The LIAISON PLEX Respiratory Flex (RSP Flex) Assay is a multiplexed qualitative test for the simultaneous in vitro detection and identification of multiple bacterial and viral nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals with clinical signs and symptoms of respiratory tract infection, including SARS-CoV-2. The test is performed on the automated LIAISON PLEX System utilizing reverse transcription (RT), polymerase chain reaction (PCR), and array hybridization to detect specific nucleic acid gene sequences of the following organism types and subtypes:

Viruses: Adenovirus Human Coronavirus (HKU1, NL63, OC43, and 229E not differentiated) Human Enterovirus/Rhinovirus (not differentiated) Human Metapneumovirus, Influenza A Influenza A (subtype H1) Influenza A (subtype H3) Influenza B Parainfluenza 1 Parainfluenza 2 Parainfluenza 3 Parainfluenza 4 Respiratory Syncytial Virus Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2)

Bacteria: Bordetella holmesii Bordetella parapertussis Bordetella pertussis Chlamydia pneumoniae Mycoplasma pneumoniae

Nucleic acids from the bacterial and viral organisms identified by this test are generally detectable in NPS specimens during the acute phase of infection. Detecting and identifying specific bacterial and viral nucleic acids from individuals exhibiting signs and symptoms of respiratory infection aids in the diagnosis of respiratory infection, if used in conjunction with other clinical, epidemiological, and laboratory findings. The results of this test should not be used as the sole basis for diagnosis, treatment, or patient management decisions.

Negative results in the presence of a respiratory illness may be due to infection with pathogens that are not detected by this test or due to lower respiratory that is not detected by an NPS specimen. Conversely, positive results do not rule out infection or co-infection with organisms not detected by the LIAISON PLEX Respiratory Flex (RSP Flex) Assay. The agent(s) detected may not be the definite cause of disease.

The use of additional laboratory testing (e.g., bacterial and viral culture, immunofluorescence, and radiography), may be necessary when evaluating a patient with possible respiratory tract infection.

Device Description

The LIAISON PLEX® Respiratory Flex Assay is a multiplexed nucleic acid test system composed of the LIAISON PLEX Instrument, the LIAISON PLEX® System Software (preinstalled on the LIAISON PLEX Instrument), the LIAISON PLEX® Respiratory Flex Assay cartridge, and the LIAISON PLEX® Respiratory Flex Assay File. The LIAISON PLEX® Respiratory Flex Assay cartridge contains the reagents to perform nucleic acid extraction and purification, reverse transcription, PCR, and array hybridization. Specifically, the LIAISON PLEX® Respiratory Flex Assay detects bacteria and viruses from nasopharyngeal swab (NPS) specimens collected from individuals with signs and symptoms of respiratory infection.

The LIAISON PLEX System consists of a touchscreen user interface that includes the software for running and analyzing assay results, one to six processing/imaging LIAISON PLEX modules, and a handheld barcode reader. Each LIAISON PLEX module processes one sample at a time under the control of the LIAISON PLEX System software.

LIAISON PLEX® automates the sample processing through analysis within a single cartridge. Processing steps include 1.) Sample Preparation: Nucleic acid extraction from organisms by chemical and mechanical means and isolation of nucleic acid on magnetic beads 2.) Target Amplification: Multiplex PCR and RT-PCR based amplification of extracted nucleic acid to generate target specific amplicons 3.) Hybridization: Amplicons hybridize with their target specific DNA probe arranged in a microarray format and that are attached to mediator and gold nanoparticles 4.) Analysis: Gold nanoparticles specifically bound to target amplicons are silver enhanced and the light scatter from microarray spot is measured and analyzed to confirm presence (Detected) or absence (not Detected) of a target.

The LIAISON PLEX Respiratory Flex Assay has the option of creating and processing results for custom panels using Flex® Software. Flex Software allows users to randomly select and group targets in tiers for result processing. Up to 7 targets may be selected for the initial test tier. After the first tier, each additional tier requires a specific number of credits. Flex™ credits allow the end-user to create custom panels and pay for a smaller subset of results tailored to the individual patient's clinical presentation. Alternatively, a laboratory may choose the fixed price option where all target results are processed at the same time.

AI/ML Overview

The LIAISON PLEX Respiratory Flex Assay is a multiplexed qualitative test for the simultaneous in vitro detection and identification of multiple bacterial and viral nucleic acids in nasopharyngeal swabs (NPS) from individuals with clinical signs and symptoms of respiratory tract infection, including SARS-CoV-2.

Here's an analysis of the acceptance criteria and study proving its performance:

1. Table of Acceptance Criteria and Reported Device Performance

The provided document doesn't explicitly state "acceptance criteria" with numerical thresholds for performance metrics. However, regulatory bodies like the FDA typically expect high sensitivity (Positive Percent Agreement - PPA) and specificity (Negative Percent Agreement - NPA) for diagnostic assays. Based on the clinical performance summary, we can infer the achieved performance.

Infered Acceptance Criteria and Reported Device Performance (Summary for Key Analytes):

Analyte (Overall Performance)	Infered Acceptance Criteria (Typical)	Reported Device Performance (PPA)	Reported Device Performance (NPA)
Adenovirus	High PPA, High NPA	100% (96.1-100% CI)	95.7% (94.7-96.6% CI)
Bordetella parapertussis	High PPA, High NPA	80.0% (37.6-96.4% CI)	99.8% (99.5-99.9% CI)
Human Coronavirus	High PPA, High NPA	90.0% (83.6-94.1% CI)	99.5% (99.1-99.8% CI)
Enterovirus/Rhinovirus	High PPA, High NPA	93.7% (90.5-95.8% CI)	97.8% (96.9-98.4% CI)
Human Metapneumovirus (hMPV)	High PPA, High NPA	95.4% (90.4-97.9% CI)	99.6% (99.2-99.8% CI)
Influenza A	High PPA, High NPA	100% (97.1-100% CI)	99.1% (98.5-99.4% CI)
Influenza A Subtype H1	High PPA, High NPA	100% (90.6-100% CI)	99.9% (99.7-100% CI)
Influenza A Subtype H3	High PPA, High NPA	97.2% (92.1-99.0% CI)	99.8% (99.4-99.9% CI)
Influenza B	High PPA, High NPA	100% (67.6-100% CI)	100% (99.8-100% CI)
Parainfluenza 1	High PPA, High NPA	91.7% (64.6-98.5% CI)	100% (99.8-100% CI)
Parainfluenza 2	High PPA, High NPA	92.3% (66.7-98.6% CI)	100% (99.8-100% CI)
Parainfluenza 3	High PPA, High NPA	93.2% (81.8-97.7% CI)	99.9% (99.7-100% CI)
Parainfluenza 4	High PPA, High NPA	88.9% (56.5-98.0% CI)	99.9% (99.7-100% CI)
Respiratory Syncytial Virus (RSV)	High PPA, High NPA	95.9% (90.8-98.3% CI)	100% (99.8-100% CI)
SARS-CoV-2	High PPA, High NPA	96.5% (93.4-98.1% CI)	99.5% (99.0-99.7% CI)

Note: For analytes with 0/0 positive cases (Bordetella holmesii, Bordetella pertussis, Chlamydia pneumoniae, Mycoplasma pneumoniae in the prospective study), performance is "Not Evaluable" (NE) but the NPA is 100%. These were supplemented with contrived specimens.

2. Sample Size and Data Provenance

Prospective Clinical Study:

Sample Size (Test Set): 1843 unique clinical specimens initially enrolled, with 1832 specimens yielding valid results after retests.
Data Provenance: Prospectively collected between October 2022 to April 2023 from six geographically diverse clinical sites within the United States. Specimens were remnant and de-identified, collected from pediatric and adult patients.

Archived Specimen Testing (Retrospective):

Sample Size (Test Set): 256 pre-selected, left-over, frozen, de-identified specimens, all yielding valid results after retests.
Data Provenance: Retrospectively collected from November 2013 through June 2023 from four sites/vendors in the United States.

Contrived Specimen Testing:

Sample Size (Test Set): 300 contrived specimens, all yielding valid results after retests.
Data Provenance: Artificially created samples to cover low prevalence targets. Tested at two US sites (details about origin of base matrix not specified beyond "simulated NPS matrix").

3. Number of Experts and Qualifications for Ground Truth

The document does not specify the number of experts used and their exact qualifications (e.g., "radiologist with 10 years of experience") for establishing ground truth. Instead, it indicates that the ground truth was established by comparator methods, which are themselves FDA-cleared molecular panels or analytically validated assays. These methods inherently rely on expertise for their development and validation but do not require additional human expert adjudication for each case in this study.

4. Adjudication Method for the Test Set

Specimens that obtained discordant results between the LIAISON PLEX Respiratory Flex Assay and the comparator method underwent additional testing for investigation.

For targets typically compared against FDA-cleared molecular respiratory panels, discordant samples were re-tested with an FDA-cleared molecular respiratory panel or PCR/BDS.
For Bordetella holmesii, Bordetella parapertussis, Bordetella pertussis, comparator performance was based on "well-validated Fragment Analysis (FA) assays followed by PCR/Bi-Directional Sequencing (PCR/BDS) assays."

This implies an adjudication method involving a third, more definitive or confirmatory test (often a "tie-breaker" or "gold standard" method like PCR/BDS) for discordant results. This is a common practice in diagnostic device studies. The document does not specify a "2+1" or "3+1" structure as those typically refer to multiple human readers or interpretations.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not done. This study evaluates the performance of a diagnostic assay itself for detecting nucleic acids, not the impact of AI assistance on human readers' interpretation of images or other data. Therefore, there is no effect size reported for how much human readers improve with AI vs. without AI assistance.

6. Standalone (Algorithm Only) Performance

Yes, a standalone performance was done. The entire clinical performance study (prospective, archived, and contrived) evaluates the LIAISON PLEX Respiratory Flex Assay as an algorithm-only or device-only diagnostic tool without human-in-the-loop interpretation being part of the primary evaluation. The results presented for PPA and NPA are based solely on the device's output compared to the ground truth.

7. Type of Ground Truth Used

The ground truth used was primarily established through comparator laboratory methods:

FDA-cleared molecular respiratory panels for most viral and bacterial targets.
FDA-cleared molecular SARS-CoV-2 assay for SARS-CoV-2.
Analytically Validated Fragment Analysis (FA) assays followed by PCR/Bi-Directional Sequencing (PCR/BDS) assays for Bordetella species.
For discordant results, additional testing with FDA-cleared molecular respiratory panels or PCR/BDS was performed for investigation.

This combination of highly sensitive and specific molecular diagnostic assays, with confirmatory testing for discordance, serves as the ground truth.

8. Sample Size for the Training Set

The document does not explicitly state the sample size for a separate "training set" for the LIAISON PLEX Respiratory Flex Assay. This is characteristic of molecular diagnostic assays which are typically developed and optimized through analytical studies (e.g., limit of detection, inclusivity, exclusivity) and then validated in clinical performance studies without a distinct "AI training set" in the common machine learning sense. The performance characteristics (analytical and clinical) presented are for the final, locked-down assay.

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

Since a distinct "training set" in the AI/machine learning context is not specified, the method for establishing its ground truth is not detailed. However, the development of such assays involves extensive analytical testing using characterized isolates, strains, and clinical samples to define reactivity, specificity, and sensitivity. The ground truth for this analytical development would typically be based on:

Known concentrations of purified nucleic acids or organisms.
Well-characterized reference materials and clinical specimens with confirmed presence/absence of targets via established methods (e.g., culture, sequencing, reference PCR).

Summary

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March 1, 2024

Image /page/0/Picture/1 description: The image shows the logo of the U.S. Food and Drug Administration (FDA). On the left is the Department of Health & Human Services logo. To the right of that is the FDA logo, which is a blue square with the letters "FDA" in white. To the right of the blue square is the text "U.S. FOOD & DRUG ADMINISTRATION" in blue.

Luminex Corporation Tara Viviani Sr. Director Molecular Regulatory Affairs 4088 Commercial Avenue Northbrook, Illinois 60062

Re: K233410

Trade/Device Name: LIAISON PLEX Respiratory Flex Assay Regulation Number: 21 CFR 866.3981 Regulation Name: Device To Detect And Identify Nucleic Acid Targets In Respiratory Specimens From Microbial Agents That Cause The SARS-Cov-2 Respiratory Infection And Other Microbial Agents When In A Multi-Target Test Regulatory Class: Class II Product Code: QOF, OEM, OOU, OTG, OZE, OZX, OZY, OZZ, OCC, NSU Dated: October 6, 2023 Received: October 6, 2023

Dear Tara Viviani:

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 (the 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. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database available at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. 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.

Additional information about changes that may require a new premarket notification are provided in the FDA guidance documents entitled "Deciding When to Submit a 510(k) for a Change to an Existing Device"

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(https://www.fda.gov/media/99812/download) and "Deciding When to Submit a 510(k) for a Software Change to an Existing Device" (https://www.fda.gov/media/99785/download).

Your device is also subject to, among other requirements, the Quality System (QS) regulation (21 CFR Part 820), which includes, but is not limited to, 21 CFR 820.30. Design controls; 21 CFR 820.90. Nonconforming product; and 21 CFR 820.100, Corrective and preventive action. Please note that regardless of whether a change requires premarket review, the OS regulation requires device manufacturers to review and approve changes to device design and production (21 CFR 820.30 and 21 CFR 820.70) and document changes and approvals in the device master record (21 CFR 820.181).

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR Part 803) for devices or postmarketing safety reporting (21 CFR Part 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safetyreporting-combination-products); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR Part 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR Parts 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely.

Joseph Briggs -S

Joseph Briggs, Ph.D. Deputy Branch Chief Division of Microbiology Devices OHT7: Office of In Vitro Diagnostics Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K233410

Device Name LIAISON PLEX Respiratory Flex Assay

Indications for Use (Describe)

Bacteria: Bordetella holmesii Bordetella parapertussis Bordetella pertussis Chlamydia pneumoniae Mycoplasma pneumoniae

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X Prescription Use (Part 21 CFR 801 Subpart D)

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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 21 CFR 807.92.

Preparation Date: 21-February-2024

A. 510(k) Number:

K233410

B. Purpose for Submission:

Traditional 510(k), New Device

C. Measurand:

Adenovirus, Bordetella holmesii, Bordetella parapertussis, Bordetella pertussis, Chlamydia pneumoniae, Human Coronavirus (HKU1, NL63, OC43, and 229E not differentiated), Human Enterovirus (Rhinovirus (not differentiated), Human Metapneumovirus, Influenza A (subtype H1), Influenza A (subtype H3), Influenza B, Mycoplasma pneumoniae, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, Parainfluenza 4, Respiratory Syncytial Virus (RSV), and Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) nucleic acid target sequences

D. Type of Test:

Qualitative Multiplexed Nucleic Acid Test that Utilizes Reverse Transcription, Real Time Polymerase Chain Reaction (PCR), and Array Hybridization.

E. Applicant:

Tara Viviani, Luminex Corporation 4088 Commercial Avenue Northbrook, IL 60062 (847) 400-9000

F. Proprietary and Established Names:

LIAISON PLEX® Respiratory Flex Assay

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G. Regulatory Information:

PrimaryProductCode	Classification	Regulation Section	Panel
QOF	II	21 CFR 866.3981 - Device To Detect AndIdentify Nucleic Acid Targets In RespiratorySpecimens From Microbial Agents That CauseThe SARS-CoV-2 Respiratory Infection AndOther Microbial Agents When In A Multi-Target Test	MI - Microbiology

H. Intended Use:

Intended use(s):

Viruses:

Adenovirus Human Coronavirus (HKU1, NL63, OC43, and 229E not differentiated) Human Enterovirus/Rhinovirus (not differentiated) Human Metapneumovirus, Influenza A Influenza A (subtype H1) Influenza A (subtype H3) Influenza B Parainfluenza 1 Parainfluenza 2 Parainfluenza 3 Parainfluenza 4 Respiratory Syncytial Virus Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2)

Bacteria:

Bordetella holmesii Bordetella parapertussis Bordetella pertussis Chlamydia pneumoniae Mycoplasma pneumoniae

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Negative results in the presence of a respiratory illness may be due to infection with pathogens that are not detected by this test or due to lower respiratory tract infection that is not detected by an NPS specimen. Conversely, positive results do not rule out infection or coinfection with organisms not detected by the LIAISON PLEX Respiratory Flex (RSP Flex) Assay. The agent(s) detected may not be the definite cause of disease.

Indication(s) for use:

Same as intended use.

Special conditions for use statement(s):

For prescription use only.

For in vitro diagnostic use only

Special instrument requirements:

For use with LIAISON PLEX Systems only

l. Device Description:

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Substantial Equivalence Information: J.

Predicate device name(s):

BioFire Respiratory Panel 2.1 (RP2.1)

Predicate 510(k) number(s):

DEN200031

Comparison with predicate:

The following table compares Luminex's LIAISON PLEX® Respiratory Flex Assay to the BioFire Respiratory Panel 2.1 (RP2.1) (DEN200031).

Comparison to Predicate
-------------------------	--	--	--

Comparison toPredicate Device	Predicate Device:BioFire Respiratory Panel 2.1 (RP2.1)(DEN200031)	Candidate Device:LIAISON PLEX® Respiratory FlexAssay
Product Code	QOF	QOF
Regulation Number	21 CFR 866.3981	21 CFR 866.3981
Comparison toPredicate Device	Predicate Device:BioFire Respiratory Panel 2.1 (RP2.1)(DEN200031)	Candidate Device:LIAISON PLEX® Respiratory FlexAssay
Organisms Detected	Adenovirus, Coronavirus 229E,Coronavirus HKU1, Coronavirus NL63,Coronavirus OC43, Severe AcuteRespiratory Syndrome Coronavirus(SARS-CoV-2), HumanMetapneumovirus, HumanRhinovirus/Enterovirus, Influenza A,including subtypes H1, H1-2009, andH3, Influenza B, Parainfluenza Virus 1,Parainfluenza Virus 2, ParainfluenzaVirus 3, Parainfluenza Virus 4,Respiratory Syncytial Virus, Bordetellaparapertussis (IS1001), Bordetellapertussis (ptxP), Chlamydiapneumoniae, and Mycoplasmapneumoniae	Adenovirus, Bordetella holmesii,Bordetella parapertussis, Bordetellapertussis, Chlamydia pneumoniae,Coronavirus 229E, CoronavirusHKU1, Coronavirus NL63,Coronavirus OC43,Enterovirus/Rhinovirus, HumanMetapneumovirus, Influenza A,Influenza A (subtype H1), Influenza A(subtype H3), Influenza B,Mycoplasma pneumoniae,Parainfluenza 1, Parainfluenza 2,Parainfluenza 3, Parainfluenza 4,Respiratory Syncytial Virus, andSARS-CoV-2
Measurand	Nucleic acid from Organisms detected	Nucleic acid from Organisms detected
Intended Use	The BioFire Respiratory Panel 2.1 (RP2.1)is a PCR-based multiplexed nucleic acidtest intended for use with the BioFireFilmArray 2.0 or BioFire FilmArray Torchsystems for the simultaneous qualitativedetection and identification of multiplerespiratory viral and bacterial nucleicacids in nasopharyngeal swabs (NPS)obtained from individuals suspected ofrespiratory tract infections, includingCOVID-19.The following organism types andsubtypes are identified using the BioFireRP2.1:	The LIAISON PLEX Respiratory Flex(RSP Flex) Assay is a multiplexedqualitative test for the simultaneous invitro detection and identification ofmultiple bacterial and viral nucleicacids in nasopharyngeal swabs (NPS)obtained from individuals with clinicalsigns and symptoms of respiratorytract infection, including SARS-CoV-2.The test is performed on theautomated LIAISON PLEX Systemutilizing reverse transcription (RT),polymerase chain reaction (PCR), andarray hybridization to detect specificnucleic acid gene sequences of thefollowing organism types andsubtypes:
	Adenovirus,Coronavirus 229E,Coronavirus HKU1,Coronavirus NL63,Coronavirus OC43,Severe Acute Respiratory SyndromeCoronavirus (SARS-CoV-2),Human Metapneumovirus,Human Rhinovirus/Enterovirus,Influenza A, including subtypes H1, H1-2009, and H3,Influenza B,Parainfluenza Virus 1,Parainfluenza Virus 2,Parainfluenza Virus 3,Parainfluenza Virus 4,Respiratory Syncytial Virus,Bordetella parapertussis (IS1001),	Viruses:AdenovirusHuman Coronavirus (HKU1, NL63,OC43, and 229E not differentiated)Human Enterovirus/Rhinovirus (notdifferentiated)Human Metapneumovirus,Influenza AInfluenza A (subtype H1)Influenza A (subtype H3)Influenza BParainfluenza 1Parainfluenza 2Parainfluenza 3Parainfluenza 4Respiratory Syncytial VirusSevere Acute Respiratory SyndromeCoronavirus (SARS-CoV-2)
Comparison toPredicate Device	Predicate Device:BioFire Respiratory Panel 2.1 (RP2.1)(DEN200031)	Candidate Device:LIAISON PLEX® Respiratory FlexAssay
	Bordetella pertussis (ptxP),Chlamydia pneumoniae, andMycoplasma pneumoniaeNucleic acids from the respiratory viraland bacterial organisms identified by thistest are generally detectable in NPSspecimens during the acute phase ofinfection. The detection and identificationof specific viral and bacterial nucleic acidsfrom individuals exhibiting signs and/orsymptoms of respiratory infection isindicative of the presence of the identifiedmicroorganism and aids in the diagnosisof respiratory infection if used inconjunction with other clinical andepidemiological information. The results ofthis test should not be used as the solebasis for diagnosis, treatment, or otherpatient management decisions.Negative results in the setting of arespiratory illness may be due toinfection with pathogens that are notdetected by this test, or lower respiratorytract infection that may not be detectedby an NPS specimen. Positive results donot rule out coinfection with otherorganisms. The agent(s) detected by theBioFire RP2.1 may not be the definitecause of disease. Additional laboratorytesting (e.g. bacterial and viral culture,immunofluorescence, and radiography)may be necessary when evaluating apatient with possible respiratory tractinfection.	Bacteria:Bordetella holmesiiBordetella parapertussisBordetella pertussisChlamydia pneumoniaeMycoplasma pneumoniaeNucleic acids from the bacterial andviral organisms identified by this testare generally detectable in NPSspecimens during the acute phase ofinfection. Detecting and identifyingspecific bacterial and viral nucleicacids from individuals exhibiting signsand symptoms of respiratory infectionaids in the diagnosis of respiratoryinfection, if used in conjunction withother clinical, epidemiological, andlaboratory findings. The results of thistest should not be used as the solebasis for diagnosis, treatment, orpatient management decisions.Negative results in the presence of arespiratory illness may be due toinfection with pathogens that are notdetected by this test or due to lowerrespiratory tract infection that is notdetected by an NPS specimen.Conversely, positive results do not ruleout infection or co-infection withorganisms not detected by theLIAISON PLEX Respiratory Flex (RSPFlex) Assay. The agent(s) detectedmay not be the definite cause ofdisease.The use of additional laboratory testing(e.g., bacterial and viral culture,immunofluorescence, and radiography),may be necessary when evaluating apatient with possible respiratory tractinfection.
Automated System(Sample to Answer)	Automated	Same
Instrumentation	BioFire® FilmArray® 2.0 or BioFire®FilmArray® Torch Systems	LIAISON PLEX®
Sample Types	Nasopharyngeal Swab (NPS) Specimens	Same
Technological Principles	Highly multiplexed nested nucleic acidamplification with melt analysis.	Highly multiplexed nucleic acid PCRand RT-PCR test with microarraydetection
Internal Controls	Two controls are included in each reagentpouch to control for sample processing	Multiple internal controls contained inthe cartridge monitor sampleprocessing and RT and PCR
Comparison toPredicate Device	Predicate Device:BioFire Respiratory Panel 2.1 (RP2.1)(DEN200031)	Candidate Device:LIAISON PLEX® Respiratory FlexAssay
	and both stages of PCR and melt analysis.	functions.
Bordetella SpeciesDetected	Bordetella parapertussisBordetella pertussis	Bordetella parapertussis Bordetella pertussis Bordetella holmesii
Human CoronavirusResult Reporting	Each target human coronavirus species(i.e., HKU1, OC43, 229E, NL63) isreported independently.	The human coronavirus target species(i.e., HKU1, OC43, 229E, NL63) arenot differentiated.
Influenza A Subtyping	Influenza A subtypes H1, H1-2009, and H3detected/reported.	Influenza A subtypes H1 and H3detected/reported.
Time to Result	~45 minutes	~2 hours

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K. Standards/Guidance Documents Referenced:

Standards

. CLSI. User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline -Second Edition. CLSI document EP12-A2. Wayne, PA: Clinical and Laboratory Standards Institute; 2008.
CLSI. Evaluation of Stability of In Vitro Diagnostic Reagents; Approved Guideline. CLSI document EP25-A. Wayne, PA: Clinical and Laboratory Standards Institute; 2009.
. CLSI. Interference Testing in Clinical Chemistry. 3rd ed. CLSI guideline EP07. Wayne, PA: Clinical and Laboratory Standards Institute; 2018.
ISO 14971:2019 Medical devices - Application of risk management to medical devices
IEC 62366-1:2015 Medical devices - Part 1: Application of usability engineering to medical devices
ISO 62304:2006 Medical device software - Software life-cycle processes
ISO 15223-1:2016: Medical Devices - Symbols to be used with medical device labels, labeling and information to be supplied - Part 1: General requirements
. IEC 61010-1 Ed. 3.0 2010: Safety requirements for electrical equipment for measurement, control, and laboratory use - Part 1: General requirements
EN 61010-2-101:2002/IEC 61010-2-101:2015: Safety requirements for electrical equipment for measurement, control and laboratory use - Part 2-101: Particular requirements for in vitro diagnostic (IVD) medical equipment.
IEC 60601-1-2:2014 (Edition 4.0): Medical electrical equipment - Part 1-2: General requirements for basic safety and essential performance - Collateral Standard: Electromagnetic disturbances - Requirements and tests
. ISO 13485:2016/EN ISO 13485:2016; Medical devices - Quality Management System -Requirements for regulatory purposes
ISO 20916:2019; In vitro diagnostic medical devices. Clinical performance studies using specimens from human subjects. Good study practice
. EN ISO 18113-1:2011; In vitro diagnostic medical devices - Information supplied by the manufacturer (labeling). Terms, definition and general requirements
. EN ISO 18113-2:2011; In vitro diagnostic medical devices - Information supplied by the manufacturer (labeling) – Part 2: In vitro diagnostic reagents for professional use

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. EN ISO 18113-3:2011; In vitro diagnostic medical devices - Information supplied by the manufacturer (labeling) – Part 3: In vitro diagnostic instruments for professional use
EN ISO 23640:2015; In vitro diagnostic medical devices - Evaluation of stability of in vitro
. IEC 61326-1:2012; Electrical equipment for measurement control and laboratory use -EMC requirements - Part 1: General requirements
. EN 61326-2-6:2006/IEC 61326-2-6:2012; Electrical equipment for measurement control and laboratory use - EMC requirements - Part 2-6: Particular requirements - In vitro diagnostic (IVD) medical equipment

Special Controls

Class II Special Controls as per 21 CFR 866.3981 ●

Guidance Documents

Electronic Submission Template for Medical Device 510(k) Submissions Guidance for Industry ● and Food and Drug Administration Staff (October 2, 2023).
. Respiratory Viral Panel Multiplex Nucleic Acid Assay - Class II Special Controls Guidance for Industry and FDA Staff (October 9, 2009).
. Content of Premarket Submissions for Device Software Functions - Guidance for Industry and Food and Drug Administration Staff (June 14, 2023).
. Cybersecurity in Medical Devices: Quality System Considerations and Content of Premarket Submissions - Guidance for Industry and Food and Drug Administration Staff (September 23, 2023).
Statistical Guidance on Reporting Results from Studies Evaluating Diagnostic Tests - Guidance for Industry and FDA Staff (March 13, 2007).

L. Test Principle:

The LIAISON PLEX® Respiratory Flex Assay is performed on nasopharyngeal swab (NPS) specimens collected in Copan Universal Transport Medium™ or BD™ Universal Viral Transport Media. The system consists of an instrument and a single-use, disposable test cartridge and a transfer pipette. The user loads a portion of the sample into the sample port of the LIAISON PLEX Respiratory Flex Assay Cartridge. Next, the user sets up the sample order on the LIAISON PLEX System by first entering the sample information or scanning the barcode ID located on the sample tube, then scanning the barcode ID located on the test cartridge. Last, the user inserts the test cartridge into the processing module to initiate the test. The LIAISON PLEX System identifies the assay being run and automatically initiates the proper testing protocol to process the sample, analyze the data, and generate test results.

The LIAISON PLEX System automates the LIAISON PLEX Respiratory Flex Assay sample analysis through the following steps: a) Sample Preparation: Nucleic acid extraction via mechanical and chemical cell lysis and magnetic bead- based nucleic acid isolation of prepared specimens obtained from patients; b) Target Amplification: Multiplex PCR- and RT-PCR-based amplification of the extracted nucleic acids to generate target-specific amplicons; c) Hybridization: Amplicons hybridize to target-specific capture DNA on a microarray format, and target-specific mediator and gold nanoparticle probes hybridize to captured amplicons; d) Signal Analysis: Gold nanoparticle probes bound specifically to target-containing spots in the microarray are silver-enhanced, and light scatter from the spots is measured and further

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analyzed to determine the presence (Detected) or absence (Not Detected) of a target.

M. Performance Characteristics:

1. Analytical performance:
- a. Precision/Reproducibility:

Within Laboratory Precision

Within laboratory precision of the LIAISON PLEX Respiratory Flex Assay was evaluated by testing three lots of LIAISON PLEX Respiratory Flex Assay cartridges at a single site over five non-consecutive days. Three target concentrations were prepared and tested. Targets consisted of a negative sample and a positive sample comprised of five targets (B. pertussis, Adenovirus, influenza B, human metapneumovirus, and SARS-CoV-2). All positive samples were diluted in a simulated NPS matrix to a low positive concentration (1.5X LoD) and a moderate positive concentration (5X LoD). Targets were randomized and blinded to the operators in an order that each operator tested each target (negative, 1.5X LoD) in triplicate on each of the testing days. Qualitative results of the within laboratory precision study are summarized in Table 1.

Target	Panel Concentration	% Positive	% Agreement withExpected Results(95% CI)
Bordetellapertussis	Low Positive (1.5X LoD)	93.3% (42/45)	93.3% (82.1-97.7%)
	Moderate Positive (5X LoD)	100% (45/45)	100% (92.1-100%)
	Negative	0% (0/45)	100% (92.1-100%)
Adenovirus	Low Positive (1.5X LoD)	97.8% (44/45)	97.8% (88.4-99.6%)
	Moderate Positive (5X LoD)	97.8% (44/45)	97.8% (88.4-99.6%)
	Negative	0% (0/45)	100% (92.1-100%)
Influenza B	Low Positive (1.5X LoD)	100% (45/45)	100% (92.1-100%)
	Moderate Positive (5X LoD)	100% (45/45)	100% (92.1-100%)
	Negative	0% (0/45)	100% (92.1-100%)
hMPV	Low Positive (1.5X LoD)	100% (45/45)	100% (92.1-100%)
	Moderate Positive (5X LoD)	97.8% (44/45)	97.8% (88.4-99.6%)
	Negative	0% (0/45)	100% (92.1-100%)
SARS-CoV-2	Low Positive (1.5X LoD)	100% (45/45)	100% (92.1-100%)
	Moderate Positive (5X LoD)	100% (45/45)	100% (92.1-100%)
	Negative	0% (0/45)	100% (92.1-100%)

Table 1 - Within Laboratory Precision

Note: Results are shown only for the intended targets. Panel members co-spiked with 5 different targets 3 lots and tested over 5 non-consecutive days (45 total replicates).

Reproducibility

Reproducibility of the LIAISON PLEX Respiratory Flex Assay was evaluated by testing one lot of LIAISON PLEX Respiratory Flex Assay cartridges with two operators at each of three sites over five non-consecutive days. Three target concentrations were prepared and tested across all

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sites and operators to evaluate site-to-site reproducibility. Targets consisted of a negative sample and a positive sample comprised of five assay targets (B. pertussis, Adenovirus, influenza B, human metapneumovirus, and SARS-CoV-2). All positive samples were diluted in a simulated NPS matrix to a low positive concentration (1.5X LoD) and a moderate positive concentration (5X LoD). Targets were randomized and blinded to the operators in an order that each operator tested each target (negative, 1.5X LoD, and 5X LoD) in triplicate on each of the testing days. Qualitative results of the reproducibility study are summarized in Table 2.

		% Agreement with Expected Results
Organism	TargetConcentration	Site 1	Site 2	Site 3	All Sites(95% Confidence)
Adenovirus	Low Positive(1.5X LoD)	96.7%(29/30)	100%(30/30)	96.7%(29/30)	97.8% (88/90)(92.3% - 99.4%)
	Moderate Positive(5X LoD)	100%(30/30)	100%(30/30)	100%(30/30)	100% (90/90)(95.9% - 100%)
Bordetella pertussis	Low Positive(1.5X LoD)	93.3%(28/30)	100%(30/30)	96.7%(29/30)	96.7% (87/90)(90.7% - 98.9%)
	Moderate Positive(5X LoD)	100%(30/30)	100%(30/30)	100%(30/30)	100% (90/90)(95.9% - 100%)
Influenza B	Low Positive(1.5X LoD)	93.3%(28/30)	100%(30/30)	96.7%(29/30)	96.7% (87/90)(90.7%- 98.9%)
	Moderate Positive(5X LoD)	100%(30/30)	100%(30/30)	100%(30/30)	100% (90/90)(95.9% - 100%)
HumanMetapneumovirus	Low Positive(1.5X LoD)	90.0%(27/30)	100%(30/30)	93.3%(28/30)	94.4% (85/90)(87.6% - 97.6%)
	Moderate Positive(5X LoD)	100%(30/30)	93.3%(28/30)	100%(30/30)	97.8% (88/90)(92.3% - 99.4%)
SARS-CoV-2	Low Positive(1.5X LoD)	96.7%(29/30)	100%(30/30)	100%(30/30)	98.9% (89/90)(93.9% - 99.8%)
	Moderate Positive(5X LoD)	100%(30/30)	100%(30/30)	100%(30/30)	100% (90/90)(95.9% - 100%)
Negative NPS	Negative	100%(30/30)	100%(30/30)	100%(30/30)	100% (90/90)(95.9% - 100%)

Table 2. Reproducibility Results

b. Linearity/assay reportable range:

Not applicable. The LIAISON PLEX® Respiratory Flex Assay is a qualitative assay.

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

Controls:

Each LIAISON PLEX Respiratory Flex Assay cartridge includes internal controls (extraction control, amplification control, and hybridization control) to ensure performance of sample preparation, amplification, and detection. Extraction control is automatically added to the sample prior to initiation of sample preparation and assesses extraction, nucleic acid recovery, amplification of RNA targets, and detection. Additionally, an amplification control present in the lyophilized PCR

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master mix, serves as an independent amplification and detection control for DNA targets. Finally, a post-amplification hybridization control serves as an indicator of successful hybridization. Internal control results are reported as Pass, Fail, or N/A on the printed reports (see Table 3 for detailed explanations of each control result). Internal controls must either (1) generate a signal above threshold in each internal reaction for the system to report a valid test result, or (2) the amplification or extraction control result can be below the signal threshold if a DNA or RNA target pathogen is detected, respectively.

If the Test Result is "No Call" for reasons other than failure of internal controls, the Internal Control Result is reported as "N/A" and the user should repeat the test with a new cartridge. For additional assistance regarding assay failures unrelated to internal controls, please refer to Chapter 8 (Troubleshooting Unexpected Results/Failures) of the LIAISON PLEX® System User Manual.

InternalControl Result	Explanation	Suggested Action
Pass	The hybridization control was detected, indicating successfulhybridization.The amplification control was detected, indicating successfulamplification.The extraction control was detected, indicating successfulextraction.	Review and reportresults
N/A	The hybridization control was detected, indicating successfulhybridization.A DNA pathogen target was detected, indicating successfulamplification. If a DNA pathogen target is detected, theamplification control result is ignored.The extraction control was detected, indicating successfulextraction.	Review and reportresults
N/A	The hybridization control was detected, indicating successfulhybridization.The amplification control was detected, indicating successfulamplification.A RNA pathogen target was detected, indicating successfulextraction. If a RNA pathogen target is detected, the extractioncontrol result is ignored.	Review and reportresults
Fail	The hybridization control was not detected indicating hybridizationwas not successful.The amplification control, or a DNA pathogen was detected,indicating successful amplification.The extraction control, or a RNA pathogen was detected, indicatingsuccessful extraction.	Repeat test with anew cartridge

Table 3. Interpretation of Controls on the LIAISON PLEX Respiratory Flex Assay Report

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InternalControl Result	Explanation	Suggested Action
Fail	The hybridization control was detected indicating successfulhybridization.The amplification control, or a DNA pathogen was not detected,indicating amplification was not successful.The extraction control, or a RNA pathogen was detected, indicatingsuccessful extraction.	Repeat test with anew cartridge
Fail	The hybridization control was detected indicating successfulhybridization.The amplification control, or a DNA pathogen was detected,indicating successful amplification.The extraction control, or a RNA pathogen, was not detected,indicating extraction was not successful.	Repeat test with anew cartridge

External Controls

Positive and negative external controls should be tested with each new lot or shipment of reagents, or monthly, (whichever occurs first), or in accordance with updated local, regional, state, and/or federal guidelines. Positive and negative external controls are not provided with the LIAISON PLEX Respiratory Flex Assay. Verified negative nasopharyngeal swab (NPS) specimens can be used as the negative control. Previously characterized positive samples or verified negative NPS specimens spiked with well characterized organisms may be used as the external positive control. External controls should be used in accordance with laboratory protocols and in accordance with local, state, and federal accrediting organizations, as applicable.

Stability:

Specimen Stability

Contrived specimen stability at room temperature (15°C - 30°C), refrigerated (2°C - 8°C), and frozen (<-70°C) storage was evaluated for use with the LIAISON PLEX® Respiratory Flex Assay. A representative panel of 5 Respiratory Flex target organisms (i.e., Bordetella pertussis, adenovirus, influenza B, hMPV, and SARS-CoV-2) was co-spiked into negative clinical NPS matrix at three concentrations - a low positive (2x LoD) sample, a moderate positive (5x LoD) sample, as well as the negative clinical matrix independently. Testing occurred at baseline and various time points up to 36 days for the frozen storage, up to 80 hours for refrigerated storage, and up to 9 hours for room temperature storage.

The results of this study demonstrated that specimens stored frozen (≤ -70°C) are stable for up to 30 days, specimens stored refrigerated (2°C - 8°C) are stable for up to 72 hours, and specimens stored at room temperature (15°C - 30°C) are stable for up to 8 hours.

Fresh vs. Frozen Specimen Stability

Performance of the LIAISON PLEX® Respiratory Flex Assay was assessed using contrived specimens tested fresh (i.e. unfrozen) and specimens tested frozen (stored at < -70°C).

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The effect of repeated freeze/thaw cycles was also assessed between freshly prepared contrived specimens and those that had undergone 1, 2, and 3 freeze/thaw cycles. Four contrived sample panels were prepared by co-spiking 5-6 targets into clinical negative NPS matrix at three concentrations - a low positive (2x LoD) sample, a moderate positive (5x LoD) sample, as well as the negative clinical matrix independently (see Table 4).

Panel	Organism	Panel	Organism
A	Bordetella pertussis	C	Bordetella parapertussis
	Adenovirus		RSV A
	Influenza B		Parainfluenza 2
	hMPV (A2)		Influenza A (subtype H3)
	SARS-CoV-2		hMPV (B2)
B	Bordetella holmesii	D	RSV B
	Mycoplasma pneumoniae		Chlamydia pneumoniae
	Parainfluenza 4		Parainfluenza 1
	Influenza A (subtype H1)		Human coronavirus NL63
	Parainfluenza 3		Human coronavirus OC43
	Rhinovirus A

Positive panels spiked at 2x were tested in replicates of 40 at TO (fresh) and 20 replicates following 1, 2, and 3 freeze/thaw (F/T) cycles after storage at -70°C. Positive panels spiked at 5x LoD and the negative sample were tested in replicates of 10 at TO (fresh) and following 1, 2, and 3 F/T cycles after storage at -70°C.

The results of the study support that NPS specimens in UVT/UTM can undergo up to two freeze/thaw cycles prior to testing with the LIAISON PLEX Respiratory Flex Assay.

d. Detection Limit:
A limit of detection study (LoD) was performed to evaluate the analytical sensitivity of the LIAISON PLEX RSP Flex Assay. Thirty-nine (39) strains and isolates that represent the 19 reportable targets of the LIAISON PLEX RSP Flex Assay were tested individually by serially diluting each target in NPS matrix. Testing was broken into two parts; LoD Determination and LoD Confirmation. The determined LoD concentrations were evaluated using a 3-fold dilution series and testing of at least six replicates per dilution. The determined LoD for each target was defined as the lowest concentration at which 100% of six replicates were positive for the intended reportable target. The confirmed LoD was evaluated using a dilution series around the determined LoD and testing of at least 20 replicates was performed at the confirmed LoD and the dilution below the confirmed LoD. The confirmed LoD for each organism was defined as the lowest concentration at which ≥ 95% of the 20 replicates were positive for the intended reportable target. The confirmed LoD for each target tested is listed in Table 5. The LoD for coanalyte spiked samples was also evaluated and shown to be equivalent to single spiked samples.

Table 5. LIAISON PLEX Respiratory Flex Assay Target Limit of Detection

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Target Organism	Strain / Isolate	Concentration at LoD¹
		Bacteria
Bordetella holmesii	F061	7.29E+03 copies/mL	8.68E+01 CFU/mL
Bordetella pertussis	18323 NCTC 10739	3.80E+03 copies/mL	1.98E+03 CFU/mL
Bordetella parapertussis	C510	7.90E+02 copies/mL	2.06E+01 CFU/mL
Chlamydia pneumoniae	CM-1	5.68E+02 copies/mL	1.04E+02 IFU/mL
Mycoplasma pneumoniae	M129	1.30E+03 copies/mL	4.24E+01 CCU/mL
		Viruses
	Type 31 (A)	1.76E+03 copies/mL	1.09E-02 TCID50/mL
	Type 3 (B)	6.86E+02 copies/mL	1.69E-01 TCID50/mL
Adenovirus(A, B, C, D, E, F)	Type 1 (C)	1.12E+03 copies/mL	8.97E+01 TCID50/mL
	Type 26 (D)	7.48E+02 copies/mL	1.10E-02 TCID50/mL
	Type 4 (E)	3.53E+02 copies/mL	1.08E-02 TCID50/mL
	Type 40 (F)	4.85E+02 copies/mL	2.29E-02 TCID50/mL
	229E	4.00E+02 copies/mL	9.15E-02 TCID50/mL
Human Coronavirus(HKU1, 229E, NL63,OC43)	HKU1	1.67E+03 copies/mL	N/A²
	NL63	7.64E+01 copies/mL	1.34E-02 TCID50/mL
	OC43	9.48E+03 copies/mL	9.58E-01 TCID50/mL
	(hMPV-9) A1	2.13E+03 copies/mL	2.09E-01 TCID50/mL
HumanMetapneumovirus	(hMPV-27) A2	2.04E+03 copies/mL	2.14E-01 TCID50/mL
	(hMPV-3) B1	5.00E+03 copies/mL	4.31E-01 TCID50/mL
	(hMPV-8) B2	1.50E+04 copies/mL	1.66E+00 TCID50/mL
	Brisbane/02/18Influenza AH1 Subtype	1.35E+04 copies/mL	3.97E+00 TCID50/mL
Influenza A Influenza A(subtype H1)	Guangdong-Maonan/SWL/1536/19Influenza AH1 Subtype	1.37E+04 copies/mL	5.86E+00 TCID50/mL
	HongKong/2671/19Influenza AH3 Subtype	1.59E+05 copies/mL	1.50E+01 TCID50/mL
Influenza A Influenza A(subtype H3)	A/Kansas/14/2017Influenza AH3 Subtype	1.96E+03 copies/mL	5.58E+00 TCID50/mL
	Singapore/INFUMH-16-0019/16Influenza AH3 Subtype	4.55E+03 copies/mL	1.10E+01 TCID50/mL
	Alabama/2/17 (Victoria Lineage)	3.35E+02 copies/mL	7.30E-01 TCID50/mL
Influenza B	Washington/02/19 (Victoria Lineage)	3.02E+03 copies/mL	2.79E+01 TCID50/mL
	Colorado/6/17 (Victoria Lineage)	3.02E+03 copies/mL	6.64E-01 TCID50/mL
	Wisconsin/1/10 (Yamagata Lineage)	1.01E+03 copies/mL	3.23E-01 TCID50/mL
Parainfluenza 1	N/A	7.61E+02 copies/mL	1.06E+01 TCID50/mL
Parainfluenza 2	N/A	8.46E+03 copies/mL	1.55E+01 TCID50/mL
Parainfluenza 3	N/A	1.93E+03 copies/mL	3.18E+00 TCID50/mL
Parainfluenza 4	A	5.76E+03 copies/mL	6.65E+01 TCID50/mL
Respiratory SyncytialVirus A	A (2006 Isolate)	3.83E+03 copies/mL	1.11E+00 TCID50/mL
Respiratory SyncytialVirus B	B (3/2015 Isolate #1)	1.61E+04 copies/mL	7.48E-01 TCID50/mL
Enterovirus / Rhinovirus	Human Rhinovirus 1A	8.19E+03 copies/mL	4.99E-01 TCID50/mL

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Target Organism	Strain / Isolate	Concentration at LoD1
	Human Rhinovirus B14	8.18E+03 copies/mL 1.10E+01 TCID50/mL
	Human Rhinovirus C1	1.92E+04 copies/mL N/A2
	Human EnterovirusEchovirus Type 6	2.25E+04 copies/mL 3.00E+01 TCID50/mL
SARS-CoV-2	USA-WA1/2020	8.00E+03 copies/mL 4.04E+01 TCID50/mL

4 Concentrations in copies/mL were obtained by digital-droplet PCR.

PTesting for Coronavirus HKU and Rhinovirus 10 utilized a dinical speciment due to the last of a cultured isolate. Viral concentration was deternined in RNA copies/mL by digital droplet PCR.

Limit of Detection Testing with the WHO International Standard for SARS-CoV-2 (NIBSC, 20/146)

An LoD study was performed to evaluate the analytical sensitivity of the Respiratory Flex Assay with the World Health Organization (WHO) Internal Standard for SARS-CoV-2 (Table 6). The WHO International SARS-CoV-2 standard was reconstituted then serially diluted in NPS matrix. As with the LoD study described above, testing was broken into two parts: preliminary and confirmatory LoD testing. For the preliminary LoD study, testing at multiple concentrations in triplicate was performed. The preliminary LoD was defined as the lowest concentration at which 100% of replicates were positive for SARS-CoV-2. The confirmed LoD was determined by testing a 3-fold dilution series of multiple concentrations around the preliminary LoD in replicates of 20. The confirmed LoD was defined as the lowest concentration at which ≥ 95% of the replicates were positive for the intended reportable target. To confirm the LoD, at least one dilution below the LoD was required to result in less than 95% positivity. The confirmed LoD for the Respiratory Flex Assay with the WHO International Standard was 7.7x10 IU/mL.

Table 6. Limit of Detection Results for WHO International Standard for SARS-CoV-2 (NIBSC, 20/146) Target

	Concentration Tested (IU/mL)	SARS-CoV-2 Positivity
International WHOSARS-CoV-2Standard	7.70E+05	95.0% (19/20)
	2.57E+05	65.0% (13/20)

Analytical Reactivity (Inclusivity) e. Laboratory (Wet Testing)

The analytical reactivity (inclusivity) of the LIAISON PLEX Respiratory Flex Assay was evaluated by using a collection of 181 isolates and clinical samples (34 bacteria and 147 viruses), representing the genetic diversity of the analytes in the LIAISON PLEX Respiratory Flex Assay. The organisms were diluted to a final concentration of 3X the target LoD in simulated NPS matrix and each diluted organism was tested in triplicate. In cases where 100% positivity was not achieved at 3X LoD, samples were reprepared at the same concentration and retested in triplicate. If 100% positivity was obtained during retesting, no additional testing was performed. If less than 100% positivity was obtained during retesting, the organism was prepared at a higher concentration and tested until 100% positivity was obtained.

Of the 181 strains tested, a total of 176 strains were detected with 100% positivity at 3X LoD in the

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laboratory. Four strains (influenza A Brisbane (02/18), coronavirus NL63 (NR-470), coronavirus 229E (VR-740) and SARS-CoV-2 (Stanford)) were detected at 9X LoD and one strain (B. holmesii (CIP 104396)) was detected at 27X LoD. Results of laboratory (wet testing) are shown in Table 7.

Three influenza A variant strains (H1N1v, H1N2v, and H3N2v) and two influenza A strains (H2N2 and H7N9) were included in the evaluation. The influenza A variant strains were tested in the laboratory using synthetic DNA. Based on this analysis, the three influenza A variant strains (H1N1v, H1N2v) strains are expected to be detected as influenza A positive and subtype H1, H1, and H3 positive, respectively. Influenza H2N2 and H7N9 are predicted to be detected as influenza A, H1/H3 negative.

ReportableTarget & Subtype	Serovars/Group/Species	Concentration		Concentrationcopies/mL	% Detected (#Detected/# Tested)
Bacteria
Bordetella holmesii	F061	2.60E+02	CFU/mL	2.19E+04	100% (3/3)
	CDC F5101 [CDC 84-013939]	1.95E+02	CFU/mL	2.19E+04	83.3% (5/6)
	CIP 104396	N/A1		1.97E+056	100% (3/3)
	CIP 104395 [G7702; 92A2997]	N/A1		2.19E+04	80.0% (4/5)
Bordetellaparapertussis	NCTC 5952 [522]	1.05E+02	CFU/mL	2.37E+03	100% (3/3)
	508 & 344 [NCTC 10853]	9.15E+00	CFU/mL	2.37E+03	100% (3/3)
	517	3.12E+01	CFU/mL	2.37E+03	100% (3/3)
	12822	7.68E+01	CFU/mL	2.37E+03	100% (3/3)
	509 and 609	6.02E+01	CFU/mL	2.37E+03	100% (3/3)
	PT28G	3.32E+01	CFU/mL	2.37E+03	100% (3/3)
	PT 26/28G	2.73E+01	CFU/mL	2.37E+03	100% (3/3)
	C510	6.18E+01	CFU/mL	2.37E+03	100% (3/3)

Bordetella pertussis	18323 [NCTC 10739]	4.12E+03	CFU/mL	1.14E+04	100% (3/3)
	CNCTC Hp 12/63 [623]	4.87E+03	CFU/mL	1.14E+04	100% (3/3)
	10-536	2.47E+03	CFU/mL	1.14E+04	100% (3/3)
	5 [17921]	2.99E+03	CFU/mL	1.14E+04	100% (3/3)
	Tohama I	5.71E+03	CFU/mL	1.14E+04	100% (3/3)
	MN2531	N/A1		1.14E+04	100% (3/3)
	PT9/28G [W28]	1.99E+03	CFU/mL	1.14E+04	100% (3/3)
	589	1.95E+03	CFU/mL	1.14E+04	100% (3/3)
	F	N/A1		1.14E+04	100% (3/3)
Chlamydiapneumoniae	CWL-029	1.16E+02	IFU/mL	1.71E+03	100% (3/3)
	AR-39	1.93E+02	IFU/mL	1.71E+03	100% (3/3)
	J-21	9.48E-01	TCID50/mL	1.71E+03	100% (3/3)
	2023	9.32E+01	IFU/mL	1.71E+03	100% (3/3)
Mycoplasmapneumoniae	M129	1.27E+02	CCU/mL	3.89E+03	100% (3/3)
	15531-TTR	1.23E+03	CFU/mL	3.89E+03	100% (3/3)
	Mac	1.45E+02	CCU/mL	3.89E+03	100% (3/3)
	PI 1428	1.56E+02	CCU/mL	3.89E+03	100% (3/3)
	Bru	N/A1	CFU/mL	3.89E+03	100% (3/3)
	M52	1.83E+01	CFU/mL	3.89E+03	100% (3/3)
	UTMB-10P	7.83E-01	CCU/mL	3.89E+03	100% (3/3)
	Mutant 22	9.47E+01	CFU/mL	3.89E+03	100% (3/3)
	M129-B7	1.33E-01	CFU/mL	3.89E+03	100% (3/3)
Adenovirus	A 31	1.79E-02	TCID50/mL	5.28E+03	100% (3/3)
	B 3	5.08E-01	TCID50/mL	2.06E+03	100% (3/3)
	B 7A	7.05E+00	TCID50/mL	2.06E+03	100% (3/3)
	B 21	3.60E-01	TCID50/mL	2.06E+03	100% (3/3)
	B 11	1.43E+00	TCID50/mL	2.06E+03	100% (3/3)
ReportableTarget & Subtype	Serovars/Group/Species	Concentration		Concentrationcopies/mL	% Detected (#Detected/# Tested)
	B 14	5.68E-01	TCID50/mL	2.06E+03	100% (3/3)
	B 34	1.19E+02	TCID50/mL	2.06E+03	83.3% (5/6)
	B 35	9.52E+00	TCID50/mL	2.06E+03	100% (3/3)
	C 1	2.69E+02	TCID50/mL	3.35E+03	100% (3/3)
	C 2	1.30E+02	TCID50/mL	3.35E+03	100% (3/3)
	C 5	1.10E+02	TCID50/mL	3.35E+03	100% (3/3)
	C 6	4.81E+01	TCID50/mL	3.35E+03	100% (3/3)
	D 26	3.51E-01	TCID50/mL	2.24E+03	100% (3/3)
	D 37	1.43E-01	TCID50/mL	2.24E+03	100% (3/3)
	E 4	3.23E-02	TCID50/mL	1.06E+03	83.3% (5/6)
	F 40-Dugan	7.60E-02	TCID50/mL	1.45E+03	100% (3/3)
	F 41-Tak	8.97E-03	TCID50/mL	1.45E+03	100% (3/3)
	HKU1	N/A2		5.00E+03	100% (3/3)
	HKU1			5.00E+03	100% (3/3)
	NL63Source #: NR-4705	5.41E-03	TCID50/mL	6.88E+03	100% (3/3)
Human Coronavirus	NL63Source #: 0810228CF5	4.02E-02	TCID50/mL	2.29E+02	100% (3/3)
	OC43Source #: 0810024CF5	2.87E+00	TCID50/mL	2.84E+04	100% (3/3)
	OC43Source #: VR-15585	1.02E+00	TCID50/mL	2.84E+04	100% (3/3)
	229ESource #: 0810229CF5	2.74E-01	TCID50/mL	1.20E+03	100% (3/3)
	229ESource #: VR-7405	1.51E+00	TCID50/mL	3.60E+03	100% (3/3)
	Human EnterovirusCoxsackievirus A10	3.53E+02	TCID50/mL	6.75E+04	100% (3/3)
	Human EnterovirusCoxsackievirus 71 (2003)	1.18E+00	TCID50/mL	6.75E+04	100% (3/3)
	Human EnterovirusCoxsackievirus A9	1.98E+02	TCID50/mL	6.75E+04	100% (3/3)
	Human EnterovirusCoxsackievirus B3	1.79E+01	TCID50/mL	6.75E+04	100% (3/3)
	Human EnterovirusCoxsackievirus B4	1.04E+02	TCID50/mL	6.75E+04	100% (3/3)
Enterovirus/Rhinovirus	Human EnterovirusEchovirus 6	9.01E+01	TCID50/mL	6.75E+04	100% (3/3)
	Human EnterovirusEchovirus 9	5.21E+00	TCID50/mL	6.75E+04	100% (3/3)
	Human EnterovirusEchovirus 11	1.02E+03	TCID50/mL	6.75E+04	100% (3/3)
	Human EnterovirusEchovirus 30	1.20E+02	TCID50/mL	6.75E+04	100% (3/3)
	Human EnterovirusCoxsackievirus A21	2.02E+01	TCID50/mL	6.75E+04	100% (3/3)
	Human EnterovirusCoxsackievirus A24	8.50E+00	TCID50/mL	6.75E+04	100% (3/3)
	Human Enterovirus68	1.05E+01	TCID50/mL	6.75E+04	100% (3/3)
	Human Rhinovirus A16	1.45E+02	TCID50/mL	2.46E+04	100% (3/3)
	Human Rhinovirus A2	4.89E-01	TCID50/mL	2.46E+04	100% (3/3)
	Human Rhinovirus A34	1.69E+02	TCID50/mL	2.46E+04	100% (3/3)
	Human Rhinovirus A57	1.04E+01	TCID50/mL	2.46E+04	100% (3/3)
ReportableTarget & Subtype	Serovars/Group/Species	Value	Units	Concentrationcopies/mL	% Detected (#Detected/# Tested)
	Human Rhinovirus A7	1.20E+01	TCID50/mL	2.46E+04	100% (3/3)
	Human Rhinovirus A77	2.15E-01	TCID50/mL	2.46E+04	100% (3/3)
	Human Rhinovirus A85	7.60E+02	TCID50/mL	2.46E+04	100% (3/3)
	Human Rhinovirus B14	3.31E+01	TCID50/mL	2.45E+04	100% (3/3)
	Human Rhinovirus B17	1.75E+02	TCID50/mL	2.45E+04	100% (3/3)
	Human Rhinovirus B27	5.09E+00	TCID50/mL	2.45E+04	100% (3/3)
	Human Rhinovirus B3	5.68E+01	PFU/mL	2.45E+04	100% (3/3)
	Human Rhinovirus B42	8.64E+00	TCID50/mL	2.45E+04	100% (3/3)
	Human Rhinovirus B83	1.13E+01	TCID50/mL	2.45E+04	100% (3/3)
	Human Rhinovirus C1	NA2		5.75E+04	100% (3/3)
	Human Rhinovirus C1	NA2		5.75E+04	100% (3/3)
	Human Rhinovirus C1	NA2		5.75E+04	100% (3/3)
	Human Rhinovirus C1	NA2		5.75E+04	100% (3/3)
	(hMPV-9) A1	6.26E-01	TCID50/mL	6.40E+03	100% (3/3)
	(hMPV-16) A1	4.01E+00	TCID50/mL	6.40E+03	100% (3/3)
	(hMPV-20) A2	NA2		6.12E+03	100% (3/3)
	(hMPV-27) A2	6.41E-01	TCID50/mL	6.12E+03	100% (3/3)
HumanMetapneumovirus	(hMPV-3) B1	1.29E+00	TCID50/mL	1.50E+04	100% (3/3)
	(hMPV-5) B1	9.50E+00	TCID50/mL	1.50E+04	100% (3/3)
	(hMPV-4) B2	1.37E+03	TCID50/mL	4.50E+04	100% (3/3)
	(hMPV-8) B2	4.99E+00	TCID50/mL	4.50E+04	100% (3/3)
	(hMPV-18) B2	2.78E+01	TCID50/mL	4.50E+04	100% (3/3)
	A/Wisconsin/588/2019	4.06E+01	FFU/mL	4.50E+03	Matrix: 100% (3/3)H1: 100% (3/3)
	A/Hawaii/66/2019 X-345A	2.45E+03	CEID50/mL	4.50E+03	Matrix: 100% (6/6)H1: 83.3% (5/6)
	A/Indiana/02/2020	2.06E+03	CEID50/mL	4.50E+03	Matrix: 100% (3/3)H1: 100% (3/3)
	A/Michigan/272/2017	1.86E+01	TCID50/mL	4.50E+03	Matrix: 100% (3/3)H1: 100% (3/3)
	A/Idaho/07/2018	8.73E-01	TCID50/mL	4.50E+03	Matrix: 100% (3/3)H1: 100% (3/3)
	A/Wisconsin/505/2018	5.40E+00	TCID50/mL	4.50E+03	Matrix: 100% (6/6)H1: 83.3% (5/6)
	Guangdong-Maonan/SWL1536/19	1.93E+00	TCID50/mL	4.50E+03	Matrix: 100% (6/6)H1: 83.3% (5/6)
Influenza A	H1N1	Brisbane/02/18	3.97E+00	TCID50/mL	1.35E+06	Matrix: 100% (3/3)H1: 100% (3/3)
		A/St.Petersburg/61/2015	1.82E+03	CEID50/mL	4.50E+03	Matrix: 100% (3/3)H1: 100% (3/3)
	A/Bangladesh/3002/2015	7.78E+02	CEID50/mL	4.50E+03	Matrix: 100% (3/3)H1: 100% (3/3)
	A/Denver/1/57	6.84E+02	CEID50/mL	4.50E+03	Matrix: 100% (3/3)H1: 100% (3/3)
	New Caledonia/20/99	8.71E+00	TCID50/mL	4.50E+03	Matrix: 100% (3/3)H1: 100% (3/3)
	PR/8/34	1.58E+00	TCID50/mL	4.50E+03	Matrix: 100% (3/3)H1: 100% (3/3)
	Singapore/63/04	6.23E-01	TCID50/mL	4.50E+03	Matrix: 100% (3/3)H1: 100% (3/3)
	Solomon Islands/03/06	5.00E-01	TCID50/mL	4.50E+03	Matrix: 100% (3/3)H1: 100% (3/3)

Table 7. Inclusivity of LIAISON PLEX Respiratory Flex Assay

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Reportable Concentration Concentration % Detected (# Serovars/Group/Species Target & Subtype Value Detected/# Tested) Units copies/mL Matrix: 100% (3/3) Taiwan/42/06 3.58E-01 TCIDso/mL 4.50E+03 H1: 100% (3/3) Matrix: 0% (0/3)7 A/Ohio/09/2015 (Subtype N/A3 4.50E+03 H1: 100% (3/3)7 Synthetic DNA) H1N1v A/Ohio/09/2015 (Influenza A Matrix: 100% (3/3)7 N/A3 4.50E+03 Synthetic DNA) H1: 0% (0/3)7 Matrix: 100% (3/3) H1N2 A/swine/Ohio/09SW1484E/2009 4.15E+03 TCID50/mL 4.50E+03 H1: 100% (3/3) Matrix: 0% (0/3)7 A/Minnesota/19/2011 N/A3 4.50E+03 (Subtype Synthetic DNA) H1: 100% (3/3)7 H1N2v Matrix: 100% (3/3)7 A/Minnesota/19/2011 N/A3 4.50E+03 (Influenza A Synthetic DNA) H1: 0% (0/3)7 Matrix: 100% (3/3) A/Kansas/14/2017 NYMC X-327 1.87E+03 CEID50/mL 5.89E+03 H3: 100% (3/3) Matrix: 100% (3/3) A/Texas/71/2017 4.35E+01 FFU/mL 5.89E+03 H3: 100% (3/3) Matrix: 100% (3/3) A/Wisconsin/04/2018 5.89E+03 2.38E+01 FFU/mL H3: 100% (3/3) Matrix: 100% (3/3) A/Arizona/45/2018 8.27E+01 FFU/mL 5.89E+03 H3: 100% (3/3) Matrix: 100% (3/3) A/Hong Kong/45/2019 6.09E+01 FFU/mL 5.89E+03 H3: 100% (3/3) H3N2 Matrix: 100% (3/3) 5.89E+03 A/Tasmania/503/2020 1.99E+01 FFU/mL H3: 100% (3/3) Matrix: 100% (3/3) A/Delaware/01/2021 7.01E+01 FFU/mL 5.89E+03 H3: 100% (3/3) A/Singapore/INFIMH-16-Matrix: 100% (3/3) CEID50/mL 8.93E+02 5.89E+03 0019/2016 H3: 100% (3/3) Matrix: 100% (3/3) A/California/55/2020 8.55E+01 FFU/mL 5.89E+03 H3: 100% (3/3) Matrix: 100% (3/3) CEID50/mL A/Alaska/232/2015 1.14E+03 5.89E+03 H3: 100% (3/3) Matrix: 0% (0/3)7 A/Hawaii/28/2020 (Subtype N/A3 5.89E+03 Synthetic DNA) H3: 100% (3/3)7 H3N2v Matrix: 100% (3/3)7 A/Hawaii/28/2020 (Influenza A N/A3 5.89E+03 Synthetic DNA) H3: 0% (0/3)7 Matrix: 100% (3/3)8 HA4 A/Egypt/N03072/2010 4.70E-03 5.89E+03 Subtype: 0% (0/3)8 Matrix: 100% (3/3)8 HA4 HSMI A/Hubei/1/2010 3.97E-03 5.89E+03 Subtype: 0% (0/3)8 Matrix: 100% (3/3)8 A/Anhui/01/2005 HA4 5.89E+03 1.54E-02 Subtype: 0% (0/3)8 H7N2 A/turkey/Virginia/4529/2002 4.40E-02 HA4 5.89E+03 Matrix: 100% (3/3)8

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ReportableTarget & Subtype		Serovars/Group/Species	Concentration		Concentrationcopies/mL	% Detected (#Detected/# Tested)
	H7N7	A/mallard/Netherlands/12/2000	8.03E-03	HA4	5.89E+03	Subtype: 0% (0/3)8Matrix: 100% (3/3)8
	H9N2	A/Hong Kong/33982/2009	5.06E+03	CEID50/mL	5.89E+03	Subtype: 0% (0/3)8Matrix: 100% (3/3)8
Influenza B		B/Washington/02/2019 (VictoriaLineage)	1.97E+02	CEID50/mL	1.01E+03	100% (3/3)
		B/New Hampshire/01/2021(Victoria Lineage)	9.28E-01	TCID50/mL	1.01E+03	100% (3/3)
		B/Missouri/12/2018 (NA D197E)(Victoria Lineage)	1.35E+02	TCID50/mL	1.01E+03	100% (3/3)
		B/Wisconsin/10/2016 (NAI221V) (Yamagata Lineage)	1.81E+03	TCID50/mL	1.01E+03	100% (3/3)
		B/Indiana/17/2017 (NA 1221T)(Yamagata Lineage)	1.79E+03	TCID50/mL	1.01E+03	100% (3/3)
		B/Hawaii/01/2018 (NA D197N(Victoria Lineage)	4.56E+02	TCID50/mL	1.01E+03	100% (3/3)
		B/Oklahoma/10/2018 (NAD197N) (Yamagata Lineage)	1.85E+03	TCID50/mL	1.01E+03	100% (3/3)
		B/Michigan/01/2021 (VictoriaLineage)	7.91E+00	TCID50/mL	1.01E+03	100% (3/3)
		B/Hong Kong/286/2017 (VictoriaLineage)	2.80E+00	TCID50/mL	1.01E+03	100% (3/3)
		B/Colorado/6/2017 (VictoriaLineage)	2.33E+00	TCID50/mL	1.01E+03	100% (3/3)
		B/Texas/43/2019 (VictoriaLineage)	1.55E+00	TCID50/mL	1.01E+03	100% (3/3)
		B/Wisconsin/1/10 (YamagataLineage)	3.23E-01	TCID50/mL	1.01E+03	100% (3/3)
		B/Florida/02/06 (YamagataLineage)	1.60E-01	TCID50/mL	1.01E+03	100% (3/3)
		B/Florida/07/04 (YamagataLineage)	3.71E+01	TCID50/mL	1.01E+03	100% (3/3)
Parainfluenza 1		B/Phuket/3073/13 (YamagataLineage)	6.53E-01	TCID50/mL	1.01E+03	100% (3/3)
		N/A	3.19E+01	TCID50/mL	2.28E+03	83.3% (5/6)
		C35	2.62E+00	TCID50/mL	2.28E+03	100% (3/3)
Parainfluenza 2		N/A	4.65E+00	TCID50/mL	2.54E+04	100% (3/3)
		Greer	4.81E+00	TCID50/mL	2.54E+04	100% (3/3)
Parainfluenza 3		N/A	9.54E+00	TCID50/mL	5.79E+03	100% (3/3)
		ATCC-2011-5	1.78E+02	TCID50/mL	5.79E+03	83.3% (5/6)
		C243	8.83E+02	TCID50/mL	5.79E+03	100% (3/3)
		NIH 47885	3.57E+01	TCID50/mL	5.79E+03	100% (3/3)
Parainfluenza 4		4A	6.63E+00	TCID50/mL	1.73E+04	100% (3/3)
		4A M-25	5.94E+00	TCID50/mL	1.73E+04	100% (3/3)
		4B	5.40E+00	TCID50/mL	1.73E+04	100% (3/3)
		4B CH 19503	8.79E+01	TCID50/mL	1.73E+04	100% (3/3)
Respiratory SyncytialVirus		A 2006 Isolate	3.34E+00	TCID50/mL	1.15E+04	100% (3/3)
		A2	1.10E+03	PFU/mL	1.15E+04	100% (3/3)
		A Long	1.11E+03	PFU/mL	1.15E+04	100% (3/3)
		B CH93(18)-18	1.46E+01	TCID50/mL	4.82E+04	100% (3/3)
		B WV/14617/85	2.12E+00	TCID50/mL	4.82E+04	100% (3/3)
		B 18537	3.16E+00	PFU/mL	4.82E+04	100% (3/3)
ReportableTarget & Subtype	Serovars/Group/Species	Concentration		Concentrationcopies/mL	% Detected (#Detected/# Tested)
	USA-WA1/2020	1.37E+01	TCID50/mL	2.40E+04	100% (3/3)
	B.1.1.529 BA.1: USA/MD-HP20874/2021 (Omicron)	9.59E-01	TCID50/mL	2.40E+04	100% (3/3)
	Italy-INMI1	1.44E+02	TCID50/mL	2.40E+04	100% (3/3)
	Hong Kong/VM20001061/2020	2.37E+00	TCID50/mL	2.40E+04	100% (3/3)
SARS-CoV-2	B.1_2020: USA/NY-Wadsworth-103677-01/2020	9.74E-01	TCID50/mL	2.40E+04	100% (3/3)
	B.1.1.7:England/204820464/2020(Alpha)	1.53E+01	TCID50/mL	2.40E+04	100% (3/3)
	B.1.1.7:USA/CA_CDC_5574/2020 (Alpha)	6.61E+00	TCID50/mL	2.40E+04	100% (3/3)
	B.1.351: South Africa/KRISP-K005325/2020 (Beta)	5.84E+00	TCID50/mL	2.40E+04	100% (3/3)
	P1: Japan/TY7-503/2021(Gamma)	5.65E+00	TCID50/mL	2.40E+04	100% (3/3)
	P2_2021: NY-Wadsworth-21006055-01/2021 (Zeta)	8.82E+00	TCID50/mL	2.40E+04	100% (3/3)
	B.1.526_2021: USA/NY-Wadsworth-21025952-01/2021Isolate 1 (Lota)	1.39E+01	TCID50/mL	2.40E+04	100% (3/3)
	B.1.617.1: USA/CA-Stanford-15_S02/2021 (Kappa)	1.22E+01	TCID50/mL	7.20E+046	100% (3/3)
	B.1.617.2: USA/PHC658/2021(Delta)	9.42E+00	TCID50/mL	2.40E+04	100% (3/3)

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4No supplier concentration listed.

2Clinical samples quantified in copies/mL based on digital droplet PCR (ddPCR) analysis.

3 Synthetic DNA quantified in copies/mL based on spectrophotometric analysis and optical density measurements.

4Titer determined through hemagglutination assay using 0.5% turkey red blood cells.

් No additional strain information was provided by the supples tested within the same species were distinguished using the vendor catalog number.

် Four strains (influenza A Brisbane (02/18), coronavirus 229E (VR-740) and SARS-CoV-2 (Stanford) were detected at 9X LoD and one strain (B. holmesii (CIP 104396)) was detected at 27X LoD.

'Subtype synthetic DNA and influenza A synthetic DNA were expected to the subtype and influenza A targets, respectively.

³Matrix positive, subtype negative results for infl2, H7N2, H7N2, H7N7, and H9N2 are expected results based on the assay design.

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In Silico Inclusivity for SARS-CoV-2 and Influenza

In addition to wet testing, in silico analyses for SARS-CoV-2 and Influenza were performed.

SARS-CoV-2:

For SARS-CoV-2, 5,622,325 sequences in GISAID (as of July 31, 2023) were included in the analysis. The LIAISON PLEX Respiratory Flex Assay targets three SARS-CoV-2 gene regions (E, ORF1ab, and ORF3a). The Respiratory Flex Assay result logic states that if at least 1 of these targets is detected, SARS-CoV-2 is positive. This same result logic was implemented for the in silico inclusivity assessment. Of the sequences included in this evaluation, 99.94% (5,619,069/5,622,325) have no mismatch in at least one gene oligo set and thus are predicted to be detected by the Respiratory Flex Assay. Of the 0.06% (3,256/5,622,325) of sequences with mismatches in at least one oligo binding region in all 3 SARS-CoV-2 target genes, a Tm analysis revealed that amplification/hybridization were expected to occur. Thus, it is expected that 100% of SARS-CoV-2 sequences evaluated in this study will be detected by the assay.

Influenza:

For influenza A, influenza A H1, influenza A H3, and influenza B, sequences uploaded to GISAID between September 1, 2015 and July 5, 2023 were included in the analysis. The following number of sequences were included in the evaluation of influenza A, A H1, A H3, and influenza B: 112,056, 54,364, 104,428, 26,470. Of the 26,470 influenza B sequences, 66.1% (17,509/26,470) were Victoria lineage, 30.9% (8,167/26,470) were Yamagata lineage, and 3.0% (794/26,470) were of unknown/unclassified lineage. The influenza in silico inclusivity analysis results are shown in Table 8. Based on the reactivity criteria (>90% homology), 99.9% (112,034/112,056) of influenza A sequences are expected to be detected, 98.9% (53,778/54,364) of influenza A H1 sequences are expected to be detected, 99.9% (104,315/104,428) of influenza A H3 sequences are expected to be detected, and 99.9% (26,433/26,470) of influenza B sequences are expected to be detected.

ReportableTarget	Target Gene	of Sequences inAlignment	# of Sequences withPercent Oligo Identify>90%
Influenza A	Matrix protein (MP)	112,056	112,0341
Influenza A H1	HA	54,364	53,778
Influenza A H3	HA	104,428	104,315
Influenza B	Non-structural protein (NS)	26,470	26,4332

Table 8. Influenza In Silico Inclusivity Results

1 Analysis included influenza A subtype H0, H1, H3, H5, H7, H9, and H10 strains.

2 Analysis included 17,509 Victoria lineage strains, 8,167 Yamagata lineage strains of unknown lineage.

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f. Analytical specificity
Cross-Reactivity and Microbial Interference:

Off-Panel Cross Reactivity

Laboratory (Wet Testing)

Cross-reactivity was assessed in the laboratory by wet testing 60 off-panel viral, fungal, and bacterial organisms that may be found in a respiratory tract clinical specimen. The potential cross-reacting organisms were spiked into simulated NPS matrix that was negative for all targets on the assay. Bacteria were tested at concentrations ≥ 1x10° CFU/mL (or equivalent) and viruses were tested at ≥ 1x105 TCID50/mL (or equivalent), or the highest available concentration. For Mycobacterium tuberculosis, genomic DNA (quantified in ng/uL) was evaluated to minimize pathogenic exposure to the test operator.

Of the 60 potential cross-reacting off-panel organisms tested in the laboratory (Table 9), 59 organisms yielded negative results for all targets and are considered non-reactive with the LIAISON PLEX Respiratory Flex Assay. Mycoplasma qenitalium cross-reacted with the Mycoplasma pneumoniae assay at a concentration of 4x10 CCU/mL. No cross-reactivity was observed when Mycoplasma genitalium was tested at a lower concentration of 1x10f CCU/mL.

Organism	Conc./Unit	Organism	Conc./Unit
Acinetobacter baumannii	1x106 CFU/mL	Legionella pneumophila	4x105 CFU/mL3
Aspergillus flavus	1x106 CFU/mL	Listeria innocua	1x106 CFU/mL
Aspergillus fumigatus	4x105 CFU/mL3	Listeria monocytogenes	1x106 CFU/mL
Bordetella avium	1x106 CFU/mL	Measles	1x105 TCID50/mL
Bordetella bronchiseptica	1x106 CFU/mL	MERS-CoV	NA2
Bordetella hinzii	1x106 CFU/mL	Moraxella catarrhalis	1x106 CFU/mL
Bordetella petrii	1x106 CFU/mL	Mumps Virus	1x105 TCID50/mL
Bordetella trematum	1x106 CFU/mL	Mycobacteriumtuberculosis(H37Rv gDNA)	2.88 ng/uL
Bordetella parapertussisBpp5 (synthetic DNA)1	1x106 copies/mL	Mycoplasmagenitalium3	4x106 CCU/mL
			1x106 CCU/mL
			4x105 CCU/mL
Candida albicans	1x106 CFU/mL	Mycoplasma hominis	1x106 CFU/mL
Candida glabrata	1x106 CFU/mL	Nasal Wash (pooled)	NA2
Chlamydia trachomatisSerovar D	1x106 IFU/mL	Neisseria elongata	1x106 CFU/mL
Coronavirus-SARS	NA2	Neisseria gonorrhoeae	1x106 CFU/mL
Corynebacteriumdiphtheriae	1x106 CFU/mL	Neisseria lactamica	1x106 CFU/mL
Corynebacteriumpseudodiphtheriticum	1x106 CFU/mL	Neisseria meningitidis	1x106 CFU/mL
Corynebacterium striatum	1x106 CFU/mL	Neisseria mucosa	1x106 CFU/mL
Cytomegalovirus	1x105 TCID50/mL	Neisseria sicca	1x106 CFU/mL
Epstein Barr Virus	1x105 copies/mL	Pneumocystis jiroveci	1x106 CFU/mL

Table 9. Organisms Tested for Potential Cross-Reactivity (Off-Panel)

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Organism	Conc./Unit	Organism	Conc./Unit
Escherichia coli	1x106 CFU/mL	Proteus vulgaris	1x106 CFU/mL
Fluoribacter bozemanae	4x106 CFU/mL	Pseudomonasaeruginosa	1x106 CFU/mL
Fusobacteriumnecrophorum	1x106 CFU/mL	Serratia marcescens	1x106 CFU/mL
Haemophilus influenzae	1x106 CFU/mL	Staphylococcus aureus	1x106 CFU/mL
Haemophilusparainfluenzae	1x106 CFU/mL	Staphylococcusepidermidis	1x106 CFU/mL
Herpes Simplex VirusType 1	1x105 TCID50/mL	Staphylococcushaemolyticus	1x106 CFU/mL
Klebsiella pneumoniae	1x106 CFU/mL	Streptococcusagalactiae	1x106 CFU/mL
Lactobacillus acidophilus	1x106 CFU/mL	Streptococcuspneumoniae	1x106 CFU/mL
Lactobacillus plantarum	1x106 CFU/mL	Streptococcuspyogenes	1x106 CFU/mL
Legionella anisa	1x106 CFU/mL	Streptococcussalivarius	1x106 CFU/mL
Legionella feeleii	1x106 CFU/mL	Ureaplasmaurealyticum	1x106 CCU/mL
Legionella longbeachae	1x106 CFU/mL	Varicella-Zoster Virus	2.34x104TCID50/mL3

CFU = Colony Forming Units; CCU = Colony Changing Units; TCID50 = Median Tissue Culture Infectious Dose.

14 portion of the B. parapertussis Bpp5 genome was identified by in-silico analysis as potentially cross-reactive with B. pertussis. Synthetic DNA was tested that matched the region of high homology in the assay. Testing was included in the off-panel testing since the targeted sequence was not expected to be detected as B. parapertussis by the assay.

2No concentration provided by the supplier.

3The highest possible concentration was tested.

In Silico Cross-Reactivity

In silico analysis of assay specificity/exclusivity was performed by conducting a BLAST comparison of the assay's oligos sequences to the GenBank nt sequence database, as of July 14, 2023. Sequences for 83 off-panel organisms (68 bacteria/fungi and 15 viruses) that can be found in a respiratory specimen were included. Additionally, sequences for all on-panel organisms were included to evaluate intra-panel cross-reactivity. A summary of the results from the analysis is provided in Table 10. The LIAISON PLEX Respiratory Flex assays were shown to be specific for their respective analytes with the following exceptions:

Cross-reaction of the Adenovirus assays with closely related Adenovirus G (serotype 52) strains.
Cross-reaction of the SARS-CoV-2 assays with closely related bat and pangolin coronavirus sequences;
Cross-reaction of the B. parapertussis assay with strains of B. bronchiseptica that carry IS1001;
Cross-reaction of the influenza A H1 subtyping assay with 3 swine H3N2 strains and 1 avian H6N1 strain;
Cross-reaction of the influenza A H3 subtyping assay with 59 swine H1N1 and swine H1N2 strains, 1 duck H5N2 strain, 1 ostrich H7N1 strain, 1 avian H7N9 strain, 1 avian H8N4 strain, and 1 avian H11N9 strain.

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Reportable Target	Predicted Cross-Reaction
Adenovirus	Adenovirus G (serotype 52) - strains
SARS-CoV-2	Bat coronavirus and Bat SARS-like coronavirus (accessions MG772933,MG772934, and MN996532)1
Bordetella parapertussis	Bordetella bronchiseptica containing IS1001 element (accessions JX013523to JX013527 and CP022962)²
Influenza A H1	H5N1 (accession CY110922)3;swine H3N2 (accessions KM110061, KM110062, KM110063, andOM935891);avian H6N1 (accession OP888980)
Influenza A H3	swine H1N1 and swine H1N2 – 59 strains;duck H5N2 (accession OK103962);ostrich H7N1 (accession AF202244);avian H7N9 (accession KP413675);avian H8N4 (accession OK103964);avian H11N9 (accession OK103956)

Table 10. Organisms Predicted by In Silico Analyses to Cross-React with the Respiratory Flex Assay.

1 is unlikely that Bat coronavirus and Bat SARS-like coronavirus would be present in human clinical NPS specimens; but if present, the cross-reactive product(s) produced by the LIAISON PLEX Respiratory Flex Assay will be reported as SARS-CoV-2.

²The LIAISON PLEX Respiratory Flex Assay contains primers designed to target the B. parapertussis IS1001 insertion sequence. Some strains of Bordetella bronchiseptica, which is rarely isolated from humans, carry the same sequence that is targeted by LIALSON PLEX Respiratory Flex primers for B. parapertussis. If present, the LIAISON PLEX Respiratory Flex Assay will report these specimens as B. parapertussis.

³This H5N1 human strain sequence containing H1N1 sequence fragments. Therefore, detection of this sequence by the H1 oligos is not considered a cross-reaction.

On-Panel Cross Reactivity

Potential intra-panel cross-reactivity was evaluated using 28 on-panel organisms. The potential cross-reacting on-panel organisms were spiked into simulated NPS matrix that was negative for all targets on the assay. Bacteria were tested at concentrations ≥ 1x10 CFU/mL (or equivalent) and viruses were tested at ≥ 1x105 TCID50/mL (or equivalent), or the highest available concentration.

All 28 on-panel organisms tested (Table 11) yielded the expected on-panel result and did not cross-react with other target assays of the LIAISON PLEX Respiratory Flex Assay.

Organism	Conc./Unit
Adenovirus	1x105 TCID50/mL
Bordetella holmesii	1x106 CFU/mL
Bordetella parapertussis	1x106 CFU/mL
Bordetella pertussis	1x106 CFU/mL
Chlamydia pneumoniae	1x106 IFU/mL
Human Coronavirus 229E	1x105 TCID50/mL
Human Coronavirus HKU1	6.62x104 copies/mL
Human Coronavirus NL63	1x105 TCID50/mL
Human Coronavirus OC43	1x105 TCID50/mL

Table 11. Organisms Tested for Potential Cross-Reactivity (On-Panel)

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Conc./Unit Organism Echovirus 1x105 TCID50/mL (Enterovirus/Rhinovirus) Human Metapneumovirus 1x105 TCID50/mL Influenza B (Washington/02/2019/Victoria 1x105 TCID50/mL Lineage) Influenza B (Phuket/3073/13/Yamagata 1x105 TCID50/mL Lineage) 1x106 CCU/mL Mycoplasma pneumoniae Parainfluenza 1 1x105 TCID50/mL Parainfluenza 2 1x105 TCID50/mL Parainfluenza 3 1x108 TCID50/mL Parainfluenza 4 1x105 TCID50/mL RSV A 1x105 PFU/mL RSV B 1x10> TCID50/mL Influenza A H1N1 1x105 TCID50/mL Influenza A H3N2 1x10 TCID50/mL Influenza A H5N11 2x107 copies/mL Influenza A H7N21 6x106 copies/mL Influenza A H7N71 2x107 copies/mL Influenza A H9N21 4x108 copies/mL Influenza A H1N2² 3x107 copies/mL 1x105 TCID50/mL SARS-CoV-2

LIAISON PLEX® Respiratory Flex Assay Traditional 510(k) Submission

CFU = Colony Forming Units; IFU = Inclusion Forming Units; TCID50 = Median Tissue Culture Infectious Dose; PFU = Plaque Forming Units

"These influenza A non-subtype H1/H3 strains are expected to be inclusive to the influenza A matrix target, only (i.e., are expected to be reported as influenza A positive, subtype H3 negative). All were negative for both subtype H1 and subtype H3, as anticipated.

²This influenza A H1N2 strain is expected to be influenza A matrix target and influenza A subtype H1 target. This strain was positive for both the influenza A matrix target and influenza A subtype H1 target, as expected.

Microbial Interference

The impact of 16 potentially interfering non-panel microbial organisms commonly found in nasopharyngeal swab samples were tested (Table 12) in the presentative assay panel targets. Each potentially interfering non-panel organism was tested at a high concentration of ≥ 1x10° CFU/mL (or equivalent) for bacteria and 1x107 TCIDsg/mL (or equivalent) for viruses, or the highest concentration available, in the presence of a multi-analyte panel consisting of five assay targets (B. pertussis, Adenovirus, influenza B, human metapneumovirus, and SARS-CoV-2) at a low concentration (3X LoD).

None of the potentially interfering organisms tested at high concentration interfered with detection for panel targets at a low concentration, except for Streptococcus pyogenes at 1x10° CFU/mL and Legionella pneumophilia at 4x105 CFU/mL, which both interfered with detection of Adenovirus in 1 of 6 replicates (83.3% positivity).

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Candida albicans	Pseudomonas aeruginosa
Corynebacterium diphtheriae	Streptococcus pneumoniae
Cytomegalovirus	Streptococcus pyogenes
Haemophilus influenzae	SARS1
Herpes Simplex Virus 1	Legionella pneumophilia2
MERS1	Measles Virus
Neisseria meningitidis	Moraxella catarrhalis
Staphylococcus aureus	Mumps

Table 12. Organisms Tested for Potential Microbial Interference

'No concentration was provided for material. Material was tested at the highest available concentration by diluting stock material directly into representative multi-analyte panel preparation.

2Tested at the highest available concentration of 4x105 CFU/mL

Interfering Substances:

The potential inhibitory effect of non-microbial substances (endogenous and exogenous) expected to be found in nasopharyngeal swab (NPS) specimens or introduced during sample handling, were evaluated for the LIAISON PLEX Respiratory Flex Assay. Potential interference from 36 interfering substances were evaluated in the presence and absence of a multi-analyte panel of targets (Table 13). Each interfering substance tested was diluted to a clinically relevant concentration and tested in the presence of a positive and negative target in triplicate. The positive target was a multianalyte panel consisting of five assay targets (B. pertussis, Adenovirus, influenza B, human metapneumovirus, and SARS-CoV-2) at a low concentration (3X LoD), while the negative target was a simulated NPS matrix. Interference was observed for the substances/concentrations shown in Table 14.

Substance/Class	Description/Active Ingredient	Concentration Tested
Nasal Corticosteroid	Beclomethasone dipropionate	25 µg/mL
Anesthetic	Benzocaine	10% w/v
Nasal Corticosteroid	Budesonide	$3.4x10^{-2}$ µmol/L
Nasal Corticosteroid	Dexamethasone	30.6 µmol/L
Nasal Corticosteroid	Flunisolide	25 µg/mL
FLONASE Sensimist Allergy Relief	Fluticasone furoate	$2.84x10^{-3}$ µmol/L
Fluticasone Propionate NasalSpray	Fluticasone propionate	$2.84x10^{-3}$ µmol/L
DNA	Human DNA	20 ng/µL
Nasal Wash	Human Nasal Wash	9.1%
Sputum/Mucus	Human Sputum/Mucus	1 swab/1mL sample1
	Human Sputum/Mucus	1 swab/2mL sample2
Human Blood	Human Whole Blood	5.0% v/v
	Human Whole Blood	4.5% v/v
	Human Whole Blood	4.0% v/v
Human Cells	Leukocytes	1000 cells/µL
	Leukocytes	666.7 cells/µL
	Leukocytes	333.3 cells/µL
Oral Anesthetic and Analgesic	Menthol	1% w/v
Nasal Corticosteroid	Mometasone furoate	$8.63x10^{-4}$ µmol/L
Mucin	Mucin, bovine submaxillary Type I-S	100 µg/mL
Mucin	Mucin, porcine stomach Type II	100 µg/mL
Mucin	Mucin, porcine stomach Type III	100 µg/mL

Table 13: Interfering Substances Tested

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Substance/Class	Description/Active Ingredient	Concentration Tested
Antibiotic, Nasal Ointment	Mupirocin	3.0 µmol/L
Anti-viral	Oseltamivir Phosphate	1.28 µmol/L
Afrin Nasal Spray	Oxymetazoline	1% v/v
Nasal Decongestant	Phenylephrine	$1.79x10^{-1}$ µmol/L
Saline Nasal Spray	Sodium Chloride	1% v/v
Nasal Corticosteroid	Triamcinolone acetonide	25 µg/mL
Antibiotic	Tobramycin	76.0 µmol/L
Anti-viral	Zanamivir	100 µg/mL
Anti-viral	Zinc	5% v/v
ZICAM Nasal Spray	Galphimia GlaucaHistaminum HydrochloricumLuffa operculataSulfur	1% v/v
NPS Swab	Nylon swab (Copan)	NA
Transport Media	Universal Transport Medium (Copan)	100%

4 nylon nasopharyngeal swab was fully coated with human sputum/mucus and then eluted into 1 mL of simulated NPS matrix, containing 5 representative target organisms at 3x LoD. The eluent was subsequently tested with the Respiratory Flex Assay.

3 nylon nasopharyngeal swab was fully coated with human sputum/mucus and then eluted into 2 mL of simulated NPS matrix, containing 5 representative target organisms at 3x LoD. The eluent was subsequently tested with the Respiratory Flex Assay.

Active	Test Conc.	% Positivity (# Detected/# Tested)
Ingredient		Adenovirus	B. pertussis	HumanMetapneumovirus	Influenza B	SARS-CoV-2
HumanSputum/Mucus	1 swab/1mLsample	100%(6/6)	100%(6/6)	33.3%(2/6)1	66.7%(4/6)1	83.3%(5/6)1
	1 swab/2mLsample	100%(3/3)	100%(3/3)	100%(3/3)	100%(3/3)	100%(3/3)
Human WholeBlood	5.0% v/v	100%(6/6)	83.3%(5/6)1	66.7% (4/6)1	83.3%(5/6)1	100%(6/6)
	4.5% v/v	100%(3/3)	100%(3/3)	66.7% (2/3)1	100%(3/3)	100%(3/3)
	4.0% v/v	100%(3/3)	100%(3/3)	100%(3/3)	100%(3/3)	100%(3/3)
Leukocytes	1000 cells/µL	100%(3/3)	100%(3/3)	33.3%(1/3)2	100%(3/3)	66.7%(2/3)2
	666.7 cells/µL	100%(3/3)	100%(3/3)	33.3%(1/3)	33.3%(1/3)	33.3%(1/3)
	333.3 cells/µL	100%(3/3)	100%(3/3)	100%(3/3)	100%(3/3)	100%(3/3)
Mupirocin	3.0 µmol/L	100%(6/6)	100%(6/6)	100%(6/6)	83.3%(5/6)3	100%(6/6)
Tobramycin	76.0 µmol/L	100%(5/5)	100%(5/5)	80%(4/5)4	100%(5/5)	80%(4/5)4

Table 14 Substances that Interfered with Detection of at Least One Target Organism

1 Unexpected negative results were obtained during original and repeat testing was performed at more dilute concentrations until 100% detection occurred.

² The original three replicates tested resulted in 33.3% (1/2) replicates being invalid and 50% (1/2) positivity (2/2) for Adenovirus, B. pertussis, Flu B, and SARS-CoV-2. New test material was prepared and tested, resulting in 66.6% (2/3) invalid results, and 0% (0/1) positivity for hMPV and SARS-CoV-2. Therefore, testing was performed at more dilute concentrations until 100% detection occurred. ?The original three replicates tested resulted in 66.7% (2/3) positivity for influenza B. New test material was prepared and tested, resulting in 100% (3/3) positivity for influenza B.

"The original three replicates tested resulted in 33.3% (1/3) replicates being invalid and 50% (1/2) positivity for hMPV and SARS-CoV-2. New

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test material was prepared and tested, resulting in 100% (3/3) positivity for hMPV and SARS-CoV-2.

Competitive Inhibition/Co-infection:

Competitive inhibition of the LIAISON PLEX Respiratory Flex Assay was assessed by testing 27 pairings of clinically prevalent co-infections, as listed in Table 12. A single pairing consisted of one target at a high concentration with another target at a low concentration (Table 15). All low concentration targets were tested at 3X LoD. All high concentration targets were tested at 1x105 TCID50/mL (or equivalent), or the highest available concentration. Testing of each combination was performed in triplicate, at a minimum. Of the 54 combinations tested, 48 combinations did not show evidence of inhibition and generated the expected results for both targets tested. Interference was observed for the following co-infections:

. Parainfluenza 3 (low concentration) in the presence of human coronavirus OC43 (high concentration of 1x10 TCIDsg/mL). Competitive interference was no longer observed when the human coronavirus OC43 concentration was decreased to 5x104 TCID50/mL.
. Parainfluenza 3 (low concentration) in the presence of adenovirus 37D (high concentration of 1x10 ° TCID50/mL).
RSV A (low concentration) in the presence of adenovirus 37D (high concentration of 1x105 TCIDsofmL). Competitive interference was no longer observed when the adenovirus 37D concentration was decreased to 5x104 TCID50/mL.
Flu A H3N2 (low concentration) in the presence of adenovirus 37D (high concentration of 1x10 TCID30 mL). Specifically, detection of influenza A (matrix) was decreased in the presence of adenovirus 37D at a high concentration of 1x105 TCIDso/mL. Competitive interference was no longer observed when the adenovirus 37D concentration was decreased to 5x104 TCID50/mL.
. Human coronavirus 229E (low concentration) in the presence of SARS-CoV-2 (high concentration of 1x105 TCID50/mL).
SARS-CoV-2 (low concentration) in the presence of human coronavirus OC43 (high concentration of 1x10 TCID50/mL).

Target 1 (High Conc.)	Conc. (TCID50/mL)2	Target 2(Low Conc.)1	% Detected(# Detected/ # Tested)
Organism		Organism	Target 1	Target 2
Adenovirus 37D	1x105	Rhinovirus	100% (3/3)	100% (3/3)
Rhinovirus	1x105	hMPV	100% (3/3)	100% (3/3)
Adenovirus 37D	1x105	Coronavirus NL63	100% (3/3)	100% (3/3)
Rhinovirus	1x105	RSV A	100% (3/3)	100% (3/3)
Coronavirus OC43	1x105	PIV-3	100% (6/6)	66.7% (4/6)3
Coronavirus OC43	5x104	PIV-3	100% (3/3)	100% (3/3)
Rhinovirus	1x105	Coronavirus NL63	100% (3/3)	100% (3/3)
Adenovirus 37D	1x105	hMPV	100% (3/3)	100% (3/3)
Rhinovirus	1x105	Flu A H3N2	100% (3/3)	Matrix:100% (3/3)Subtype H3:100% (3/3)
Adenovirus 37D	1x105	PIV-3	100% (7/7)	85.7% (6/7)4
Coronavirus NL63	1x105	hMPV	100% (3/3)	100% (3/3)
Target 1 (High Conc.)		Target 2(Low Conc.)¹	% Detected(# Detected/ # Tested)
Organism	Conc. (TCID₅₀/mL)²	Organism	Target 1	Target 2
Rhinovirus	1x10⁵	SARS-CoV-2	100% (3/3)	100% (3/3)
Rhinovirus	1x10⁵	PIV-3	100% (3/3)	100% (3/3)
Adenovirus 37D	1x10⁵	RSV A	100% (6/6)	66.7% (4/6)⁵
Adenovirus 37D	5x10⁴	RSV A	100% (3/3)	100% (3/3)
Rhinovirus	1x10⁵	PIV-4	100% (3/3)	100% (3/3)
Rhinovirus	1x10⁵	PIV-1	100% (3/3)	100% (3/3)
Adenovirus 37D	1x10⁵	Flu A H3N2	100% (6/6)	Matrix:66.7% (4/6)⁶Subtype H3:100% (6/6)
Adenovirus 37D	5x10⁴	Flu A H3N2	100% (3/3)	Matrix:100% (3/3)Subtype H3:100% (3/3)
Rhinovirus	1x10⁵	Flu A H1N1	100% (3/3)	Matrix:100% (3/3)Subtype H1:100% (3/3)
SARS-CoV-2	1x10⁵	Flu A H3N2	100% (6/6)	Matrix:100% (3/3)Subtype H3:100% (3/3)
SARS-CoV-2	1x10⁵	Flu B	100% (3/3)	100% (3/3)
SARS-CoV-2	1x10⁵	Coronavirus 229E	100% (6/6)	83.3% (5/6)⁷
SARS-CoV-2	1x10⁵	Coronavirus NL63	100% (3/3)	100% (3/3)
SARS-CoV-2	1x10⁵	Coronavirus OC43	100% (3/3)	100% (3/3)
SARS-CoV-2	1x10⁵	Coronavirus HKU1	100% (3/3)	100% (3/3)
SARS-CoV-2	1x10⁵	RSV A	100% (3/3)	100% (3/3)
SARS-CoV-2	1x10⁵	Adenovirus 3B	100% (3/3)	100% (3/3)
SARS-CoV-2	1x10⁵	Adenovirus 4E	100% (3/3)	100% (3/3)
SARS-CoV-2	1x10⁵	Adenovirus 7A	100% (3/3)	100% (3/3)
Rhinovirus	1x10⁵	Adenovirus 37D	100% (3/3)	100% (3/3)
hMPV	1x10⁵	Rhinovirus	100% (3/3)	100% (3/3)
Coronavirus NL63	1x10⁵	Adenovirus 37D	100% (3/3)	100% (3/3)
RSV A	1x10⁵ (PFU/mL)	Rhinovirus	100% (3/3)	100% (3/3)
PIV-3	1x10⁵	Coronavirus OC43	100% (3/3)	100% (3/3)
Coronavirus NL63	1x10⁵	Rhinovirus	100% (3/3)	100% (3/3)
hMPV	1x10⁵	Adenovirus 37D	100% (3/3)	100% (3/3)
Flu A H3N2	1x10⁵ (CEID₅₀/mL)	Rhinovirus	100% (3/3)	100% (3/3)
PIV-3	1x10⁵	Adenovirus 37D	100% (3/3)	100% (3/3)
hMPV	1x10⁵	Coronavirus NL63	100% (3/3)	100% (3/3)
SARS-CoV-2	1x10⁵	Rhinovirus	100% (3/3)	100% (3/3)
PIV-3	1x10⁵	Rhinovirus	100% (3/3)	100% (3/3)
Target 1 (High Conc.)		Target 2(Low Conc.)1	% Detected(# Detected/ # Tested)
Organism	Conc. (TCID50/mL)2	Organism	Target 1	Target 2
RSV A	1x105 (PFU/mL)	Adenovirus 37D	100% (3/3)	100% (3/3)
PIV-4	1x105	Rhinovirus	100% (3/3)	100% (3/3)
PIV-1	1x105	Rhinovirus	100% (3/3)	100% (3/3)
Flu A H3N2	1x105 (CEID50/mL)	Adenovirus 37D	Matrix: 100%(3/3)Subtype H3:100% (3/3)	100% (3/3)
Flu A H1N1	1x105 (CEID50/mL)	Rhinovirus	Matrix: 100%(3/3)Subtype H1:100% (3/3)	100% (3/3)
Flu A H3N2	1x105 (CEID50/mL)	SARS-CoV-2	Matrix: 100%(3/3)Subtype H3:100% (3/3)	100% (3/3)
Influenza B	1x105	SARS-CoV-2	100% (3/3)	100% (3/3)
Coronavirus 229E	1x105	SARS-CoV-2	100% (3/3)	100% (3/3)
Coronavirus NL63	1x105	SARS-CoV-2	100% (3/3)	100% (3/3)
Coronavirus OC43	1x105	SARS-CoV-2	100% (6/6)	83.3% (5/6)8
Coronavirus HKU1	1.31x104 (copies/mL)	SARS-CoV-2	100% (3/3)	100% (3/3)
RSV A	1x105 (PFU/mL)	SARS-CoV-2	100% (3/3)	100% (3/3)
Adenovirus 3B	1x105	SARS-CoV-2	100% (3/3)	100% (3/3)
Adenovirus 4E	1x105	SARS-CoV-2	100% (3/3)	100% (3/3)
Adenovirus 7A	1x105	SARS-CoV-2	100% (3/3)	100% (3/3)

Table 15. Summary of Competitive Inhibition Results

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1Low concentration target organisms were prepared at 3x LoD.

2Concentrations are in TCID50/mL, unless otherwise noted.

³ Inexpected negative results were obtained and repeat testing for parainfluenza 3, therefore testing was performed at more dilute coronavirus concentrations until 100% detection of parainfluenza 3 occurred.

"The original three replicates tested resulted in 66.7% (2/3) positivenza 3. New test material was prepared and tested, resulting in 100% (4/4) positivity for parainfluenza 3. Four replicates were performed during rather than three because a single SARS-Col-2 result was obtained during retesting. ් Unexpected negative results were obtained during original and repeat testing was performed at more dilute adenovirus concentrations until 100% detection of RSV A occurred.

็ Unexpected negative results were obtainal and repeat testing for influenza A (matrix), therefore testing was performed at more diute adenovirus concentrations until 100% detection of influenza A (matrix) occurred.

7The original three replicates tested resulted in 66.7% (2/3) positivity for coronavirus. New tested, resulting in 100% (3/3) positivity for coronavirus.

°The original three replicates tested resulted in 66.7% (2/3) positivity for SARS-CoV-2. New test material was prepared and tested resulting in 100% (3/3) positivity for SARS-CoV-2.

Carry-Over and Cross-Contamination:

Carry-over and cross contamination for the LIAISON PLEX Respiratory Flex Assay was evaluated by testing positive and negative samples in an alternating series. A multi-analyte panel consisting of five assay targets (B. pertussis, Adenovirus, influenza B, human metapneumovirus, and SARS-CoV-2) at a high concentration (B. pertussis at 1x10 CFU/mL and the viral targets at 1x10 TCIDso/mL) was prepared in simulated NPS matrix and used for positive samples. Simulated negative NPS matrix was used for negative samples. Positive samples were tested in modules adjacent to negative samples in order to evaluate possible cross contamination. Immediately following the testing of a positive sample, a negative sample was run in the

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same module to evaluate possible carry-over contamination. This alternating series of positive and negative samples was continued across five consecutive runs using two LIAISON PLEX Systems for a total of 30 positive and 30 negative tests. No carry-over or cross contamination was observed.

g. Assay cut-off
The specific assay parameters for the LIAISON PLEX® Respiratory Flex Assay are considered confidential and proprietary.

Comparison Studies:

h. Method comparison with predicate device:
Refer to Section 2 Clinical Performance.
i. Matrix Comparison:
Not applicable

2. Clinical Performance:

Prospective Clinical Evaluation

A multi-site prospective clinical study established the clinical performance of the LIAISON PLEX Respiratory Flex Assay for the detection and identification of bacteria and viral targets from nasopharyngeal swab (NPS) specimens transported in Copan Universal Transport Medium™ or BD™ Universal Viral Transport Media, collected from patients exhibiting clinical signs and symptoms of respiratory tract infection (RTI). The clinical performance of the LIAISON PLEX Respiratory Flex Assay was evaluated using NPS clinical specimens prospectively collected between October 2022 to April 2023 from six geographically diverse clinical sites within the United States. The clinical study included remnant, de-identified specimens collected from pediatric and adult patients exhibiting clinical signs and symptoms of respiratory tract infections. Specimens were stored refrigerated at 2-8°C for up to 72-hours before testing (i.e., Category I specimens) or if they could not be tested within 72-hours, after freezing at -70℃ (Category II specimens).

A total of 1911 unique prospective specimens that met the pre-determined inclusion criteria were enrolled in the study. Clinical runs and re-runs using LIAISON PLEX Respiratory Flex Assay were tested on the LIAISON PLEX System by trained operators at four clinical sites. Out of the 1911 specimens enrolled in the prospective study, 68 specimens were disqualified and removed from further analysis. Most of the specimen exclusions were due to non-compliance with the study protocol or due to not meeting the inclusion criteria after enrollment. This left 1843 clinical specimens for evaluation. Of these 1843 specimens, 66.3% (1221/1843) were tested fresh, while 33.7% (622/1843) were tested frozen. Patient demographic information for the 1843 prospectively collected NPS specimens is presented in Table 16.

Table 16. Prospective Study Demographic Details (N=1843)

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	# Specimens (%)
Gender
Male	839 (45.5%)
Female	1004 (54.5%)
Total	1843 (100.0%)
Age (years)
0-1	350 (19.0%)
>1-5	274 (14.9%)
>5-21	447 (24.3%)
>21-65	535 (29.0%)
> 65	237 (12.9%)
Total	1843 (100.0%)
Subject Status
Outpatient	590 (32.0%)
Hospitalized	317 (17.2%)
Emergency Room	913 (49.5%)
Unknown	23 (1.2%)
Total	1843 (100.0%)

The LIAISON PLEX Respiratory Flex Assay was evaluated for prospective clinical performance by comparing to an FDAcleared molecular respiratory panel for all analytes, except the following: SARS-COV-2, B. holmesii, B. pard B. pertussis. Performance for SARS-CoV-2 was evaluated by comparing to an FDA-cleared molecular SARS-CoV-2 assay. Performance for the denoted Bordetella species was based on comparison to well-validated Fragment Analysis (FA) assays followed by PCR/Bi-Directional Sequencing (PCR/BDS) assays (see Table 17).

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Table 17. Comparator Methods for the LIAISON PLEX Respiratory F/ex Assay Clinical Study

LIAISON PLEX Respiratory Flex Target	Comparator Method
Adenovirus (inclusive to A, B, C, D, E, and F)	FDA-Cleared MolecularRespiratory Panel
Chlamydia pneumoniae
Human Coronavirus (inclusive to HKU1, NL63, OC43, and 229E)
Enterovirus/Rhinovirus
Human Metapneumovirus
Influenza A
Influenza A subtype H1
Influenza A subtype H3
Influenza B
Mycoplasma pneumoniae
Parainfluenza 1
Parainfluenza 2
Parainfluenza 3
Parainfluenza 4
RSV (inclusive to RSV A and RSV B)
SARS-CoV-2	FDA-Cleared Molecular SARS-CoV-2 Assay, Cleared Under 21CFR 866.3981
Bordetella holmesii	Analytically Validated FragmentAnalysis Assays Followed by
Bordetella parapertussis
Bordetella pertussis	PCR/Bi-Directional Sequencing

Out of the 1843 prospective clinical specimens included in the prospective study analysis, 95.2% (1755/1843) generated valid Respiratory Flex Assay results (i.e., detected or not detected) on the first attempt, for an initial invalid rate of 4.8% (88/1843). Of the 88 specimens with initial invalid results, 77 produced valid results on repeat, 6 specimens remained invalid on repeat, and 5 specimens were not retested, resulting in a final invalid rate of 0.6% (11/1843). This left 1832 specimens with valid Respiratory Flex Assay results. The final number of evaluable results varied by target based on the number of valid comparator method results obtained.

Clinical Performance (Positive Percent Agreement, Negative Percent Agreement, and 95% confidence interval) of the LIAISON PLEX Respiratory Flex Assay vs the comparator method(s)is summarized in Table 18 for prospective specimens. Positive Percent Agreement (PPA) was calculated as 100% × (TP / (TP + FN)). True positive (TP) indicates that both the Respiratory Flex Assay and the comparator method had a positive result for the specific analyte, and false negative (FN) indicates that the Respiratory Flex Assay was negative while the comparator result was positive. Negative Percent Agreement (NPA) was calculated as 100% × (TN / (TN + FP)). True negative (TN) indicates that both the Respiratory Flex Assay and the comparator method had negative results, and false positive (FP) indicates that the Respiratory Flex Assay was positive while the comparator result was negative. Specimens that obtained discordant results underwent additional testing with either an FDA-cleared molecular respiratory panel or PCR/BDS for investigation.

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Table 18. Prospective Clinical Performance of the LIAISON PLEX Respiratory Flex Assay with NPS Specimens

	SpecimensPositive Percent Agreement			Negative Percent Agreement
Analyte		TP/(TP+FN)	%	95% CI	TN/(TN+FP)	%	95% CI
Adenovirus	Fresh	75/75	100	95.1-100	1074/1129	95.1	93.7-96.2
	Frozen	19/19	100	83.2-100	578/597	96.8	95.1-98.0
	Overall	94/94	100	96.1-100	1652/17261	95.7	94.7-96.6
Bordetella holmesii	Fresh	0/0	NE	NE	1127/1127	100	99.7-100
	Frozen	0/0	NE	NE	603/603	100	99.4-100
	Overall	0/0	NE	NE	1730/1730	100	99.8-100
Bordetellaparapertussis	Fresh	4/4	100	51.0-100	1161/1163	99.8	99.4-100
	Frozen	0/1	0	0-79.3	604/605	99.8	99.1-100
	Overall	4/5	80.0	37.6-96.4	1765/17682	99.8	99.5-99.9
Bordetella pertussis	Fresh	0/0	NE	NE	1146/1146	100	99.7-100
	Frozen	0/0	NE	NE	607/607	100	99.4-100
	Overall	0/0	NE	NE	1753/1753	100	99.8-100
Chlamydiapneumoniae	Fresh	0/0	NE	NE	1204/1204	100	99.7-100
	Frozen	0/0	NE	NE	616/616	100	99.4-100
	Overall	0/0	NE	NE	1820/1820	100	99.8-100
Human Coronavirus	Fresh	90/97	92.8	85.8-96.5	1100/1107	99.4	98.7-99.7
	Frozen	27/33	81.8	65.6-91.4	582/583	99.8	99.0-100
	Overall	117/1303	90.0	83.6-94.1	1682/16904	99.5	99.1-99.8
Enterovirus/Rhinovirus	Fresh	230/242	95.0	91.5-97.1	937/962	97.4	96.2-98.2
	Frozen	81/90	90.0	82.1-94.6	518/526	98.5	97.0-99.2
	Overall	311/3325	93.7	90.5-95.8	1455/14886	97.8	96.9-98.4
hMPV	Fresh	113/118	95.8	90.5-98.2	1080/1086	99.4	98.8-99.7
	Frozen	12/13	92.3	66.7-98.6	603/603	100	99.4-100
	Overall	125/1317	95.4	90.4-97.9	1683/16898	99.6	99.2-99.8
Influenza A	Fresh	18/18	100	82.4-100	1185/1186	99.9	99.5-100
	Frozen	111/111	100	96.7-100	490/505	97.0	95.2-98.2
	Overall	129/129	100	97.1-100	1675/16919	99.1	98.5-99.4
Influenza ASubtype H1	Fresh	16/16	100	80.6-100	1187/1188	99.9	99.5-100
	Frozen	21/21	100	84.5-100	595/595	100	99.4-100
	Overall	37/37	100	90.6-100	1782/178310	99.9	99.7-100
Influenza ASubtype H3	Fresh	2/3	66.7	20.8-93.9	1200/1201	99.9	99.5-100
	Frozen	102/104	98.1	93.3-99.5	509/512	99.4	98.3-99.8
	Overall	104/10711	97.2	92.1-99.0	1709/171312	99.8	99.4-99.9
Influenza B	Fresh	4/4	100	51.0-100	1200/1200	100	99.7-100
	Frozen	4/4	100	51.0-100	612/612	100	99.4-100
	Overall	8/8	100	67.6-100	1812/1812	100	99.8-100
Mycoplasmapneumoniae	Fresh	0/0	NE	NE	1204/1204	100	99.7-100
	Frozen	0/0	NE	NE	616/616	100	99.4-100
	Overall	0/0	NE	NE	1820/1820	100	99.8-100
Analyte		Positive Percent Agreement			Negative Percent Agreement
		TP/(TP+FN)	%	95% CI	TN/(TN+FP)	%	95% CI
Parainfluenza 1	Fresh	7/8	87.5	52.9-97.8	1196/1196	100	99.7-100
	Frozen	4/4	100	51.0-100	612/612	100	99.4-100
	Overall	11/1213	91.7	64.6-98.5	1808/1808	100	99.8-100
Parainfluenza 2	Fresh	9/10	90	59.6-98.2	1194/1194	100	99.7-100
	Frozen	3/3	100	43.9-100	613/613	100	99.4-100
	Overall	12/13	92.3	66.7-98.6	1807/1807	100	99.8-100
Parainfluenza 3	Fresh	37/39	94.9	83.1-98.6	1164/1165	99.9	99.5-100
	Frozen	4/5	80	37.6-96.4	611/611	100	99.4-100
	Overall	41/4414	93.2	81.8-97.7	1775/177615	99.9	99.7-100
Parainfluenza 4	Fresh	4/4	100	51.0-100	1199/1200	99.9	99.5-100
	Frozen	4/5	80.0	37.6-96.4	611/611	100	99.4-100
	Overall	8/916	88.9	56.5-98.0	1810/181117	99.9	99.7-100
RespiratorySyncytial Virus	Fresh	37/38	97.4	86.5-99.5	1166/1166	100	99.7-100
	Frozen	81/85	95.3	88.5-98.2	531/531	100	99.3-100
	Overall	118/12318	95.9	90.8-98.3	1697/1697	100	99.8-100
SARS-CoV-2	Fresh	178/183	97.3	93.8-98.8	996/1000	99.6	99.0-99.8
	Frozen	68/72	94.4	86.6-97.8	521/525	99.2	98.1-99.7
	Overall	246/25519	96.5	93.4-98.1	1517/152520	99.5	99.0-99.7

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TP - true positive; FN - false negative; TN - true negative; FP - false positive; NE - not evaluable

²Of the 74 specimens with false positive adenovirus results by the Respiratory Flex Assay, 21 were positive by an FDA-cleared molecular respiratory panel, 21 were negative, and 32 were not tested.

² Of the 3 specimens with false positive bordertusis results by the Respiratory Flex Assay, 1 was negative by an FDA-cleared molecular respiratory panel and 2 were not tested.

? Of the 13 specimens with false negative coronavirus results by the Respiratory Flex Assay, 3 were positive, and 1 was not tested.

*Of the 8 specimens with false positive coronavirus by the Respiratory Flex Assay, 5 were negative, and 1 was not tested.

fof the 21 specimens with false negative enterovirus results by the Respiratory Flex Assay, 9 were negative, and 4 were not tested

°Of the 33 specimens with false positive results by the Respiratory Flex Assay, 4 were positive by PCR/BDS, 27 were net t tested.

? Of the 6 specimens with false negative hPMV results by the Respiratory Flex Assay, 4 were positive by PCR/BDS and 2 were negative.

® Of the 6 specimens with false positive hPMV results by the Respiratory Flex Assay, 4 were positive by PCR/BDS and 2 were negative.

9 Of the 16 specimens with false positive influenza A results by the Respiratory Flex Assay, 7 were negative.

10The 1 specimen with a false positive influenza A subtype H1 result by the Respiratory Flex Assay was negative by PCR/BDS.

11The 3 specimens with false negative influenza A subtype H3 results by the Respiratory Flex Assay were all negative by PCR/BDS.

14The 4 specimens with false positive influenza A subtype H3 results by the Respiratory Flex Assay were all negative by PCR/BDS.

13The 1 specimen with a false negative parainfluenza 1 result by the Respiratory Flex Assay was positive by PCR/BDS.

440f the 3 specimens with false negative parainfluenza 3 results by the Respiratory Flex Assay, 2 were negative by PCR/BDS and 1 was not tested.

15The 1 specimen with a false positive parainfluenza 3 result by the Respiratory Flex Assay was negative by PCR/BDS.

16The 1 specimen with a false negative parainfluenza 4 result by the Respiratory Flex Assay was negative by PCR/BDS.

17The 1 specimen with a false positive parainfluenza 4 result by the Respiratory Flex Assay was negative by PCR/BDS.

40 f the 5 specimens with false negative RSV results by the Rssay, 1 was negative by PCR/BDS, and 3 were negative by an FDA-cleared molecular Flu/RSV assay.

40 the 9 specimens with false negative SARS-CoV-2 results by PCR/BDS, 2 were positive by PCR/BDS, 2 were not tested. 20 the 8 specimens with false positive SARS-COV-2 results by the Respiratory Flex Assay, 5 were negative, and 1 was not tested.

The LIAISON PLEX Respiratory Flex Assay reported multiple organism detections in a total of 176 prospective specimens as shown in Table 19. Of these 176 specimens, comparator results were unavailable for at least 1 of the organisms identified in the co-infection for 3 specimens, which were excluded from further analysis. The remaining 173 coinfections represent 14.6% (173/1187) of positive prospective specimens and 9.4% (173/1840) of all prospective specimens. The majority of co-infections, 87.3% (151/173), contained two organisms, while 11.6% (20/173) of coinfections contained three organisms, and 1.2% (2/173) contained 4 organisms. Out of the 173 specimens with multiple

{40}------------------------------------------------

detections, 45.5% (77/173) contained one or more organisms that were not detected by the comparator method(s) (Table 19). Co-infections identified by the comparator methods which were not reported by the LIAISON PLEX Respiratory Flex Assay are illustrated in Table 20.

				Number of Specimens
Analyte 1	Analyte 2	Analyte 3	Analyte 4	Total	LIAISONPLEXRespiratoryFlex AssayFalsePositives	LIAISON PLEXRespiratory FlexAssay FalsePositiveAnalyte(s)
Adenovirus	Bordetellaparapertussis	Enterovirus/Rhinovirus		2	2	Adenovirus (2),Bordetellaparapertussis (1)
Adenovirus	HumanCoronavirus			10	5	Adenovirus
Adenovirus	HumanCoronavirus	Enterovirus/Rhinovirus		1	1	Adenovirus
Adenovirus	HumanCoronavirus	hMPV		1	1	hMPV
Adenovirus	HumanCoronavirus	Parainfluenza3		1	0	NA
Adenovirus	Enterovirus/Rhinovirus			35	18	Adenovirus (17),Enterovirus/Rhinovirus (1)
Adenovirus	Enterovirus/Rhinovirus	hMPV		3	1	Adenovirus (1),Enterovirus/Rhinovirus (1)
Adenovirus	Enterovirus/Rhinovirus	hMPV	SARS-CoV-2	1	1	Adenovirus (1),Enterovirus/Rhinovirus (1)
Adenovirus	Enterovirus/Rhinovirus	Influenza A &Influenza A(subtype H1)		1	1	Adenovirus
Adenovirus	Enterovirus/Rhinovirus	Parainfluenza1		1	1	Adenovirus
Adenovirus	Enterovirus/Rhinovirus	RespiratorySyncytial Virus		1	1	Adenovirus,Enterovirus/Rhinovirus
Adenovirus	Enterovirus/Rhinovirus	SARS-CoV-2		1	0	NA
Adenovirus	hMPV			7	4	Adenovirus
				Number of Specimens
Analyte 1	Analyte 2	Analyte 3	Analyte 4	Total	LIAISONPLEXRespiratoryFlex AssayFalsePositives	LIAISON PLEXRespiratory FlexAssay FalsePositiveAnalyte(s)
Adenovirus	hMPV	Parainfluenza2		1	0	NA
Adenovirus	hMPV	SARS-CoV-2		1	1	Adenovirus
Adenovirus	Influenza A &Influenza A(subtype H1)			2	2	Adenovirus
Adenovirus	Influenza A &Influenza A(subtype H3)			2	2	Adenovirus
Adenovirus	Influenza B			1	1	Adenovirus
Adenovirus	Parainfluenza1	RespiratorySyncytial Virus		1	1	Adenovirus
Adenovirus	Parainfluenza2			1	1	Adenovirus
Adenovirus	Parainfluenza3			5	4	Adenovirus
Adenovirus	RespiratorySyncytial Virus			4	3	Adenovirus
Adenovirus	SARS-CoV-2			2	2	Adenovirus
BordetellaparapertussiS	HumanCoronavirus			1	1	Bordetellaparapertussis
BordetellaparapertussiS	Enterovirus/Rhinovirus			2	1	Bordetellaparapertussis
BordetellaparapertussiS	hMPV			1	0	NA
BordetellaparapertussiS	Parainfluenza3			1	0	NA
HumanCoronavirus	Enterovirus/Rhinovirus			8	2	HumanCoronavirus (1),Enterovirus/Rhinovirus (1)
HumanCoronavirus	Enterovirus/Rhinovirus	hMPV		1	0	NA
				Number of Specimens
Analyte 1	Analyte 2	Analyte 3	Analyte 4	Total	LIAISONPLEXRespiratoryFlex AssayFalsePositives	LIAISON PLEXRespiratory FlexAssay FalsePositiveAnalyte(s)
HumanCoronavirus	Enterovirus/Rhinovirus	SARS-CoV-2		1	0	NA
HumanCoronavirus	hMPV			6	2	HumanCoronavirus
HumanCoronavirus	Influenza A &Influenza A(subtype H1)			1	0	NA
HumanCoronavirus	Parainfluenza3			3	0	NA
HumanCoronavirus	RespiratorySyncytial Virus			2	0	NA
HumanCoronavirus	SARS-CoV-2			4	1	HumanCoronavirus
Enterovirus/Rhinovirus	hMPV			11	1	Enterovirus/Rhinovirus
Enterovirus/Rhinovirus	hMPV	Influenza A(subtype H3)	SARS-CoV-2	1	1	Influenza A H3,Influenza A,Enterovirus/Rhinovirus, SARS-CoV-2
Enterovirus/Rhinovirus	hMPV	SARS-CoV-2		1	1	Enterovirus/Rhinovirus
Enterovirus/Rhinovirus	Influenza A &Influenza A(subtype H1)			2	0	NA
Enterovirus/Rhinovirus	Influenza A &Influenza A(subtype H3)			7	2	Enterovirus/Rhinovirus
Enterovirus/Rhinovirus	Influenza A &Influenza A(subtype H3)	RespiratorySyncytial Virus		2	2	Influenza A H3,Influenza A
Enterovirus/Rhinovirus	Parainfluenza1			3	1	Enterovirus/Rhinovirus
Enterovirus/Rhinovirus	Parainfluenza3			5	2	Enterovirus/Rhinovirus
Enterovirus/Rhinovirus	Parainfluenza4			4	1	Parainfluenza 4
Enterovirus/Rhinovirus	RespiratorySyncytial Virus			9	0	NA
				Number of Specimens
Analyte 1	Analyte 2	Analyte 3	Analyte 4	Total	LIAISONPLEXRespiratoryFlex AssayFalsePositives	LIAISON PLEXRespiratory FlexAssay FalsePositiveAnalyte(s)
Enterovirus/Rhinovirus	SARS-CoV-2			5	2	Enterovirus/Rhinovirus
hMPV	RespiratorySyncytial Virus			1	1	hMPV
Influenza A& InfluenzaA (subtypeH1)	Influenza A(subtype H3)			1	0	NA
Influenza A& InfluenzaA (subtypeH1)	SARS-CoV-2			1	1	SARS-CoV-2
Influenza A& InfluenzaA (subtypeH3)	RespiratorySyncytial Virus			1	1	Influenza A
Parainfluenza 2	SARS-CoV-2			1	0	NA
Parainfluenza 3	RespiratorySyncytial Virus			1	0	NA
Parainfluenza 3	SARS-CoV-2			1	1	SARS-CoV-2
Total				173	77
Total Double Infections				151	62
Total Triple Infections				20	13
Total Quadruple Infections				2	2

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{42}------------------------------------------------

{43}------------------------------------------------

Table 20. Co-infections Identified by the Comparator Methods which were Not Reported by the LIAISON PLEX Respiratory Flex Assay in the Prospective Study

			Number of Specimens
Analyte 1	Analyte 2	Analyte 3	Total	LIAISON PLEXRespiratory FlexAssay FalseNegatives	LIAISON PLEXRespiratory Flex AssayFalse Negative Analyte(s)
Adenovirus	Human Coronavirus		7	1	Human Coronavirus
Adenovirus	Enterovirus/		18	2	Enterovirus/
Analyte 1	Analyte 2	Analyte 3	Total	LIAISON PLEXRespiratory FlexAssay FalseNegatives	LIAISON PLEXRespiratory Flex AssayFalse Negative Analyte(s)
	Rhinovirus				Rhinovirus
Adenovirus	Enterovirus/Rhinovirus	Parainfluenza 2	1	1	Parainfluenza 2
Adenovirus	hMPV		3	1	hMPV
Adenovirus	hMPV	SARS-CoV-2	1	1	SARS-CoV-2
Adenovirus	SARS-CoV-2		1	1	SARS-CoV-2
HumanCoronavirus	Enterovirus/Rhinovirus		8	1	Human Coronavirus
HumanCoronavirus	Enterovirus/Rhinovirus	SARS-CoV-2	2	1	Human Coronavirus
HumanCoronavirus	hMPV		6	2	Human Coronavirus
HumanCoronavirus	Influenza A &Influenza A(subtype H3)		1	1	Human Coronavirus
HumanCoronavirus	Parainfluenza 3		5	2	Human Coronavirus (1),Parainfluenza 3 (1)
HumanCoronavirus	RespiratorySyncytial Virus		3	1	Human Coronavirus
Influenza A &Influenza A(subtype H3)	Parainfluenza 4		1	1	Parainfluenza 4
Influenza A &Influenza A(subtype H3)	RespiratorySyncytial Virus		1	1	Respiratory SyncytialVirus
hMPV	SARS-CoV-2		4	1	hMPV
RespiratorySyncytial Virus	hMPV		1	1	hMPV
RespiratorySyncytial Virus	SARS-CoV-2		1	1	Respiratory SyncytialVirus
Enterovirus/Rhinovirus	BordetellaParapertussis		3	1	Bordetellaparapertussis
Enterovirus/Rhinovirus	hMPV		11	1	hMPV
Enterovirus/Rhinovirus	Influenza A &Influenza A(subtype H1)		6	3	Enterovirus/Rhinovirus
Enterovirus/Rhinovirus	Influenza A(subtype H3)		1	1	Influenza A (subtype H3)
Enterovirus/Rhinovirus	Influenza A &Influenza A(subtype H3)	SARS-CoV-2	1	1	SARS-CoV-2
Enterovirus/Rhinovirus	RespiratorySyncytial Virus		13	3	Enterovirus/Rhinovirus
Enterovirus/Rhinovirus	SARS-CoV-2		5	3	Enterovirus/Rhinovirus(2), SARS-CoV-2 (1)
			Number of Specimens
Analyte 1	Analyte 2	Analyte 3	Total	LIAISON PLEXRespiratory FlexAssay FalseNegatives	LIAISON PLEXRespiratory Flex AssayFalse Negative Analyte(s)
Parainfluenza 3	Enterovirus/Rhinovirus	RespiratorySyncytial Virus	1	1	Parainfluenza 3 (1),Enterovirus/Rhinovirus (1)
Total			105	34
Total Double Infections			99	29
Total Triple Infections			6	5

{44}------------------------------------------------

{45}------------------------------------------------

Testing of Preselected Archived Specimens

A number of analytes on the LIAISON PLEX Respiratory Flex Assay were of low prevalence during the prospective study and were not encountered in large enough numbers to adequately demonstrate system performance. To supplement the results of the prospective clinical study, an evaluation of preselected archived retrospective NPS specimens was performed.

A total of 256 pre-selected left-over frozen, de-identified specimens (Category III specimens) sourced from four sites/vendors in the United States were obtained and tested at three US sites. Pre-selected specimen collection dates ranged from November 2013 through June 2023. Preselected specimens were characterized by the same comparator methods as the prospective study (described above). The pre-selected specimens were tested in a randomized, blinded manner with negative specimens. A summary of the available demographic information of the tested specimens is provided in Table 21. Out of the 256 specimens included in the pre-selected study analysis, 241 (94.1%) generated valid Respiratory Flex Assay results (i.e., Detected or Not Detected) on the first attempt. There were 15 specimens (5.9%) with invalid results on the initial run that required retesting. Of the specimens with initial invalid results, all 15 specimens generated valid Respiratory Flex Assay results after retest for a final success rate of 100% (256/256). The results of the LIAISON PLEX Respiratory Flex Assay performance for these archived specimens are shown in Table 22.

	# Specimens (%)
Gender
Male	117(45.7%)
Female	124(48.4%)
Unknown	15(5.9%)
Total	256(100.0%)

Table 21. Archived Specimen Demographic Details (N=256)

{46}------------------------------------------------

Age (years)
0-1	44(17.2%)
>1-5	53(20.7%)
>5-21	69(27.0%)
>21-65	44(17.2%)
> 65	32(12.5%)
Unknown	14(5.5%)
Total	256(100.0%)
Subject Status
Outpatient	0 (0.0%)
Hospitalized	0 (0.0%)
EmergencyRoom	0 (0.0%)
Unknown	256 (100.0%)
Total	256 (100.0%)

Table 22. LIAISON PLEX Respiratory Flex Assay Archived Performance Summary for NPS Specimens

	Positive Percent Agreement			NegativePercent Agreement
Analyte	TP/(TP+FN)	%	95% CI	TN/(TN+FP)	%	95% CI
Adenovirus	6/6	100	61.0-100	241/2501	96.4	93.3-98.1
Bordetella holmesii	0/0	NE	NE	234/234	100	98.4-100
Bordetella parapertussis	8/8	100	67.6-100	233/236	98.7	96.3-99.6
Bordetella pertussis	23/23	100	85.7-100	214/217	98.6	96.0-99.5
Chlamydia pneumoniae	13/14	92.9	68.5-98.7	241/242	99.6	97.7-99.9
Human Coronavirus	4/4	100	51.0-100	249/252	98.8	96.6-99.6
Enterovirus/Rhinovirus	24/272	88.9	71.9-96.1	223/2293	97.4	94.4-98.8
hMPV	1/1	100	20.7-100	255/255	100	98.5-100
Influenza A	1/1	100	20.7-100	254/2554	99.6	97.8-99.9
Influenza A subtype H1	1/1	100	20.7-100	254/2554	99.6	97.8-99.9
Influenza A subtype H3	0/0	NE	NE	256/256	100	98.5-100
Influenza B	23/23	100	85.7-100	232/233	99.6	97.6-99.9
Mycoplasma pneumoniae	23/24	95.8	79.8-99.3	226/232	97.4	94.5-98.8
Parainfluenza 1	18/18	100	82.4-100	237/238	99.6	97.7-99.9
Parainfluenza 2	19/20	95.0	76.4-99.1	235/236	99.6	97.6-99.9
Parainfluenza 3	2/2	100	34.2-100	254/254	100	98.5-100
Parainfluenza 4	23/23	100	85.7-100	230/2335	98.7	96.3-99.6
Respiratory Syncytial Virus	9/9	100	70.1-100	246/247	99.6	97.7-99.9

TP – true positive; FN – false negative; TN – true negative; FP – false positive; NE – not evaluable

{47}------------------------------------------------

¹Of the 9 specimens with false positive adenovirus results by the Respiratory Flex Assay, seven were negative by PCR/BDS and two were not tested.

? Of the 3 specimens with false negative enterovirus results by the Respiratory Flex Assay, one was negative by PCR/BDS, one was positive by PCR/BDS, and one was not tested.

3 Of the 6 specimens with false positive enterovirus results by the Respiratory Flex Assay, four were negative by PCR/BDS and two were not tested.

4The 1 specimen with a false positive influenza A/influenza A H1 result by the Respiratory Flex Assay was negative for influenza A by PCR/BDS and not tested for influenza A H1.

50f the 3 specimens with false positive parainfluenza 4 results, one was negative by PCR/BDS and two were not tested.

Contrived Specimen Testing

Contrived specimens were tested to supplement the positive clinical specimens in the prospective and pre-selected study cohorts for low prevalence targets, including Bordetella holmesii, Bordetella parapertussis, Bordetella pertussis, Chlamydia pneumoniae, and Mycoplasma pneumoniae. Positive contrived specimens for influenza A H1N1 pdm09 were prepared and tested prior to completion of the prospective clinical study, in anticipation of potentially low prevalence for influenza A H1. The prospective clinical study ended up yielding an adequate number of influenza A H1 positive specimens to demonstrate performance, however since the contrived data was already acquired, it's presented here . A total of 300 specimens were contrived, blinded, randomized and tested along with negative specimens at two testing sites during August 2023.

Out of the 300 specimens included in the contrived study analysis, 291 specimens (97.0%) generated valid RSP Flex Assay results (i.e., Detected or Not Detected) on the first attempt. There were 9 specimens (3.0%) with an invalid result on the initial run. Of the 9 specimens retested, all 9 generated a valid result after a single retest for a final success rate of 100% (300/300).

Results from contrived specimen testing with the LIAISON PLEX Respiratory Flex Assay are shown in Table 23.

	Target	Positive Percent Agreement			Negative Percent Agreement
Analyte	Conc.(xLoD)	TP/(TP+FN)	%	95% Cl	TN/(TN+FP)	%	95% Cl
	2x	25/25	100	86.7-100	125/125	100	97.0-100
Bordetella	10x	13/13	100	77.2-100	65/65	100	94.4-100
holmesii	100x	12/12	100	75.8-100	60/60	100	94.0-100
	Combined	50/50	100	92.9-100	250/250	100	98.5-100
	2x	25/25	100	86.7-100	125/125	100	97.0-100
Bordetella	10x	12/13	92.3	66.7-98.6	65/65	100	94.4-100
parapertussis	100x	12/12	100	75.8-100	60/60	100	94.0-100
	Combined	49/50	98.0	89.5-99.6	250/250	100	98.5-100
	2x	25/25	100	86.7-100	125/125	100	97.0-100
Bordetella	10x	13/13	100	77.2-100	65/65	100	94.4-100
pertussis	100x	12/12	100	75.8-100	60/60	100	94.0-100
	Combined	50/50	100	92.9-100	250/250	100	98.5-100
Chlamydia	2x	25/25	100	86.7-100	125/125	100	97.0-100

Table 23. LIAISON PLEX Respiratory Flex Assay Performance with Contrived Specimens

{48}------------------------------------------------

	Target	Positive Percent Agreement			Negative Percent Agreement
Analyte	Conc.(xLoD)	TP/(TP+FN)	%	95% CI	TN/(TN+FP)	%	95% CI
pneumoniae	10x	13/13	100	77.2-100	65/65	100	94.4-100
	100x	12/12	100	75.8-100	60/60	100	94.0-100
	Combined	50/50	100	92.9-100	250/250	100	98.5-100
	2x	24/25	96.0	80.5-99.3	125/125	100	97.0-100
Influenza A	10x	13/13	100	77.2-100	65/65	100	94.4-100
H1N1 pdm09	100x	12/12	100	75.8-100	60/60	100	94.0-100
	Combined	49/50	98.0	89.5-99.6	250/250	100	98.5-100
	2x	24/25	96.0	80.5-99.3	125/125	100	97.0-100
Mycoplasma	10x	13/13	100	77.2-100	64/65	98.5	91.8-99.7
pneumoniae	100x	12/12	100	75.8-100	60/60	100	94.0-100
	Combined	49/50	98.0	89.5-99.6	249/250	99.6	97.8-99.9

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.

Regulation Number and Section

§ 866.3981 Device to detect and identify nucleic acid targets in respiratory specimens from microbial agents that cause the SARS-CoV-2 respiratory infection and other microbial agents when in a multi-target test.

(a)
Identification. A device to detect and identify nucleic acid targets in respiratory specimens from microbial agents that cause the SARS-CoV-2 respiratory infection and other microbial agents when in a multi-target test is an in vitro diagnostic device intended for the detection and identification of SARS-CoV-2 and other microbial agents when in a multi-target test in human clinical respiratory specimens from patients suspected of respiratory infection who are at risk for exposure or who may have been exposed to these agents. The device is intended to aid in the diagnosis of respiratory infection in conjunction with other clinical, epidemiologic, and laboratory data or other risk factors.(b)
Classification. Class II (special controls). The special controls for this device are:(1) The intended use in the labeling required under § 809.10 of this chapter must include a description of the following: Analytes and targets the device detects and identifies, the specimen types tested, the results provided to the user, the clinical indications for which the test is to be used, the specific intended population(s), the intended use locations including testing location(s) where the device is to be used (if applicable), and other conditions of use as appropriate.
(2) Any sample collection device used must be FDA-cleared, -approved, or -classified as 510(k) exempt (standalone or as part of a test system) for the collection of specimen types claimed by this device; alternatively, the sample collection device must be cleared in a premarket submission as a part of this device.
(3) The labeling required under § 809.10(b) of this chapter must include:
(i) A detailed device description, including reagents, instruments, ancillary materials, all control elements, and a detailed explanation of the methodology, including all pre-analytical methods for processing of specimens;
(ii) Detailed descriptions of the performance characteristics of the device for each specimen type claimed in the intended use based on analytical studies including the following, as applicable: Limit of Detection, inclusivity, cross-reactivity, interfering substances, competitive inhibition, carryover/cross contamination, specimen stability, precision, reproducibility, and clinical studies;
(iii) Detailed descriptions of the test procedure(s), the interpretation of test results for clinical specimens, and acceptance criteria for any quality control testing;
(iv) A warning statement that viral culture should not be attempted in cases of positive results for SARS-CoV-2 and/or any similar microbial agents unless a facility with an appropriate level of laboratory biosafety (
e.g., BSL 3 and BSL 3+, etc.) is available to receive and culture specimens; and(v) A prominent statement that device performance has not been established for specimens collected from individuals not identified in the intended use population (
e.g., when applicable, that device performance has not been established in individuals without signs or symptoms of respiratory infection).(vi) Limiting statements that indicate that:
(A) A negative test result does not preclude the possibility of infection;
(B) The test results should be interpreted in conjunction with other clinical and laboratory data available to the clinician;
(C) There is a risk of incorrect results due to the presence of nucleic acid sequence variants in the targeted pathogens;
(D) That positive and negative predictive values are highly dependent on prevalence;
(E) Accurate results are dependent on adequate specimen collection, transport, storage, and processing. Failure to observe proper procedures in any one of these steps can lead to incorrect results; and
(F) When applicable (
e.g., recommended by the Centers for Disease Control and Prevention, by current well-accepted clinical guidelines, or by published peer-reviewed literature), that the clinical performance may be affected by testing a specific clinical subpopulation or for a specific claimed specimen type.(4) Design verification and validation must include:
(i) Detailed documentation, including performance results, from a clinical study that includes prospective (sequential) samples for each claimed specimen type and, as appropriate, additional characterized clinical samples. The clinical study must be performed on a study population consistent with the intended use population and compare the device performance to results obtained using a comparator that FDA has determined is appropriate. Detailed documentation must include the clinical study protocol (including a predefined statistical analysis plan), study report, testing results, and results of all statistical analyses.
(ii) Risk analysis and documentation demonstrating how risk control measures are implemented to address device system hazards, such as Failure Modes Effects Analysis and/or Hazard Analysis. This documentation must include a detailed description of a protocol (including all procedures and methods) for the continuous monitoring, identification, and handling of genetic mutations and/or novel respiratory pathogen isolates or strains (
e.g., regular review of published literature and periodic in silico analysis of target sequences to detect possible mismatches). All results of this protocol, including any findings, must be documented and must include any additional data analysis that is requested by FDA in response to any performance concerns identified under this section or identified by FDA during routine evaluation. Additionally, if requested by FDA, these evaluations must be submitted to FDA for FDA review within 48 hours of the request. Results that are reasonably interpreted to support the conclusion that novel respiratory pathogen strains or isolates impact the stated expected performance of the device must be sent to FDA immediately.(iii) A detailed description of the identity, phylogenetic relationship, and other recognized characterization of the respiratory pathogen(s) that the device is designed to detect. In addition, detailed documentation describing how to interpret the device results and other measures that might be needed for a laboratory diagnosis of respiratory infection.
(iv) A detailed device description, including device components, ancillary reagents required but not provided, and a detailed explanation of the methodology, including molecular target(s) for each analyte, design of target detection reagents, rationale for target selection, limiting factors of the device (
e.g., saturation level of hybridization and maximum amplification and detection cycle number, etc.), internal and external controls, and computational path from collected raw data to reported result (e.g., how collected raw signals are converted into a reported signal and result), as applicable.(v) A detailed description of device software, including software applications and hardware-based devices that incorporate software. The detailed description must include documentation of verification, validation, and hazard analysis and risk assessment activities, including an assessment of the impact of threats and vulnerabilities on device functionality and end users/patients as part of cybersecurity review.
(vi) For devices intended for the detection and identification of microbial agents for which an FDA recommended reference panel is available, design verification and validation must include the performance results of an analytical study testing the FDA recommended reference panel of characterized samples. Detailed documentation must be kept of that study and its results, including the study protocol, study report for the proposed intended use, testing results, and results of all statistical analyses.
(vii) For devices with an intended use that includes detection of Influenza A and Influenza B viruses and/or detection and differentiation between the Influenza A virus subtypes in human clinical specimens, the design verification and validation must include a detailed description of the identity, phylogenetic relationship, or other recognized characterization of the Influenza A and B viruses that the device is designed to detect, a description of how the device results might be used in a diagnostic algorithm and other measures that might be needed for a laboratory identification of Influenza A or B virus and of specific Influenza A virus subtypes, and a description of the clinical and epidemiological parameters that are relevant to a patient case diagnosis of Influenza A or B and of specific Influenza A virus subtypes. An evaluation of the device compared to a currently appropriate and FDA accepted comparator method. Detailed documentation must be kept of that study and its results, including the study protocol, study report for the proposed intended use, testing results, and results of all statistical analyses.
(5) When applicable, performance results of the analytical study testing the FDA recommended reference panel described in paragraph (b)(4)(vi) of this section must be included in the device's labeling under § 809.10(b) of this chapter.
(6) For devices with an intended use that includes detection of Influenza A and Influenza B viruses and/or detection and differentiation between the Influenza A virus subtypes in human clinical specimens in addition to detection of SARS-CoV-2 and similar microbial agents, the required labeling under § 809.10(b) of this chapter must include the following:
(i) Where applicable, a limiting statement that performance characteristics for Influenza A were established when Influenza A/H3 and A/H1-2009 (or other pertinent Influenza A subtypes) were the predominant Influenza A viruses in circulation.
(ii) Where applicable, a warning statement that reads 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.
(iii) Where the device results interpretation involves combining the outputs of several targets to get the final results, such as a device that both detects Influenza A and differentiates all known Influenza A subtypes that are currently circulating, the device's labeling must include a clear interpretation instruction for all valid and invalid output combinations, and recommendations for any required followup actions or retesting in the case of an unusual or unexpected device result.
(iv) A limiting statement that if a specimen yields a positive result for Influenza A, but produces negative test results for all specific influenza A subtypes intended to be differentiated (
i.e., H1-2009 and H3), this result requires notification of appropriate local, State, or Federal public health authorities to determine necessary measures for verification and to further determine whether the specimen represents a novel strain of Influenza A.(7) If one of the actions listed at section 564(b)(1)(A) through (D) of the Federal Food, Drug, and Cosmetic Act occurs with respect to an influenza viral strain, or if the Secretary of Health and Human Services determines, under section 319(a) of the Public Health Service Act, that a disease or disorder presents a public health emergency, or that a public health emergency otherwise exists, with respect to an influenza viral strain:
(i) Within 30 days from the date that FDA notifies manufacturers that characterized viral samples are available for test evaluation, the manufacturer must have testing performed on the device with those influenza viral samples in accordance with a standardized protocol considered and determined by FDA to be acceptable and appropriate.
(ii) Within 60 days from the date that FDA notifies manufacturers that characterized influenza viral samples are available for test evaluation and continuing until 3 years from that date, the results of the influenza emergency analytical reactivity testing, including the detailed information for the virus tested as described in the certificate of authentication, must be included as part of the device's labeling in a tabular format, either by:
(A) Placing the results directly in the device's labeling required under § 809.10(b) of this chapter that accompanies the device in a separate section of the labeling where analytical reactivity testing data can be found, but separate from the annual analytical reactivity testing results; or
(B) In a section of the device's label or in other labeling that accompanies the device, prominently providing a hyperlink to the manufacturer's public website where the analytical reactivity testing data can be found. The manufacturer's website, as well as the primary part of the manufacturer's website that discusses the device, must provide a prominently placed hyperlink to the website containing this information and must allow unrestricted viewing access.