 100% | 100% |
| | 100% | 100% |
| | 100% | 100% |
| Polyester | 100% | 100% |
| | 100% | 100% |
| | 100% | 100% |
| Rayon | 100% | 100% |
| | 100% | 100% |
| | 100% | 100% |

Table 11-15: ARIES® Flu A/B & RSV Assay NPS Equivalency Results

Clinical Performance:

The clinical performance of the ARIES® Flu A/B & RSV Assay was evaluated using leftover, de-identified, nasopharyngeal swab (NPS) specimens prospectively collected from pediatric or adult patients suspected of having respiratory tract infection during the 2014/2015 and 2015/2016 flu seasons. In the first phase of the prospective study (2014/2015 flu season) specimens were collected at 3 clinical sites located in the United States and Canada from 18-January-2015 to 20-March-2015. Clinical specimens accrued during the second phase of the prospective study (2015/2016 flu season) were collected from 02-November-2015 to 29-February-2016 at 4 clinical sites located in the United States and Canada. The clinical specimen collection sites were chosen based on the

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types of patients usually referred to them, and the prevalence of respiratory pathogens in order to ensure a broad coverage of respiratory organisms, patient ages, and geographical regions.

A total of 2504 nasopharyngeal swab specimens from subjects suspected of having respiratory tract infection were collected in the prospective study. Twenty-five (25) specimens were excluded based on inclusion criteria violation or protocol deviation leaving a total of 2479 eligible unique specimens for subsequent data analysis. Of these, 1017 were collected during the 2014/2015 Flu season while the remaining 1462 specimens were enrolled during the 2015/2016 Flu season.

All 2479 eligible clinical specimens were tested by an FDA-cleared molecular comparator and the ARIES® Flu A/B & RSV Assav. The comparator assay was performed in accordance with manufacturer's instructions at a centralized testing facility. Clinical runs and re-runs using ARIES® Flu A/B & RSV Assay were carried out by trained operators at the testing sites on clinical specimens that were either stored frozen at -65°C to -80°C (N=1316; 53.1%) or kept refrigerated at 2°C to 8°C (N=1163; 46.9%) prior to testing.

Out of the 2479 clinical specimens included in the prospective analysis, 2458 (2458/2479; 99.2%) generated valid results with ARIES® Flu A/B & RSV Assay during initial testing. There were 21 specimens (21/2479, 0.8%) that were re-tested with ARIES® Flu A/B & RSV Assay because they yielded initial invalid results. All 21 specimens in question generated valid ARIES results upon repeat testing.

ARIES® Flu A/B & RSV Assay Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) for Influenza A, Influenza B and RSV are summarized in the tables below. Discordant specimens where the ARIES® Flu A/B & RSV Assay results were different from the comparator assay result were further assessed by bi-directional sequencing using analytically validated primers that targeted genomic regions distinct from the ARIES® Flu A/B & RSV Assay. Results from discordant testing analysis were not included in the calculation of Positive Percent and Negative Percent Agreement for each target. These results are, however, included as footnotes in the performance evaluation tables for each analyte.

ARIES Flu A/B & RSV Assay		Reference
	Positive	Negative	No Call	TOTAL
Positive	299	342	1	334
Negative	131	2131	1	2145
No Call	0	0	0	0
TOTAL	312	2165	2	2479
		95% CI

Table 11-16: ARIES Flu A/B & RSV Assay Performance for Influenza A (N=2479)

Confidential & Restricted

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Positive Percent Agreement	95.8%	93.0% - 97.8%
Negative Percent Agreement	98.4%	97.8% - 98.9%

් Seven (7) ARIES® Flu A/B & RSV Assay negative specimens that were positive by the comparator method (i.e. False Negative by bi-directional sequencing analytically validated primers that targeted genomic regions distinct from the ARES® Flu A/B & RSV Assav.

² Four (4) ARIES® Flu A/B & RSV Assay positive by the comparator method (i.e. False Positive) tested positive by bi-directional sequencing analytically validated primers that targeted genomic regions distinct from the ARIES® Flu A/B & RSV Assay.

Table 11-17: ARIES Flu A/B & RSV Assay Performance for Influenza B (N=2479)

ARIES Flu A/B & RSV Assay		Reference
	Positive	Negative	No Call	TOTAL
Positive	45	142	0	59
Negative	31	2417	0	2420
No Call	0	0	0	0
TOTAL	48	2431	0	2479
		95% CI
Positive Percent Agreement	93.8%	82.8% - 98.7%
Negative Percent Agreement	99.4%	99.0% - 99.7%

² Two (2) ARIES® Flu A/B & RSV Assay negative specimens that were positive by the comparator method (i.e. False Negative by bi-directional sequencing analytically validated primers that targeted genomic regions distinct from the ARIES® Flu A/B & RSV Assay.

1 Three (3) ARIES® Flu A/B & RSV Assay positive speciment that were negative by the comparator method (i.e. False Positive by bi-directional sequencing analytically validated primers that targeted genomic regions distinct from the ARIES® Flu A/B & RSV Assay.

ARIES Flu A/B & RSV Assay
	Positive	Negative	No Call	TOTAL
Positive	270	362	0	306
Negative	81	2165	0	2173
No Call	0	0	0	0
TOTAL	278	2201	0	2479
		95% CI
Positive Percent Agreement	97.1%	94.4% - 98.7%
Negative Percent
Agreement	98.4%	97.7% - 98.9%

Table 11-18: ARIES Flu A/B & RSV Assay Performance for RSV (N=2479)

2 ଠne (1) ARIES® Flu A/B & RSV Assay negative specitive by the comparator method (i.e. False Negative) tested negative by bidirectional sequencing analytically validated primers that targeted genomic regions distinct from the ARIES® Flu A/B & RSV Assay.

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4 Thirty-two (32) ARIES® Flu A/B & RSV Assay positive specimens that were negative by the comparator method (i.e. False Positive) tested positive by bi-directional sequencing analytically validated primers that targeted genomic regions distinct from the ARES® Flu A/B & RSV Assay.

Due to the low prevalence of Influenza B observed in the prospective study, the sample set was supplemented with 40 banked (pre-selected) Influenza B positive specimens collected at a single clinical laboratory (collection site) in Canada. The presence of the expected target (Influenza B) in each of the pre-selected specimens was confirmed by the comparator assay. In order to minimize bias, the pre-selected positive specimens were tested along with 40 unique negative clinical specimens in a randomized, blinded fashion at 4 external testing sites. Of the 80 Influenza B positive and negative specimens included in this study, 78 (78/80; 97.5%) generated valid results with ARIES® Flu A/B & RSV Assay on the first attempt. Invalid results were generated for 2 specimens (2/80; 2.5%) tested. Both specimens in question yielded valid ARIES results upon repeat testing.

The results from pre-selected specimens were analyzed separately from those of the prospective data set. ARIES® Flu A/B & RSV Assay accurately detected all 40 Influenza B positive specimens tested (100%; 95% confidence interval, 91.2% - 100%).

Based on the data collected from the clinical study, Positive Percent Agreement acceptance criteria of 90% with a lower bound 95% confidence interval of at least 80% were achieved for Influenza A, Influenza B and RSV. Negative Percent Agreement acceptance criteria of 90% with a lower bound 95% confidence interval of at least 90% were also achieved for all targets probed by the ARIES® Flu A/B & RSV Assay.

The results generated from this clinical study demonstrate that the diagnostic accuracy of the ARIES® Flu A/B & RSV Assay is acceptable for the detection of Influenza A, Influenza B and RSV in nasopharyngeal swabs collected from patients suspected of respiratory tract infection (RTI).

4. Expected values:

ARIES® Flu A/B & RSV Assay positive results (expected values) after allowable re-runs for each individual target are summarized for each of the enrollment periods, per age groups and per site in Tables 11-19 to 11-22 below.

Table 11-19: Expected Values (As determined by ARIES® Flu A/B & RSV) – Summary by Age Groups for the ARIES® Flu A/B & RSV Prospective Clinical Study (January 2015 – March 2015)

--------	---------------------	---------------------	----------------------	------------------------	-------------------------	----------------------

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(Analyte)	No.	Expected Value	No.	Expected Value	No.	Expected Value	No.	Expected Value	No.	Expected Value	No.	Expected Value
Influenza A	170	16.7%	8	4.2%	13	14.1%	25	19.1%	53	17.3%	71	23.9%
Influenza B	31	3.0%	1	0.5%	3	3.3%	11	8.4%	10	3.3%	6	2.0%
RSV	104	10.2%	50	26.3%	14	15.2%	4	3.1%	15	4.9%	21	7.1%

Table 11-20: Expected Values (As determined by ARIES® Flu A/B & RSV) – Summary by Age Groups for the ARIES® Flu A/B & RSV Prospective Clinical Study (November 2015 – February 2016)

| | Overall
(n=1462) | | 0-1 year
(n=244) | | >1-5 years
(n=131) | | >5-21 years
(n=114) | | >21-65 years
(n=538) | | >65 years
(n=435) | |
|---------------------|---------------------|----------------|---------------------|----------------|-----------------------|----------------|------------------------|----------------|-------------------------|----------------|----------------------|----------------|
| Target
(Analyte) | No. | Expected Value | No. | Expected Value | No. | Expected Value | No. | Expected Value | No. | Expected Value | No. | Expected Value |
| Influenza A | 164 | 11.2% | 9 | 3.7% | 17 | 13.0% | 15 | 13.2% | 98 | 18.2% | 25 | 5.7% |
| Influenza B | 28 | 1.9% | 3 | 1.2% | 2 | 1.5% | 9 | 7.9% | 9 | 1.7% | 5 | 1.1% |
| RSV | 202 | 13.8% | 83 | 34.0% | 33 | 25.2% | 10 | 8.8% | 24 | 4.5% | 52 | 12.0% |

Table 11-21: Expected Values (As determined by ARIES® Flu A/B & RSV) – Summary by Site for the ARIES® Flu A/B & RSV Prospective Clinical Study (January 2015 – March 2015)

Target (Analyte)	Overall (n=1017)		Site 01 (n=238)		Site 02 (n=225)		Site 04 (n=554)
	No.	Expected Value	No.	Expected Value	No.	Expected Value	No.	Expected Value
Influenza A	170	16.7%	42	17.6%	39	17.3%	89	16.1%
Influenza B	31	3.0%	2	0.8%	17	7.6%	12	2.2%
RSV	104	10.2%	12	5.0%	20	8.9%	72	13.0%

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	Overall (n=1462)		Site 01 (n=573)		Site 02 (n=102)		Site 03 (n=387)		Site 04 (n=400)
Target
(Analyte)	No.	Expected
Value	No.	Expected
Value	No.	Expected
Value	No.	Expected
Value	No.	Expected
Value
Influenza A	164	11.2%	74	12.9%	12	11.8%	16	4.1%	62	15.5%
Influenza B	28	1.9%	8	1.4%	4	3.9%	3	0.8%	13	3.3%
RSV	202	13.8%	58	10.1%	12	11.8%	53	13.7%	79	19.8%

Table 11-22: Expected Values (As determined by ARIES® Flu A/B & RSV) – Summary by Site for the ARIES® Flu A/B & RSV Prospective Clinical Study (November 2015 – February 2016)

N. Proposed Labeling:

The labeling provided in the submission satisfies the requirements of 21 CFR 809.10.

O. Conclusion:

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