The NxTAG® Respiratory Pathogen Panel v2 (NxTAG® RPP v2) is a multiplexed polymerase chain reaction (PCR) test intended for the simultaneous, qualitative detection of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swab specimens obtained from individuals with signs and symptoms of respiratory tract infection, including COVID-19.

The following organism types and subtypes are identified and differentiated using the NxTAG RPPv2:

Viral Targets: Influenza A, Influenza A H1, Influenza A H1pdm09, Influenza B, Respiratory Syncytial Virus A, Respiratory Syncytial Virus B, SARS-CoV-2, Coronavirus 229E, Coronavirus OC43, Coronavirus NL63, Coronavirus HKU1, Human Metapneumovirus, Rhinovirus, Adenovirus, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, Parainfluenza 4

Bacterial Targets: Chlamydia pneumoniae, Mycoplasma pneumoniae

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

Negative results in the setting of a respiratory illness may be due to infection with pathogens that are not detected by this test, or lower respiratory tract infection that may not be detected by a nasopharyngeal swab specimen. Positive results do not rule out coinfection with other organisms. The agent(s) detected by the NxTAG RPP v2 may not be the definite cause of disease.

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

The NxTAG® Respiratory Pathogen Panel v2 is indicated for use with the Luminex® MAGPIX® Instrument and xPONENT® and SYNCTTM software.

Device Description

The NxTAG® Respiratory Pathogen Panel v2 (NxTAG® RPP v2) is designed to simultaneously detect and identify 21 different potential pathogens of respiratory tract infections, including the novel coronavirus SARS-CoV-2, from a single NPS specimen in transport medium. NxTAG® RPP v2 is compatible with Luminex's MAGPIX Instrument, and xPONENT® and SYNCT™ software. It incorporates multiplex Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) with the Luminex® proprietary universal tag sorting system on the Luminex platform to easily detect the 21 respiratory pathogen targets.

Samples are extracted using the IVD-labeled bioMérieux NucliSENS® easyMag® or EMAG® extraction systems. Extracted total nucleic acid is then added to the sealed 96-well micro plate by piercing the seal with pipette tips. Each reaction well is pre-plated with two Lyophilized Bead Reagents (LBRs) that contain all the required reagents including primer mixes, bead mix, and enzyme buffer systems. Once the LBRs are resuspended, the reaction wells are re-sealed using the foils provided in the kit. The sealed plate can be placed inside the thermocycler. The reaction is amplified via RT-PCR and the reaction product undergoes near simultaneous bead hybridization within the sealed reaction wells. The hybridized, tagged beads are then sorted and read on the Luminex® MAGPIX® instrument. The MAGPIX® instrument generates a signal in the form of a median fluorescence intensity (MFI) value for each bead population.

The signals are analyzed using the NxTAG® Respiratory Pathogen Panel v2 Assay File for SYNCT™ Software, providing a reliable, qualitative call for each of the 21 targets and internal controls within each reaction well.

AI/ML Overview

Here's a breakdown of the acceptance criteria and the study that proves the device meets them, based on the provided FDA 510(k) summary for the Luminex NxTAG® Respiratory Pathogen Panel v2 (NxTAG® RPP v2):

The NxTAG® Respiratory Pathogen Panel v2 (NxTAG® RPP v2) is a multiplexed polymerase chain reaction (PCR) test intended for the simultaneous, qualitative detection of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swab specimens from individuals with signs and symptoms of respiratory tract infection, including COVID-19.

Acceptance Criteria and Reported Device Performance

The acceptance criteria for the NxTAG® RPP v2 assay are implied through the robust analytical and clinical performance studies, specifically focusing on sensitivity (Limit of Detection, LoD, and Positive Percent Agreement, PPA) and specificity (Analytical Specificity, Cross-Reactivity, and Negative Percent Agreement, NPA), as well as reproducibility and stability.

Here's a summary of the performance as reported in the document:

Table 1: Acceptance Criteria and Reported Device Performance (Key Metrics)

Metric	Acceptance Criteria (Implied/Standard)	Reported Device Performance (Overall/Range)	Section in Document
Analytical Performance
Site-to-Site Reproducibility	High overall agreement	99.90% overall percent agreement across 3 sites, 2 operators, 5 days, 4 replicates each (n=22,560 data points)	"Site-to-Site Reproducibility" (Table 1)
Lot-to-Lot Reproducibility	High overall agreement	99.95% overall percent agreement across 3 kit lots, 1 operator, 1 site, 10 replicates each (n=10,680 data points)	"Lot-to-Lot Reproducibility" (Table 2)
Limit of Detection (LoD)	≥ 95% detection at specified LoD	All targets demonstrated ≥ 95% detection (typically 19/20 or 20/20) at their confirmed LoD concentrations ranges from 5.60E+01 to 7.21E+03 copies/mL, with some higher for PIV4 and Adenovirus	"Detection Limit" (Table 3)
Analytical Reactivity (Inclusivity)	100% detection of tested strains	100% detection for 193 pathogen strains/isolates, with some exceptions (e.g., A/Denver/1/57 H1) which only detected matrix gene. In silico analysis: ~99% for Influenza A/B, ≥96% for other targets (92% for PIV2, 94% for untyped PIV4)	"Analytical Reactivity (Inclusivity)" (Table 5), "Analytical Reactivity In Silico Analysis"
Analytical Specificity (Exclusivity)	No cross-reactivity	No cross-reactivity for 60/63 off-panel organisms tested, except for one Enterovirus D strain (false positive for Influenza A H3 at high titer, but not at 1E+02 TCID50/mL). No cross-reactivity for on-panel organisms. In silico analysis predicts some SARS-related coronavirus sequences detection by SARS-CoV-2 oligos and one bat 229E-like coronavirus sequence detection by Coronavirus 229E oligos.	"Analytical Specificity (Exclusivity)" (Table 6, 7), "Analytical Cross-Reactivity In Silico Analysis"
Microbial Interference	No interference	No interference observed from 11 off-panel organisms on 22 on-panel organisms (Table 9)	"Microbial Interference" (Table 9)
Competitive Interference	No interference	No interference observed from high positive on-panel organisms on low-moderate positive on-panel organisms (Table 10, 11)	"Competitive Interference (Co-infection)" (Table 10, 11)
Interfering Substances	No interference	No interference from 18 of 20 tested substances. Menthol interfered with Coronavirus OC43 at 1%w/v (not at 0.5%w/v). FluMist® generated expected positive calls for Influenza A/B due to vaccine content.	"Interfering Substances" (Table 12)
Carry-Over/Cross-Contamination	No false positives/contamination	Two false positives observed initially (SARS-CoV-2 target), but resolved upon re-preparation and re-testing of samples and extracts. This suggests a process/handling issue rather than inherent device flaw.	"Carry-Over/Cross-Contamination"
Clinical Performance (PPA/NPA)	High PPA and NPA (typically >90%)	Prospective Cohort (Overall): PPA ranged from 86.7% (RSV A) to 100% (many targets). NPA generally >99% (Table 19). Pre-Selected Cohort: PPA generally 90-100%. NPA generally 98-100% (Table 20). Contrived Cohort: PPA 98-100% (Table 21).	"Clinical Performance" (Table 19, 20, 21)

Study to Prove Device Meets Acceptance Criteria

The device's performance was evaluated through a combination of analytical and clinical studies.

Sample sizes used for the test set and data provenance:
- Analytical Studies (Reproducibility):
  - Site-to-Site: 9-member reproducibility panel tested in 4 replicates on 5 non-consecutive days by 2 operators at 3 sites (30 runs x 4 replicates = 120 data points per panel member). Total of 22,560 data points.
  - Lot-to-Lot: 17-member reproducibility panel tested in 10 replicates on each of 3 assay kit lots by 1 operator at 1 site (30 data points per panel member). Total of 10,680 data points.
  - LoD: 20 replicates for each target.
  - Analytical Reactivity/Specificity/Interference: Generally, 3 replicates per strain/substance.
- Clinical Studies (Test Set):
  - Prospective (Arm 1): 1820 prospectively collected de-identified specimens (after retest resolving invalid results). Data provenance: Five geographically diverse clinical sites within the United States. Type: Prospective.
  - Pre-selected (Arm 2): 308 pre-selected, de-identified leftover specimens (after retest resolving invalid results). Data provenance: Six sites in the United States. Type: Retrospective (leftover specimens).
  - Contrived (Arm 3): 199 contrived specimens (spiked into negative human nasopharyngeal specimens). Type: Laboratory-generated.
Number of experts used to establish the ground truth for the test set and the qualifications of those experts:
- The document does not explicitly state the number or qualifications of experts involved in establishing the ground truth.
- For the clinical study, the ground truth was established by:
  - An FDA-cleared molecular assay (predicate device or other cleared assays).
  - PCR followed by bi-directional sequencing (BDS) for specific targets (Influenza A subtyping, and to confirm pre-selected specimens and indeterminate results from the molecular comparator).
- The implication is that these are either validated laboratory methods or the expertise is inherent in the design and validation of the comparator FDA-cleared molecular assay. For PCR/BDS, it would typically be performed by trained molecular diagnosticians or laboratory personnel.
Adjudication method for the test set:
- The document describes a comparator method algorithm: The NxTAG® RPP v2 results were compared to an FDA-cleared molecular assay. For influenza A subtyping, and potentially for other targets where initial molecular testing was insufficient, PCR/bi-directional sequencing (BDS) was used.
- Composite reference method: For cases where the initial FDA-cleared molecular assay provided certain results (e.g., in some cases of false positives/negatives for Adenovirus, Human Metapneumovirus, Influenza A, RSV A, and SARS-CoV-2 in the prospective cohort, as noted in the footnotes to Table 19), further investigation with molecular SoC assay or BDS was conducted to determine the final truth. This represents a form of adjudication, where a "truth" panel (FDA-cleared assay + PCR/BDS) is used to establish the final ground truth.
If a multi reader multi case (MRMC) comparative effectiveness study was done, if so, what was the effect size of how much human readers improve with AI vs without AI assistance:
- This question is not applicable to this device. The NxTAG® RPP v2 is an in vitro diagnostic (IVD) PCR test kit for detecting nucleic acids, not an AI-assisted diagnostic imaging or human-in-the-loop (HITL) system. The output is qualitative (positive/negative for each pathogen), and does not involve human "readers" in the context of image interpretation that would be augmented by AI.
If a standalone (i.e. algorithm only without human-in-the-loop performance) was done:
- This question is not entirely applicable in the AI sense, as it’s a molecular diagnostic test. However, the analytical performance studies (e.g., LoD, inclusivity, exclusivity, precision) can be considered "standalone" performance of the assay itself, demonstrating its inherent accuracy in detecting specific pathogens under controlled conditions, without the variability of actual clinical human samples that might contain inhibitors or mixed infections. The outputs are generated by the Luminex® MAGPIX® Instrument and xPONENT® and SYNCT™ software, which provides a "reliable, qualitative call for each of the 21 targets and internal controls." This part of the system operates without continuous human "in-the-loop" interpretation of the raw data for diagnosis on a case-by-case basis.
The type of ground truth used:
- Clinical Study:
  - Reference molecular methods: An FDA-cleared molecular assay was the primary comparator.
  - Confirmatory sequencing: PCR followed by bi-directional sequencing (BDS) was used for specific targets (e.g., influenza A subtyping) and to confirm pre-selected specimens prior to enrollment, and potentially to adjudicate discrepancies.
- Analytical Studies:
  - Known concentrations: For LoD and analytical reactivity/specificity, samples were prepared with known concentrations of highly characterized viral or bacterial strains (cultured material, clinical specimens of known titer, or WHO standards).
  - In silico analysis: For inclusivity and exclusivity, genetic sequences from public databases (GISAID EpiCoV, GISAID EpiFlu, GenBank) were analyzed, assuming complete detection if probe binding matched certain criteria.
The sample size for the training set:
- The document describes a marketing submission (510(k)) for a medical device that has completed its development and validation. Therefore, it focuses on the test set (clinical validation and analytical verification) rather than a "training set" which is typically associated with machine learning model development. For an IVD such as this, the "training" (i.e., development and optimization) of the assay's reagents and parameters would have occurred internally during product development, using various internal samples and iterative refinement. The document does not specify the sample size of any development/training set.
How the ground truth for the training set was established:
- As noted above, a "training set" in the context of this 510(k) submission would refer to the internal development and optimization work, not typically disclosed in detail for regulatory submissions like this. The ground truth during that phase would have been established through a combination of purified nucleic acid standards, well-characterized clinical samples, and comparison to established reference methods or culture, depending on the stage of development.

Summary

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March 11, 2024

Image /page/0/Picture/1 description: The image shows the logo for the U.S. Food and Drug Administration (FDA). On the left is the Department of Health and 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 Molecular Diagnostics, Inc. Kate Goscha Senior Manager, Regulatory Affairs 439 University Avenue Toronto, Ontario M5G 1Y8 Canada

Re: K231758

Trade/Device Name: NxTAG Respiratory Pathogen Panel v2 (NxTAG RPP v2) 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: OOF Dated: June 15, 2023 Received: June 16, 2023

Dear Kate Goscha:

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 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

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Indications for Use

510(k) Number (if known) K231758

Device Name

NxTAG® Respiratory Pathogen Panel v2 (NxTAG® RPP v2)

Indications for Use (Describe)

The following organism types and subtypes are identified and differentiated using the NxTAG RPPv2:

Bacterial Targets: Chlamydia pneumoniae, Mycoplasma pneumoniae

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

The NxTAG® Respiratory Pathogen Panel v2 is indicated for use with the Luminex® MAGPIX® Instrument and xPONENT® and SYNCTTM software.

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

X Prescription Use (Part 21 CFR 801 Subpart D)

Over-The-Counter Use (21 CFR 801 Subpart C)

CONTINUE ON A SEPARATE PAGE IF NEEDED.

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10.0 510(k) Summary

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

Date Prepared: February 27, 2024

A. 510(k) Number:

K231758

B. Purpose for Submission:

Traditional 510(k)

C. Measurand:

The assay detects and identifies nucleic acids of the following respiratory pathogens: Influenza A, Influenza A H1, Influenza A H1pdm09, Influenza A H3, Influenza B, Respiratory Syncytial Virus A, Respiratory Syncytial Virus B, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2), Coronavirus 229E, Coronavirus OC43, Coronavirus NL63, Coronavirus HKU1, Human Metapneumovirus, Rhinovirus/Enterovirus, Adenovirus, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, Parainfluenza 4, Chlamydia pneumoniae, and Mycoplasma pneumoniae.

D. Type of Test:

A multiplexed nucleic acid test intended for use with the Luminex® MAGPIX® Instrument, and xPONENT® and SYNCT™ software, for the qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals with clinical signs and symptoms of a respiratory tract infection, including COVID-19.

E. Applicant:

Kate Goscha 439 University Avenue Toronto, Ontario M5G 1Y8 Canada (608) 203-8909 Luminex Molecular Diagnostics, Inc.

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F. Proprietary and Established Names:

NxTAG® Respiratory Pathogen Panel v2 (NxTAG® RPP v2)

G. Regulatory Information:

1. Regulation Section:
  21 CFR 866.3981
1. Classification:
  Class II (special controls)
1. Product Code(s):
  QOF
1. Panel:
  Microbiology (83)

H. Indications for Use:

1. Indication(s) for use:
  The NxTAG® Respiratory Pathogen Panel v2 (NxTAG® RPP v2) is a multiplexed polymerase chain reaction (PCR) test intended for the simultaneous, qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swab specimens obtained from individuals with signs and symptoms of respiratory tract infection, including COVID-19.

The following organism types and subtypes are identified and differentiated using the NxTAG RPP v2:

Viral Targets
Influenza A	Coronavirus NL63
Influenza A H1	Coronavirus HKU1
Influenza A H1pdm09	Human Metapneumovirus

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Viral Targets
Influenza A H3	Rhinovirus/Enterovirus
Influenza B	Adenovirus
Respiratory Syncytial Virus A	Parainfluenza 1
Respiratory Syncytial Virus B	Parainfluenza 2
SARS-CoV-2	Parainfluenza 3
Coronavirus 229E	Parainfluenza 4
Coronavirus OC43
Bacterial Targets
Chlamydia pneumoniae	Mycoplasma pneumoniae

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

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

The NxTAG® Respiratory Pathogen Panel v2 is indicated for use with the Luminex® MAGPIX® Instrument and xPONENT® and SYNCT™ software.

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

For in vitro diagnostic use.

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3. Special instrument requirements:

. Sample Processing:
- o Extraction: bioMérieux NUCLISENS® easyMAG® or EMAG® nucleic acid extraction systems
- o Amplification: IVD-labeled Thermal Cycler required
. Sample Analysis: Luminex® MAGPIX® Instrument using xPONENT® and SYNCT™ software

Device Description: l.

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Substantial Equivalence Information: J.

1. Predicate device name(s):
  BioFire Respiratory Panel 2.1 (RP2.1)

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

DEN200031

3. Comparison with predicate:

Device & Predicate Device(s):	K231758	DEN200031
Device Trade Name	NxTAG Respiratory PathogenPanel v2	BioFire Respiratory Panel 2.1
General Device CharacteristicSimilarities
Intended Use/Indications ForUse	The Luminex NxTAG RespiratoryPathogen Panel v2 (NxTAG RPPv2) is a multiplexed polymerasechain reaction (PCR) testintended for the simultaneous,qualitative detection andidentification of multiplerespiratory viral and bacterialnucleic acids in nasopharyngealswab specimens obtained fromindividuals with signs andsymptoms of respiratory tractinfection, including COVID-19.The following organism typesand subtypes are identified anddifferentiated using the NxTAGRPP v2:Viral targets:	The BioFire Respiratory Panel2.1 (RP2.1) is a PCR-basedmultiplexed nucleic acid testintended for use with theBioFire FilmArray 2.0 or BioFireFilmArray Torch systems for thesimultaneous qualitativedetection and identification ofmultiple respiratory viral andbacterial nucleic acids innasopharyngeal swabs (NPS)obtained from individualssuspected of respiratory tractinfections, including COVID-19.The following organism typesand subtypes are identifiedusing the BioFire RP2.1:Adenovirus

Influenza A	Coronavirus 229E
Influenza A H1	Coronavirus HKU1
Influenza A H1pdm09	Coronavirus NL63
Influenza A H3	Coronavirus OC43
Influenza B	Severe Acute Respiratory
Respiratory Syncytial Virus A	Syndrome Coronavirus (SARS-CoV-2)
Respiratory Syncytial Virus B	Human Metapneumovirus
SARS-CoV-2	Human Rhinovirus/Enterovirus
Coronavirus 229E	Influenza A, including subtypes
Coronavirus OC43	H1, H1-2009, and H3
Coronavirus NL63	Influenza B
Coronavirus HKU1	Parainfluenza Virus 1
Human Metapneumovirus	Parainfluenza Virus 2
Rhinovirus/Enterovirus	Parainfluenza Virus 3
Adenovirus	Parainfluenza Virus 4
Parainfluenza Virus 1	Respiratory Syncytial Virus
Parainfluenza Virus 2	Bordetella parapertussis
Parainfluenza Virus 3	(IS1001)
Parainfluenza Virus 4	Bordetella pertussis (prxP)
Bacterial targets:	Chlamydia pneumoniae and
Chlamydia pneumoniae	Mycoplasma pneumoniae
Mycoplasma pneumoniae	Nucleic acids from the
Nucleic acids from the viral andbacterial organisms identifiedby this test are generallydetectable in nasopharyngealspecimens during the acutephase of infection. Thedetection and identification ofspecific viral and bacterialnucleic acids from individuals	respiratory viral and bacterialorganisms identified by this testare generally detectable in NPSspecimens during the acutephase of infection. Thedetection and identification ofspecific viral and bacterialnucleic acids from individualsexhibiting signs and/or

exhibiting signs and/orsymptoms of respiratoryinfection are indicative of thepresence of the identifiedmicroorganism and aids indiagnosis if used in conjunctionwith other clinical andepidemiological information,and laboratory findings. Theresults of this test should not beused as the sole basis fordiagnosis, treatment, or otherpatient management decisions.Negative results in the settingof a respiratory illness may bedue to infection with pathogensthat are not detected by thistest, or lower respiratory tractinfection that may not bedetected by a nasopharyngealswab specimen. Positive resultsdo not rule out coinfection withother organisms. The agent(s)detected by the NxTAG RPPv2may not be the definite causeof disease.Additional laboratory testing(e.g., bacterial and viral culture,immunofluorescence, andradiography) may be necessarywhen evaluating a patient withpossible respiratory tractinfection.The NxTAG RespiratoryPathogen Panel v2 is indicatedfor use with the LuminexMAGPIX Instrument andxPONENT and SYNCT software.	symptoms of respiratoryinfection is indicative of thepresence of the identifiedmicroorganism and aids in thediagnosis of respiratoryinfection if used in conjunctionwith other clinical andepidemiological information.The results of this test shouldnot be used as the sole basis fordiagnosis, treatment, or otherpatient management decisions.Negative results in the settingof a respiratory illness may bedue to infection with pathogensthat are not detected by thistest, or lower respiratory tractinfection that may not bedetected by an NPS specimen.Positive results do not rule outcoinfection with otherorganisms. The agent(s)detected by the BioFire RP2.1may not be the definite causeof disease. Additionallaboratory testing (e.g. bacterialand viral culture,immunofluorescence, andradiography) may be necessarywhen evaluating a patient withpossible respiratory tractinfection.
Specimen Type	Same	Nasopharyngeal swabs
Patient Population	Individuals with signs andsymptoms of respiratory tractinfection, including COVID-19	Individuals suspected ofrespiratory tract infections,including COVID-19
Organisms Detected	Same except for:a) addition of assay fordifferentiation ofRespiratory Syncytial Virus Aand Bb) omission of assays forBordetella pertussis andBordetella parapertussis	Viruses:AdenovirusCoronavirus 229ECoronavirus HKU1Coronavirus NL63Coronavirus OC43Severe Acute RespiratorySyndrome Coronavirus 2 (SARS-CoV-2)Human MetapneumovirusHuman Rhinovirus/EnterovirusInfluenza A VirusSubtypes: H1, H3, H1-2009Influenza B VirusParainfluenza Virus 1Parainfluenza Virus 2Parainfluenza Virus 3Parainfluenza Virus 4Respiratory Syncytial VirusBacteria:Bordetella parapertussisBordetella pertussisChlamydia pneumoniaeMycoplasma pneumoniae
Technology	Same	PCR amplification
General Device CharacteristicDifferences
System	Separate instruments fornucleic acid extraction, PCRamplification and detection	Integrated nucleic acidextraction, amplification, anddetection in a sealed vessel.
Assay Read	MAGPIX Instrument	BIOFIRE FilmARRAY 2.0 orBIOFIRE FilmArray TorchSystems
Detection	Hybridization of amplifiedproducts with fluorescently	Array-based melt curve analysis
labeled beads, sorting of tagged products

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Confidential & Restricted

510(k) Summary

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Image /page/11/Picture/0 description: The image shows the Luminex logo. The logo is in black and white and features the word "Luminex" in a bold, sans-serif font. There is a gray dot above the "i" in Luminex. The registered trademark symbol is to the right of the "x".

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Image /page/12/Picture/0 description: The image shows the logo for Luminex. The logo is in black and white, with the word "Luminex" in a bold, sans-serif font. There is a gray dot above the "i" in Luminex. The logo is simple and modern.

K. Standards/Guidance Documents Referenced:

Guidance

Highly Multiplexed Microbiological/Medical Countermeasure In Vitro Nucleic Acid Based Diagnostic Devices Guidance for Industry and Food and Drug Administration Staff - August 2014
. Respiratory Viral Panel Multiplex Nucleic Acid Assay – Class II Special Controls Guidance for Industry and FDA Staff - October 9, 2009
Guidance for Industry and FDA Staff Statistical Guidance on Reporting Results from Studies Evaluating Diagnostic Tests, March 13, 2007
Guidance on Informed Consent for In Vitro Diagnostic Device Studies using Leftover Human Specimens that are Not Individually Identifiable, January 2006
. Policy for Coronavirus Disease-2019 Tests during the Public Health Emergency (Revised). Immediately in Effect Guidance for Clinical Laboratories, Commercial Manufacturers, and Food and Drug Administration Staff, May 11, 2020

Standards

CLSI, EP05-A3, Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline - Third Edition
CLSI, EP07, Interference Testing in Clinical Chemistry; 3rd Edition
CLSI, EP12-A2, User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline – Second Edition
CLSI, EP17-A2, Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline – Second Edition
CLSI, EP24-A2, Assessment of the Diagnostic Accuracy of Laboratory Tests Using Receiver Operating Characteristic Curves; Approved Guideline—Second Edition
. CLSI, EP25-A, Evaluation of Stability of In Vitro Diagnostic Reagents; Approved Guideline
CLSI, EP37, Supplemental Tables for Interference Testing in Clinical Chemistry – First Edition
CLSI, MM03, Molecular Diagnostic Methods for Infectious Diseases; Approved Guideline - Third Edition
CLSI, MM09-A2, Nucleic Acid Sequencing Methods in Diagnostic Laboratory ● Medicine: Approved Guideline -- Second Edition
CLSI, MM17, Validation and Verification of Multiplex Nucleic Acid Assays – Second Edition

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Image /page/13/Picture/0 description: The image shows the word "Luminex" in a bold, sans-serif font. The word is black, and there is a gray dot above the "i" in "Luminex". A registered trademark symbol is located to the right of the "x".

CLSI, MM18-A, Interpretive Criteria for Identification of Bacteria and Fungi by DNA Target Sequencing; Approved Guideline
ISO 14971:2019, Medical Devices – Application of Risk Management to Medical Devices
EN ISO 23640:2015, In vitro diagnostic medical devices Evaluation of stability of in ● vitro diagnostics reagents

L. Test Principle:

The NxTAG® Respiratory Pathogen Panel v2 (NxTAG® RPP v2) incorporates multiplex Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) with the Luminex® proprietary universal tag sorting system on the Luminex platform to detect respiratory pathogen targets. Extracted total nucleic acid is added to pre-plated, Lyophilized Bead Reagents (LBRs), and mixed to resuspend the reaction reagents. The reaction is amplified via RT-PCR and the reaction product undergoes near simultaneous microsphere hybridization within the sealed reaction well. The hybridized, tagged microspheres are then sorted and read on the MAGPIX® instrument. The generated signals are analyzed using the NxTAG® Respiratory Pathogen Panel v2 Assay File for SYNCT™ Software, providing a reliable, qualitative call for each of the targets and internal controls within each reaction well.

M. Performance Characteristics:

1. Analytical performance:

a. Precision (Reproducibility and Repeatability):

Site-to-Site Reproducibility

A site-to-site reproducibility study was performed to assess the total variability of the NxTAG® Respiratory Pathogen Panel (NxTAG RPP v2) assay across study sites, operators, testing days, and instruments. Two operators at each of the 3 sites tested a 9-member reproducibility panel in 4 replicates on 5 non-consecutive days, for a total of 30 runs (3 sites x 2 operators x 5 days). For each member of the 9-member panel, a total of 120 data points (30 runs x 4 replicates) were generated using 1 assay kit lot. The reproducibility panel comprised of a negative sample and 4 multi-analyte (MA) samples prepared in negative simulated matrix (NSM) at two concentrations, Low Positive (1.5x - 3x LoD) and Moderate Positive (5x – 9x LoD). The test concentration for Influenza A H3 (in sample MA2) is based on the LoD for the subtype. Since the Influenza A H3 matrix LoD is 3-fold less sensitive than the subtype, the expected results for the matrix at 1.5x of the subtype LoD was either "positive" or "negative

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Lu

(i.e. both results were acceptable based on the respective LoDs of the Influenza A and H3 targets). Therefore, the Influenza A matrix result in sample MA2 could not be assessed and was excluded from data analysis. The results demonstrated reproducibility of the NxTAG RPP v2 assay across 3 sites with an overall percent agreement of 99.90%. The summary of results is shown in Table 1.

Sample	Target	Sample Type	Agreement with Expected Results				Overall(All Sites)
	Influenza AH1pdm09	Low Positive	40/40(100%)	40/40(100%)	40/40(100%)	120/120(100%)	240/240(100%)
	Influenza AH1pdm09	Moderate Positive	40/40(100%)	40/40(100%)	40/40(100%)	120/120(100%)	240/240(100%)
MA1	RespiratorySyncytial Virus A	Low Positive	40/40(100%)	40/40(100%)	40/40(100%)	120/120(100%)	240/240(100%)
	RespiratorySyncytial Virus A	Moderate Positive	40/40(100%)	40/40(100%)	40/40(100%)	120/120(100%)	240/240(100%)
	Rhinovirusa	Low Positive	40/40(100%)	40/40(100%)	40/40(100%)	120/120(100%)	240/240(100%)
	Rhinovirusa	Moderate Positive	40/40(100%)	40/40(100%)	40/40(100%)	120/120(100%)	240/240(100%)
	All othertargets	Low Positive	719/720(99.86)	720/720(100%)	720/720(100%)	2159/2160(99.95%)	4316/4320(99.91%)
	All othertargets	Moderate Positive	718/720(99.72%)	720/720(100%)	719/720(99.86%)	2157/2160(99.86%)	4316/4320(99.91%)
MA2	Influenza A H3	Low Positive	39/40(97.50%)	40/40(100%)	40/40(100%)	119/120(99.17%)	239/240(99.58%)
	Influenza A H3	Moderate Positive	40/40(100%)	40/40(100%)	40/40(100%)	120/120(100%)	239/240(99.58%)
	RespiratorySyncytial Virus B	Low Positive	40/40(100%)	40/40(100%)	40/40(100%)	120/120(100%)	240/240(100%)
	RespiratorySyncytial Virus B	Moderate Positive	40/40(100%)	40/40(100%)	40/40(100%)	120/120(100%)	240/240(100%)
	All othertargets	Low Positive	717/720(99.58%)	720/720(100%)	720/720(100%)	2157/2160(99.86%)	4434/4440b(99.86%)
	All othertargets	Moderate Positive	757/760(99.61%)	760/760(100%)	760/760(100%)	2277/2280(99.87%)	4434/4440b(99.86%)
MA3	Influenza B	Low Positive	39/40(97.50%)	40/40(100%)	40/40(100%)	119/120(99.17%)	239/240(99.58%)
	Influenza B	Moderate Positive	40/40(100%)	40/40(100%)	40/40(100%)	120/120(100%)	239/240(99.58%)
	Parainfluenzavirus 3	Low Positive	39/40(97.50%)	40/40(100%)	40/40(100%)	119/120(99.17%)	239/240(99.58%)
	Parainfluenzavirus 3	Moderate Positive	40/40(100%)	40/40(100%)	40/40(100%)	120/120(100%)	239/240(99.58%)
	Mycoplasmapneumoniae	Low Positive	39/40(97.50%)	40/40(100%)	40/40(100%)	119/120(99.17%)	239/240(99.58%)
	Mycoplasmapneumoniae	Moderate Positive	40/40(100%)	40/40(100%)	40/40(100%)	120/120(100%)	239/240(99.58%)

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Sample	Target	Sample Type	Site 1	Site 2	Site 3	Overall(All Sites)
	All othertargets	Low Positive	718/720(99.72%)	720/720(100%)	720/720(100%)	2158/2160(99.91%)
	targets	Moderate Positive	719/720(99.86%)	720/720(100%)	720/720(100%)	2159/2160(99.95%)
	SARS-CoV-2	Low Positive	40/40(100%)	40/40(100%)	40/40(100%)	120/120(100%)
		Moderate Positive	40/40(100%)	40/40(100%)	40/40(100%)	120/120(100%)
MA4	HumanMetapneu-movirus	Low Positive	40/40(100%)	40/40(100%)	40/40(100%)	120/120(100%)
		Moderate Positive	40/40(100%)	40/40(100%)	40/40(100%)	120/120(100%)
	Adenovirus	Low Positive	40/40(100%)	40/40(100%)	40/40(100%)	120/120(100%)
		Moderate Positive	40/40(100%)	40/40(100%)	40/40(100%)	120/120(100%)
	All othertargets	Low Positive	716/720(99.44%)	720/720(100%)	720/720(100%)	2156/2160(99.81%)
		Moderate Positive	719/720(99.86%)	720/720(100%)	720/720(100%)	2159/2160(99.96%)
NEG	Negative	N/A	840/840(100%)	840/840(100%)	840/840(100%)	2520/2520(100%)
Overall Agreement with Expected Results(all targets and all test levels)			7499/7520(99.72%)	7520/7520 (100%)	7519/7520(99.99%)	22538/22560(99.90%)

ª Reported by NxTAG RPP v2 as Rhinovirus/Enterovirus

ካ Excludes results of Influenza A Matrix target at Low Positive concentration.

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Lot-to-Lot Reproducibility

A lot-to-lot reproducibility study was performed to assess the total variability of the NxTAG® Respiratory Pathogen Panel (NxTAG RPP v2) assay across 3 assay kit lots with unique lots of critical reagents, including enzymes, buffers, primers, MagPlex microspheres, and dNTPs. One operator at 1 site tested 17-member reproducibility panel in 10 replicates on each of the 3 assay kit lots, for a total of 30 data points (10 replicates x 3 assay lots) for each member. The reproducibility panel comprised of a negative sample and 8 multi-analyte samples prepared in negative simulated matrix (NSM) at two concentrations, Low Positive (1.5x - 3x LoD) and Moderate Positive (5x -9x LoD). The test concentration for Influenza A H3 (in sample MA2) is based on the LoD for the subtype. Since the Influenza A H3 matrix LoD is 3-fold less sensitive than the subtype, the expected results for the matrix at 1.5x of the subtype LoD was either "positive" or "negative" (i.e. both results were acceptable based on the respective LoDs of the Influenza A and H3 targets). Therefore, the Influenza A matrix result in sample MA2 could not be assessed and was excluded from data analysis. The results demonstrated reproducibility of the NxTAG RPP v2 assay across 3 assay kit lots with an overall percent agreement of 99.95%. The summary of results is shown in Table 2.

			Agreement with Expected Results
Sample	Target	Sample Type	Lot 1	Lot 2	Lot 3	Overall(All Lots)
MA1	Influenza A 2009H1N1	Low Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	60/60(100%)
		ModeratePositive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	60/60(100%)
	Respiratory SyncytialVirus A	Low Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	60/60(100%)
		ModeratePositive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)
	Rhinovirusa	Low Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	60/60(100%)
		ModeratePositive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	60/60(100%)
	All other targets	Low Positive	180/180(100%)	180/180(100%)	180/180(100%)	540/540(100%)	1080/1080(100%)
		ModeratePositive	180/180(100%)	180/180(100%)	180/180(100%)	540/540(100%)
MA2	Influenza A H3	Low Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	60/60(100%)
		ModeratePositive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	60/60(100%)
	Respiratory SyncytialVirus B	Low Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	60/60(100%)

	Table 2: NxTAG® RPP v2 Lot-to-lot Reproducibility
--	---------------------------------------------------	--

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Sample	Target	Sample Type	Agreement with Expected Results			Overall(All Lots)
			Lot 1	Lot 2	Lot 3
MA3	All other targets	Moderate Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)
		Low Positive	180/180(100%)	180/180(100%)	180/180(100%)	540/540(100%)
	All other targets	Moderate Positive	190/190(100%)	190/190(100%)	190/190(100%)	570/570(100%)	1110/1110(100%)b
		Low Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)
	Influenza B	Moderate Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	60/60(100%)
		Low Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)
MA3	Parainfluenza virus 3	Moderate Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	60/60(100%)
		Low Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)
	Mycoplasma pneumoniae	Moderate Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	60/60(100%)
		Low Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)
	All other targets	Moderate Positive	180/180(100%)	180/180(100%)	180/180(100%)	540/540(100%)	1080/1080(100%)
		Low Positive	180/180(100%)	180/180(100%)	180/180(100%)	540/540(100%)
MA4	SARS-CoV-2	Moderate Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	60/60(100%)
		Low Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)
	Human Metapneumovirus	Moderate Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	60/60(100%)
		Low Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)
	Adenovirus	Moderate Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	60/60(100%)
		Low Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)
MA4	All other targets	Moderate Positive	180/180(100%)	180/180(100%)	180/180(100%)	540/540(100%)	1080/1080(100%)
		Low Positive	180/180(100%)	180/180(100%)	180/180(100%)	540/540(100%)
	Influenza A Matrix	Moderate Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	60/60(100%)
		Low Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)
	Coronavirus NL63	Moderate Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	60/60(100%)
		Low Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)
			Agreement with Expected Results
Sample	Target	Sample Type	Lot 1	Lot 2	Lot 3	Overall(All Lots)
		Positive	(100%)	(100%)	(100%)	(100%)
	Coronavirus HKU1	Low Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	60/60
		ModeratePositive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	(100%)
	All other targets	Low Positive	180/180(100%)	179/180(99.44%)	180/180(100%)	539/540(99.81%)	1079/1080
		ModeratePositive	180/180(100%)	180/180(100%)	180/180(100%)	540/540(100%)	(99.90%)
	Influenza A H1	Low Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	60/60
		ModeratePositive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	(100%)
	Parainfluenza virus 1	Low Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	60/60
		ModeratePositive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	(100%)
MA6	Chlamydiapneumoniae	Low Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	60/60
		ModeratePositive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	(100%)
	All other targets	Low Positive	180/180(100%)	180/180(100%)	180/180(100%)	540/540(100%)	1079/1080
		ModeratePositive	180/180(100%)	179/180(99.44%)	180/180(100%)	539/540(99.81%)	(99.91%)
	Parainfluenza virus 2	Low Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	60/60
		ModeratePositive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	(100%)
	Parainfluenza virus 4(subtype 4B)c	Low Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	60/60
MA7		ModeratePositive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	(100%)
	Coronavirus 229E	Low Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	60/60
		Low Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	(100%)
	All other targets	ModeratePositive	180/180(100%)	180/180(100%)	179/180(99.44%)	539/540(99.81%)	1079/1080
		Low Positive	180/180(100%)	180/180(100%)	180/180(100%)	540/540(100%)	(99.91%)
	Parainfluenza virus 4	ModeratePositive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	60/60
MA8	(subtype 4A)€	Low Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	(100%)
Sample	Target	Sample Type	Agreement with Expected Results			Overall(All Lots)
			Lot 1	Lot 2	Lot 3
	Coronavirus OC43	ModeratePositive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)	60/60(100%)
		Low Positive	10/10(100%)	10/10(100%)	10/10(100%)	30/30(100%)
	All other targets	ModeratePositive	190/190(100%)	190/190(100%)	190/190(100%)	570/570(100%)	1140/1140(100%)
		Low Positive	190/190(100%)	190/190(100%)	190/190(100%)	570/570(100%)
NEG	Negative	N/A	208/210(99.04%)	210/210(100%)	210/210(100%)	628/630 (99.68%)
Overall Agreement with Expected Results(all targets and all test levels)			3558/3560(99.94%)	3558/3560(99.94%)	3559/3560(99.97%)	10675/10680 (99.95%)

Confidential & Restricted

510(k) Summary

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Confidential & Restricted

510(k) Summary

Page 15 of 60

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ª Reported by NxTAG RPP v2 as Rhinovirus/Enterovirus

b Excludes results of Influenza A Matrix target at Low Positive concentration.

^ NxTAG RPP v2 does not distinguish Parainfluenza virus subtypes 4A and 4B which are both reported as Parainfluenza virus

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Image /page/20/Picture/0 description: The image shows the logo for Luminex. The logo is in black and white, with the word "Luminex" in a bold, sans-serif font. There is a gray circle above the "i" in Luminex. The logo is simple and modern.

b. Linearity/assay reportable range:
Not applicable. The NxTAG® RPP v2 assay is a qualitative assay.
Traceability, Stability, Expected values (controls, calibrators, or methods): C.
- Controls: i)
  - (a) Internal Control

Bacteriophage MS2 is the internal control for the assay. This internal positive control is added to each specimen prior to extraction. This internal control allows the user to ascertain whether the assay is functioning properly. Failure to detect the MS2 control indicates a failure at either the extraction step, the reverse-transcription step, the PCR step, or the detection step, and may be indicative of the presence of amplification inhibitors, thereby preventing false negative results.

(b) External Controls

External positive and negative controls should be used in accordance with local, state, federal accrediting organizations, as applicable.

Positive Control Positive controls are not included in the NxTAG® Respiratory Pathogen Panel v2 assay, but are recommended to be included in every run, as a good laboratory practice.
· Negative Amplification Control (No Template Control (NTC)) The negative amplification control is RNase- free water.
Negative Extraction Control (NEC) The negative extraction control is the sample collection medium that has undergone the entire assay procedure, starting from extraction.

ii) Stability:

(a) Specimen Stability

Stability of raw specimens in Universal Transport Media (UTM), and MicroTest™ M4RT, as well as, stability of extracted specimens were evaluated at temperatures ranging between 2ºC to 8ºC and -70ºC ± 5ºC on the NxTAG®

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RPP v2 assay. For raw specimens prepared in negative clinical matrix in UTM and extracted specimens, all targets probed by the assay were evaluated with a set of 17 samples: eight multi-analyte samples contrived in negative clinical matrix at two concentrations, Low positive (1.5x - 3x LoD) and Moderate positive (5x – 9x LoD), and a negative sample (negative clinical matrix only). For raw specimens prepared in negative clinical matrix in M4RT, a subset of targets in four multi-analyte samples consisting of representative organisms and genome types, at two concentrations Low positive (1.5x – 3x LoD) and Moderate positive (5x – 9x LoD), as well as a negative sample were evaluated. Ten replicates were tested for each condition.

All samples met the acceptance criteria, and the data support a raw specimen stability claim of 7 days at 2°C to 8°C and 12 months at -70°C ± 5°C for both transport media tested, and an extracted specimen stability claim of 4 hours at 2°C to 8°C and 12 months at -70°C ± 5°C.

(b) Device Stability

A shelf-life study was conducted to evaluate the real-time stability of NxTAG® RPP v2 at the recommended storage conditions of 2 - 8°C. Real-time stability was assessed using Positive Controls that cover all analytes probed by the assay, extracted MS2 (internal control), and no template control. Results of this study demonstrated that NxTAG® RPP v2 is stable for at least 12 months when stored at 2 - 8°C.

In-use, open-pouch/vial stability of NxTAG® RPP v2 was also evaluated by mimicking in-use conditions of the kit, which was stored at the recommended storage conditions of 2 - 8°C. Stability was assessed using Positive Controls that cover all or a subset of analytes probed by the assay, MS2 (Internal Control) and a No Template Control. The study demonstrated 100% agreement between calls made with the NxTAG® RPP v2 assay on a cold block for an hour vs. the baseline and calls made with NxTAG® RPP v2 assay on a cold block that was replaced every 15 to 20 minutes vs. the baseline, and confirmed the stability of the NxTAG® RPP v2 assay after it is opened and resealed for 6 cycles over 5 weeks, at the recommended storage conditions.

d. Detection Limit:

The Limit of Detection (LoD) of the NxTAG® RPP v2 assay for each target was assessed by testing simulated samples prepared from high-titer cultured material obtained from commercial suppliers or characterized clinical specimens. For each target, a 3-fold dilution series was prepared in negative clinical matrix (NCM,

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pooled negative nasopharyngeal swabs in Universal Transport Medium), extracted using bioMérieux® EMAG® extraction system, and tested with NxTAG® RPP v2 assay. The preliminary LoD for each target was confirmed by preparing and testing 20 replicates. The LoD concentration for each target was defined as the lowest concentration at which ≥ 95% (≥ 19/20) of the replicates can reproducibly be detected. A droplet digital PCR (ddPCR) assay was performed to quantitate clinical specimens and those culture stocks where titer information in copy number was not available. A summary of the confirmed LoD for each target is listed in Table 3. In addition, confirmation of LoD was performed for targets in Multi-Analyte (MA) samples prepared in NCM. Each MA sample consisted of 2 to 4 target analytes and 8 MA samples cover all targets probed by the NxTAG® RPP v2 assay. Confirmation of the single-analyte LoD in MA samples supported the use of MA samples in NxTAG® RPP v2 analytical studies.

Target	Strain/Isolate	Supplier/Part Number	LoD Concentration		# Detected/# Tested
Influenza A(Matrix)	A/Brisbane/59/07	ZeptoMetrix0810244CF	$1.19E+02$	2.83E-02TCID50/mL	20/20
	A/NY/02/09	ZeptoMetrix0810109CFN	$3.28E+02$	3.74E-02TCID50/mL	20/20
	A/Wisconsin/67/05	ZeptoMetrix0810252CF	$1.68E+02$	6.45E-01TCID50/mL	20/20
Influenza A H1(Subtype)	A/Brisbane/59/07	ZeptoMetrix0810244CF	$1.60E+03$	3.82E-01TCID50/mL	20/20
Influenza A H1pdm09(Subtype)	A/NY/02/09	ZeptoMetrix0810109CFN	$9.84E+02$	1.12E-01TCID50/mL	19/20
Influenza A H3(Subtype)	A/Wisconsin/67/05	ZeptoMetrix0810252CF	$5.60E+01$	2.15E-01TCID50/mL	20/20
Influenza B	B/Florida/02/06	ZeptoMetrix0810037CF	$6.33E+01$	9.67E-01TCID50/mL	19/20
Respiratory SyncytialVirus A	A2	ATCC VR-1540	$4.97E+03$	3.77E+01PFU/mL	19/20
Respiratory SyncytialVirus B	18537	ATCC VR-1580	$7.21E+03$	3.20E-01PFU/mL	20/20
Parainfluenza Virus 1	N/A	ZeptoMetrix0810014CF	$6.92E+02$	7.64E-01TCID50/mL	20/20
Parainfluenza virus 2	Greer	ATCC VR-92	$3.45E+02$	7.32E-01TCID50/mL	20/20
Parainfluenza virus 3	C 243	ATCC VR-93	$1.01E+03$	1.10E+02TCID50/mL	20/20
		Supplier/Part Number	LoD Concentration
Target	Strain/Isolate		Copies/mL	In SupplierUnit	# Detected/# Tested
Parainfluenza virus 4	Type 4A	ZeptoMetrix0810060CF	1.69E+04	8.58E-01TCID50/mL	20/20
	Type 4B, CH 19503	ATCC VR-1377	7.15E+03	5.99E+01TCID50/mL	20/20
SARS-CoV-2	USA-WA1/2020	ATCC VR-1986HK	5.00E+02	7.68E+00TCID50/mL	19/20
Coronavirus 229E	N/A	ATCC VR-740	3.81E+02	1.22E-01TCID50/mL	19/20
Coronavirus NL63	N/A	ZeptoMetrix0810228CF	1.00E+02	6.45E-03TCID50/mL	20/20
Coronavirus OC43	Betacoronavirus 1	ATCC VR-1558	4.55E+03	7.32E-02TCID50/mL	20/20
Coronavirus HKU1	Genotype B	Clinical Sample	4.18E+03	N/A	19/20
HumanMetapneumovirus	hMPV-16, Type A1,IA10-2003	ZeptoMetrix0810161CF	7.15E+01	5.76E-02TCID50/mL	20/20
	hMPV-3, Type B1,Peru2-2002	ZeptoMetrix0810156CF	2.62E+02	1.78E-02TCID50/mL	20/20
Rhinovirus/Enterovirus	Rhinovirus50-525-CV54[V-192-001-021]	ATCC VR-1195	1.54E+03	6.87E+01TCID50/mL	20/20
	EnterovirusSpecies D, Type 682007 Isolate	ZeptoMetrix0810237CF	3.53E+03	2.30E+00TCID50/mL	20/20
Adenovirus	Species B, Type 142006 isolate	ZeptoMetrix0810108CF	1.42E+03	1.44E-01TCID50/mL	19/20
	Species C, Type 1	ZeptoMetrix0810050CF	2.01E+04	9.28E+01TCID50/mL	20/20
	Species E, Type 4	ZeptoMetrix0810070CF	7.33E+03	1.91E-01TCID50/mL	19/20
Chlamydiapneumoniae	TW-183	ATCC VR-2282	2.38E+02	3.74E+01IFU/mL	20/20
Mycoplasmapneumoniae	M129	ZeptoMetrix 0801579	3.23E+03	5.56E+01CCU/mL	19/20
Target	Strain	Supplier/Part Number	Concentration(IU/mL)	# Detected / #tested
SARS-CoV-2	Heat inactivatedEngland/02/2020 isolate	NIBSC20/146	7.70E+05	20/20 (100%)
Organism	Strain	Supplier / PartNumber	ConcentrationTested(Copies/mL)	# Detected/# Tested
Influenza A H1	A/Brisbane/59/07*	ZeptoMetrix0810244CF	4.81E+03	3/3
	A/New Caledonia/20/99	ZeptoMetrix0810036CF	4.81E+03	3/3
	A/Solomon Island/03/06	ZeptoMetrix0810036CFN	4.81E+03	3/3
	A/Taiwan/42/06	ZeptoMetrix0810036CF(New PN:0810247CF)	4.81E+03	3/3
	A/Denver/1/57	ATCC VR-546	4.81E+03	3/3 (Matrix)
			$5.77E+07^a$	0/3 (H1 Subtype)
	A/NY/02/09*	ZeptoMetrix0810109CFN	5.11E+03	3/3
	A/SwineNY/01/2009(New name: A/NY/01/09)	ZeptoMetrix0810109CFN(New PN:0810248CF)	5.11E+03	3/3
	A/SwineNY/03/2009(New name: A/NY/03/09)	ZeptoMetrix0810109CFN(New PN:0810249CF)	5.11E+03	3/3
	A/Swine/Canada/6294/09	ZeptoMetrix0810109CFJ	5.11E+03	3/3
	A/California/07/09	ZeptoMetrix0810165CF	5.11E+03	3/3
	A/Mexico/4108/09	ZeptoMetrix0810166CF	5.11E+03	3/3
Influenza A H1pdm09	A/Michigan/45/15	ZeptoMetrix0810538CF	5.11E+03	3/3
	A/Brisbane/02/18	ZeptoMetrix0810585CF	5.11E+03	3/3
	A/Virginia/ATCC1/2009	ATCC VR-1736	5.11E+03	3/3
	A/Netherlands/2629/2009	BEI NR-19823	5.11E+03	3/3
	A/Houston/3H/2009	BEI NR-20340	5.11E+03	3/3
	A/Brownsville/31H/2009	BEI NR-20344	5.11E+03	3/3
	A/Dominican Republic/7293/2013	IRR FR-1298	5.11E+03	3/3
	A/Massachusetts/15/2013	IRR FR-1319	5.11E+03	3/3
	A/Swine/1976/31	ATCC VR-99	4.81E+03	3/3 (Matrix)
Organism	Strain	Supplier / PartNumber	ConcentrationTested(Copies/mL)	# Detected/# Tested
Influenza A H3	A/Swine/Iowa/15/30	ATCC VR-333	1.83E+07	3/3 (H1pdm09Subtype)
	A/Swine/Iowa/15/30	ATCC VR-333	4.81E+03	3/3 (Matrix)
	A/Swine/Iowa/15/30	ATCC VR-333	2.25E+07	3/3(H1pdm09Subtype)
	A/Wisconsin/67/05*	ZeptoMetrix0810252CF	1.68E+02	3/3
	A/Brisbane/10/07	ZeptoMetrix0810138CF	5.04E+02	3/3 (Matrix)
	A/Brisbane/10/07	ZeptoMetrix0810138CF	1.68E+02	3/3 (H3 Subtype)
	A/Texas/50/12	ZeptoMetrix0810238CF	5.04E+02	3/3
	A/Perth/16/09	ZeptoMetrix0810138CF(New PN:0810251CF)	1.68E+02	3/3
	A/Hong Kong/4801/14	ZeptoMetrix0810526CF	4.54E+03	3/3 (Matrix)
	A/Hong Kong/4801/14	ZeptoMetrix0810526CF	1.51E+03	3/3 (H3 Subtype)
	A/Singapore/INFIMH-16-0019/16	ZeptoMetrix0810574CF	1.51E+03	3/3
	A/Kansas/14/17	ZeptoMetrix0810586CF	5.04E+02	3/3 (Matrix)
	A/Kansas/14/17	ZeptoMetrix0810586CF	1.68E+02	3/3 (H3 Subtype)
	A/Hong Kong/8/68	ATCC VR-544	1.68E+02	3/3 (Matrix)
	A/Hong Kong/8/68	ATCC VR-544	4.08E+04	3/3 (H3 Subtype)
	A/Alice	ATCC VR-776	1.68E+02	3/3 (Matrix)
	A/Alice	ATCC VR-776	8.04E+03	3/3 (H3 Subtype)
	A/Port Chalmers/1/73	ATCC VR-810	1.51E+03	3/3 (Matrix)
		A/Port Chalmers/1/73	ATCC VR-810	5.04E+02
	A/Sydney/5/1997	BEI NR-12278	4.54E+03	3/3 (Matrix)
		A/Sydney/5/1997	BEI NR-12278	1.68E+02
	A/Santiago/7981/2006	IRR FR-336	1.68E+02	3/3
	A/Henan/Jinshui/147/2007	IRR FR-365	1.68E+02	3/3
	A/Brisbane/9/2006	IRR FR-366	1.68E+02	3/3
Organism	Strain	Supplier / PartNumber	ConcentrationTested(Copies/mL)	# Detected/# Tested
	A/Nepal/921/2006	IRR FR-367	1.68E+02	3/3
	A/Florida/2/2006	IRR FR-368	5.04E+02	3/3 (Matrix)
	A/Florida/2/2006	IRR FR-368	1.68E+02	3/3 (H3 Subtype)
	South Australia/55/14	0810512CF	1.68E+02	3/3
	Stockholm/6/14	0810513CF	5.04E+02	3/3 (Matrix)
	Stockholm/6/14	0810513CF	1.68E+02	3/3
	Norway/466/14	0810514CF	1.68E+02	3/3
	Hong Kong/2671/19	0810609CF	5.04E+02	3/3
	A/California/2/2014	VR-1938	1.68E+02	3/3
	A/Switzerland/9715293/2013	VR-183	1.68E+02	3/3
	Clinical Sample	500-NEG-161	1.68E+02	3/3
	Clinical Sample	500-NEG-199	5.04E+02	3/3
Influenza A H5b	A/Anhui/01/2005 (H5N1)-PR8-IBCDC-RG6	IRR FR-735	5.04E+02	3/3 (Matrix only)
	A/Egypt/N03072/2010 (H5N1)-PR8-IDCDC-RG29	IRR FR-1065	5.04E+02	3/3 (Matrix only)
	A/pheasant/New Jersey/1355/1998(H5N2)-PR8-IBCDC-4	IRR FR-771	5.04E+02	3/3 (Matrix only)
	A/Hubei/1/2010 (H5N1)-PR8-IDCDC-RG30	IRR FR-1066	5.04E+02	3/3 (Matrix only)
Influenza A H7b	A/turkey/Virginia/4529/2002(H7N2) x PR8-IBCDC-5	IRR FR-772	5.04E+02	3/3 (Matrix only)
Influenza A H7b	A/mallard/Netherlands/12/2000(H7N7)/PR8-IBCDC-1	IRR FR-773	5.04E+02	3/3 (Matrix only)
Influenza A H9b	A/Hong Kong/33982/2009 (H9N2)-PR8-IDCDC-RG26	IRR FR-1068	5.04E+02	3/3 (Matrix only)
Influenza Bc(Yamagata Lineage)	B/Florida/02/06*	ZeptoMetrix0810037CF	1.90E+02	3/3
	B/Massachusetts/2/12	ZeptoMetrix0810239CF	1.90E+02	3/3
	B/Wisconsin/1/10	ZeptoMetrix0810241CF	1.90E+02	3/3
	B/Florida/04/06	ZeptoMetrix0810037CF(New PN:0810255CF)	1.90E+02	3/3
Organism	Strain	Supplier / PartNumber	ConcentrationTested(Copies/mL)	# Detected/# Tested
	B/Florida/07/04	ZeptoMetrix0810037CF (NewPN: 0810256CF)	1.90E+02	3/3
	B/Panama/45/90	ZeptoMetrix0810037CF(New PN:0810259CF)	1.90E+02	3/3
	B/Phuket/3073/13	ZeptoMetrix0810515CF	1.90E+02	3/3
	B/Bangladesh/5972/2007	IRR FR-450	1.90E+02	3/3
	B/Hubei-Wujiagang/158/2009	IRR FR-469	1.90E+02	3/3
	B/Brisbane/33/08	ZeptoMetrix0810037CF(New PN:0810253CF)	1.90E+02	3/3
	B/Brisbane/60/08	ZeptoMetrix0810037CF(New PN:0810254CF)	1.90E+02	3/3
Influenza Bᶜ(Victoria Lineage)	B/Malaysia/2506/04	ZeptoMetrix0810258CF	1.90E+02	3/3
	B/Colorado/06/17	ZeptoMetrix0810573CF	5.69E+02	3/3
	B/Hong Kong/259/2010	IRR FR-663	1.90E+02	3/3
	B/New Jersey/1/2012	IRR FR-1270	1.90E+02	3/3
	B/Texas/02/2013	IRR FR-1302	1.90E+02	3/3
RSVA	A2*	ATCC VR-1540	1.49E+04	3/3
	2006 Isolate	ZeptoMetrix0810040ACF	1.49E+04	3/3
	Long	ATCC VR-26	1.49E+04	3/3
	18537*	ATCC VR-1580	2.16E+04	3/3
RSVB	CH93(18)-18	ZeptoMetrix0810040CF	2.16E+04	3/3
	9320	ATCC VR-955	2.16E+04	3/3
	B WV/14617/85	ATCC VR-1400	2.16E+04	3/3
	B1	BEI NR-4052	2.16E+04	3/3
PIV1	Type 1*	ZeptoMetrix0810014CF	2.08E+03	3/3
Organism	Strain	Supplier / PartNumber	ConcentrationTested(Copies/mL)	# Detected/# Tested
	C35	ATCC VR-94	2.08E+03	3/3
PIV2	Greer*	ATCC VR-92	1.03E+03	3/3
	Type 2	ZeptoMetrix0810015CF	1.03E+03	3/3

	C 243*	ATCC VR-93	3.04E+03	3/3
PIV3	Type 3	ZeptoMetrix0810016CF	3.04E+03	3/3
	ATCC-2011-5	ATCC VR-1782	3.04E+03	3/3
	NIH 47885	BEI NR-3233	3.04E+03	3/3
PIV4	Type 4A*	ZeptoMetrix0810060CF	5.08E+04	3/3
	M-25; Type 4A	ATCC VR-1378	5.08E+04	3/3
	CH 19503; Type 4B*	ATCC VR-1377	2.14E+04	3/3
	Type 4B	ZeptoMetrix0810060BCF	2.14E+04	3/3
SARS-CoV-2	2019-nCoV/USA-WA1/2020*	ATCC VR-1986HK	1.50E+03	3/3
	HongKong/VM2000106/2020	ZeptoMetrix0810590CFHI	1.50E+03	3/3
	USA-WA1/2020	ZeptoMetrix0810587CFHI	1.50E+03	3/3
	BetaCoV/Germany/BavPat1/2020p.1d	EVAg 026N-03889	2.73E+03	3/3
	2019-nCoV/Italy-INMI1d	EVAg 008N-03894	2.73E+03	3/3
	England/02/2020d	BEI NR-52499	2.73E+03	3/3
	Singapore/2/2020d	BEI NR-52501	2.73E+03	3/3
	USA-IL1/2020d	BEI NR-52503	2.73E+03	3/3
	USA-CA1/2020d	BEI NR-52504	2.73E+03	3/3
	USA-AZ1/2020d	BEI NR-52505	2.73E+03	3/3
	USA-WI1/2020d	BEI NR-52506	2.73E+03	3/3
	USA-CA3/2020d	BEI NR-52507	2.73E+03	3/3
	USA-CA4/2020d	BEI NR-52508	2.73E+03	3/3
Organism	Strain	Supplier / PartNumber	ConcentrationTested(Copies/mL)	# Detected/# Tested
	USA-CA2/2020d	BEI NR-52509	2.73E+03o	3/3
	Chile/Santiago_op4d1/2020d	BEI NR-52510	2.73E+03o	3/3
	New York-PV08410/2020d	BEI NR-53518	2.73E+03o	3/3
	USA/CA_CDC_5574/2020, HeatInactivated	BEI NR-55245	1.50E+03o	3/3
	Alpha (B1.1.7)/UK Variant	Clinical Specimen	1.50E+03	3/3
	Epsilon (B1.429)/California Variant	Clinical Specimen	1.50E+03	3/3
	Epsilon (B1.429)/California Variant	Clinical Specimen	1.50E+03	3/3
	Delta (B.1.617.2)	Clinical Specimen	1.50E+03	3/3
	Delta (B.1.617.2)	Clinical Specimen	1.50E+03	3/3
	Delta (B.1.617.2)	Clinical Specimen	1.50E+03	3/3
	Delta (B.1.617.2)	Clinical Specimen	1.50E+03	3/3
	Delta (B.1.617.2)	Clinical Specimen	1.50E+03	3/3
	Omicron (B.1.1.529 and BAlineages)	Clinical Specimen	1.50E+03	3/3
	Omicron (B.1.1.529 and BAlineages)	Clinical Specimen	1.50E+03	3/3
	Omicron (B.1.1.529 and BAlineages)	Clinical Specimen	1.50E+03	3/3
	Omicron (B.1.1.529 and BAlineages)	Clinical Specimen	1.50E+03	3/3
	Omicron (B.1.1.529 and BAlineages)	Clinical Specimen	1.50E+03	3/3
Coronavirus 229E	229E*	ATCC VR-740	1.14E+03	3/3
	229E	ZeptoMetrix0810229CF	1.14E+03	3/3
Coronavirus NL63	NL63*	ZeptoMetrix0810228CF	3.00E+02	3/3
	NL63	BEI NR-470	3.00E+02	3/3
	Betacoronavirus 1*	ATCC VR-1558	1.36E+04	3/3
Coronavirus OC43	OC43	ZeptoMetrix0810024CF	1.36E+04	3/3

Table 3: NxTAG® RPP v2 Limit of Detection

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Analytical Sensitivity for the First WHO International Standard for SARS-CoV-2 e.

The Analytical Sensitivity of the NxTAG® RPP v2 assay for the WHO standard for SARS-CoV-2 was evaluated.

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The preliminary LoD was determined by preparing a 10-fold dilution series in negative clinical matrix (NCM, pooled negative nasopharyngeal swabs in Universal Transport Medium) and testing each dilution level in triplicate. The LoD was confirmed by testing 20 replicates of sample prepared at preliminary LoD, as well as 20 replicates of samples prepared 3-fold above and below this preliminary LoD.

The Analytical Sensitivity was defined as the lowest concentration at which ≥ 95% of the replicates tested generated a positive call. The summary of the results is shown in Table 4.

Table 4. Analytical Sensitivity of NxTAG® RPP v2 for the First WHO International Standard for SARS-CoV-2.

Analytical Reactivity (Inclusivity) f.

Analytical Reactivity (Inclusivity) of the NxTAG® RPP v2 assay was assessed by testing a total of 193 pathogen strains/isolates (168 reactivity strains and 25 LoD strains). The strains tested represent the diversity of the targets probed by NxTAG® RPP v2. Each strain was prepared in negative simulated matrix (NSM) and tested in triplicate on the NxTAG RPP v2 assay. A droplet digital PCR (ddPCR) assay was performed to quantitate clinical specimens and those culture stocks where titer information in copy number was not available. For influenza A results, the concentration at which both the influenza A matrix and the subtype of that strain were detected by NxTAG® RPP v2 is shown. When the influenza A matrix and the subtype were detected at different concentrations, the concentration for each target is listed separately. The NxTAG® RPP v2 assay is capable of detecting the Influenza A matrix for all strains, including Influenza A H5, H7, and H9. Specimens containing Influenza A H5, H7, and H9 strains are expected to be reported as "Influenza A" only. The applicable subtype of all strains of influenza A H1, A H1pdm09 and A H3 with the exception of Influenza A A/Denver/1/57 H1 were detected successfully. A summary of the results, including the strain identity and the concentration at which they were detected, are shown in Table 5.

Table 5. Results of NxTAG® RPP v2 Analytical Reactivity

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Confidential & Restricted

510(k) Summary

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Confidential & Restricted

510(k) Summary

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Organism	Strain	Supplier / PartNumber	ConcentrationTested(Copies/mL)	# Detected/# Tested
Coronavirus HKU1	HKU1, Genotype B*	Clinical Specimen	1.25E+04	3/3
	HKU1, Genotype B	Clinical Specimen	1.25E+04	3/3
	HKU1, Genotype A	Clinical Specimen	1.25E+04	3/3
	HKU1, Genotype A	Clinical Specimen	1.25E+04	3/3
	Type A1, IA10-2003, hMPV-16*	ZeptoMetrix0810161CF	2.15E+02	3/3
	Type A1, IA3-2002, hMPV-9	ZeptoMetrix0810160CF	2.15E+02	3/3	Type A2, IA27-2004, hMPV-27	ZeptoMetrix0810164CF	2.15E+02	3/3	Type A2, DHI 26583	Clinical Specimen	2.15E+02	3/3	HumanMetapneumovirus	Type B1, Peru2-2002, hMPV-3*	ZeptoMetrix0810156CF	7.85E+02	3/3	Type B1, Peru3-2003, hMPV-5	ZeptoMetrix0810158CF	7.85E+02	3/3	Type B2, Peru1-2002, hMPV-4	ZeptoMetrix0810157CF	7.85E+02	3/3	Type B2, Peru6-2003, hMPV-8	ZeptoMetrix0810159CF	7.85E+02	3/3	Type B2, IA18-2003, hMPV-18	ZeptoMetrix0810162CF	7.85E+02	3/3	Species A, Type 85, strain 50-525-CV54 [V-192-001-021]*	ATCC VR-1195	4.61E+03	3/3	Species A, Type 1A	ZeptoMetrix0810012CFN	4.61E+03	3/3	Species A, Type 2, strain HGP	ATCC VR-482	4.61E+03	3/3	Rhinovirusf	Species A, Type 7, strain 68-CV11	ATCC VR-1601	4.61E+03	3/3	Species A, Type 16	ZeptoMetrix0810285CF	4.61E+03	3/3	Species A, Type 34, strain 137-3	ATCC VR-1365	4.61E+03	3/3	Species A, Type 39, strain 209	ATCC VR-340	4.61E+03	3/3	Species A, Type 54, strain FO1-3774	ATCC VR-1661	4.61E+03	3/3	Species A, Type 57, strain Ch47	ATCC VR-1600	4.61E+03	3/3
	Type A1, IA3-2002, hMPV-9	ZeptoMetrix0810160CF	2.15E+02	3/3
	Type A2, IA27-2004, hMPV-27	ZeptoMetrix0810164CF	2.15E+02	3/3
	Type A2, DHI 26583	Clinical Specimen	2.15E+02	3/3
HumanMetapneumovirus	Type B1, Peru2-2002, hMPV-3*	ZeptoMetrix0810156CF	7.85E+02	3/3
	Type B1, Peru3-2003, hMPV-5	ZeptoMetrix0810158CF	7.85E+02	3/3
	Type B2, Peru1-2002, hMPV-4	ZeptoMetrix0810157CF	7.85E+02	3/3
	Type B2, Peru6-2003, hMPV-8	ZeptoMetrix0810159CF	7.85E+02	3/3
	Type B2, IA18-2003, hMPV-18	ZeptoMetrix0810162CF	7.85E+02	3/3
	Species A, Type 85, strain 50-525-CV54 [V-192-001-021]*	ATCC VR-1195	4.61E+03	3/3
	Species A, Type 1A	ZeptoMetrix0810012CFN	4.61E+03	3/3
	Species A, Type 2, strain HGP	ATCC VR-482	4.61E+03	3/3
Rhinovirusf	Species A, Type 7, strain 68-CV11	ATCC VR-1601	4.61E+03	3/3
	Species A, Type 16	ZeptoMetrix0810285CF	4.61E+03	3/3
	Species A, Type 34, strain 137-3	ATCC VR-1365	4.61E+03	3/3
	Species A, Type 39, strain 209	ATCC VR-340	4.61E+03	3/3
	Species A, Type 54, strain FO1-3774	ATCC VR-1661	4.61E+03	3/3
Species A, Type 57, strain Ch47	ATCC VR-1600	4.61E+03	3/3

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Organism	Strain	Supplier / PartNumber	ConcentrationTested(Copies/mL)	# Detected/# Tested
	Species A, Type 77, strain 130-63[V-185-001-021]	ATCC VR-1187	4.61E+03	3/3
	Species B, Type 3, strain FEB	ATCC VR-483	4.61E+03	3/3
	Species B, Type 14, strain 1059	ATCC VR-284	4.61E+03	3/3
	Species B, Type 17, strain 33342	ATCC VR-1663	4.61E+03	3/3
	Species B, Type 27, strain 5870[5870-CV28] (NIAID V-144-001-021)	ATCC VR-1137	4.61E+03	3/3
	Species B, Type 42, strain 56822	ATCC VR-338	4.61E+03	3/3
	Species B, Type 83, strain Baylor 7[V-190-001-021]	ATCC VR-1193	4.61E+03	3/3
	Type C	Clinical Specimen	4.61E+03	3/3
	Type C	Clinical Specimen	4.61E+03	3/3
	Type C	Clinical Specimen	4.61E+03	3/3
	Type C	Clinical Specimen	4.61E+03	3/3
	Type C	Clinical Specimen	4.61E+03	3/3
	Species D, Type 68, 2007 isolate*	ZeptoMetrix0810237CF	1.06E+04	3/3
	Species A, Human Enterovirus 71,strain H	ATCC VR-1432	1.06E+04	3/3
	Species A, Human Coxsackie A10,strain M.K. (Kowalik)	ATCC VR-168	1.06E+04	3/3
Enterovirus'	Species B, Human CoxsackievirusB1, strain Conn-5	ATCC VR-28	1.06E+04	3/3
	Species B, Human CoxsackievirusB4, strain J.V.B. (Benschoten)	ATCC VR-184	1.06E+04	3/3
	Species B, Human Echovirus 11,strain Gregory	ATCC VR-41	2.86E+05	3/3
	Species B, Human Echovirus 13,strain Del Carmen	ATCC VR-1054(New PN: VR-43)	1.06E+04	3/3
	Species B, Enterovirus Type 69,strain Toluca-1 [V-068-001-021]	ATCC VR-1077	1.06E+04	3/3
	Species C, Human coxsackievirusA21, strain Kuykendall	ATCC VR-850	1.06E+04	3/3
Organism	Strain	Supplier / PartNumber	ConcentrationTested(Copies/mL)	# Detected/# Tested
	Species C, Human coxsackievirusA24, strain DN-19	ATCC VR-1662	1.06E+04	3/3
	Species D, Type 68, 2014 Isolate	ZeptoMetrix0810300CF	1.06E+04	3/3
	Species D, Type 68, strainUS/MO/14-18947	ATCC VR-1823	1.06E+04	3/3
	Species D, Type 68, strain US/IL/14-18952	ATCC VR-1824	1.06E+04	3/3
	Species D, Type 68, strainUS/KY/14-18953	ATCC VR-1825	1.06E+04	3/3
	Species D, Type 68, strain Fermon	ATCC VR-1076(New PN: VR-1826)	1.06E+04	3/3
	Species D, Type 70, strain J670/71	ATCC VR-836	1.06E+04	3/3
	Species B, Type 14, 2006 isolate*	ZeptoMetrix0810108CF	7.38E+03	3/3
	Species B, Type 3	ZeptoMetrix0810062CF	7.38E+03	3/3
	Species B, Type 7, strain Gomen	ATCC VR-7	7.38E+03	3/3
	Species B, Type 7A	ZeptoMetrix0810021CF	7.38E+03	3/3
	Species B, Type 21, AV-1645 [128]	ATCC VR-1098(New PN: VR-256)	7.38E+03	3/3
Adenovirus	Species C, Type 1*	ZeptoMetrix0810050CF	6.02E+04	3/3
	Species C, Type 1, strain Adenoid71	ATCC VR-1	6.02E+04	3/3
	Species C, Type 2	ZeptoMetrix0810110CF	6.02E+04	3/3
	Species C, Type 5	ZeptoMetrix0810020CF	6.02E+04	3/3
	Species C, Type 6	ZeptoMetrix0810111CF	6.02E+04	3/3
	Species E, Type 4*	ZeptoMetrix0810070CF	2.20E+04	3/3
C. pneumoniae	TW-183*	ATCC VR-2282	7.13E+02	3/3
	TWAR (CDC/CWL-029)	ATCC VR-1310	7.13E+02	3/3
Organism	Strain	Supplier / PartNumber	ConcentrationTested(Copies/mL)	# Detected/# Tested
	TWAR 2023	ATCC VR-1356	7.13E+02	3/3
	AR-39	ATCC 53592	7.13E+02	3/3
M. pneumoniae	M129*	ZeptoMetrix0801579	1.29E+04	3/3
	[M52]	ATCC 15293	1.29E+04	3/3
	FH strain of Eaton Agent [NCTC 10119]	ATCC 15531-TTR	1.29E+04	3/3
	Mutant 22	ATCC 39505	1.29E+04	3/3

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{34}------------------------------------------------

Indicates a LoD strain.

ª Highest possible concentration.

ხ NxTAG RPP v2 does not differentiate Influenza A H5, H7 or H9, all of which are reported as Influenza A

^ NxTAG RPP v2 does not differentiate the Yamagata and Victoria lineages, both of which are reported as influenza B

d Samples were obtained as RNA. The RNA was diluted in extracted negative simulated matrix to a concentration that represented 1.50E+03 copies/mL in a raw sample.

e Concentration units for these strains are Genome equivalents/mL.

1 Reported by NxTAG RPP v2 as Rhinovirus/Enterovirus

Analytical Reactivity In Silico Analysis

Based on in silico inclusivity analysis, it is predicted that the SARS-CoV-2 sequences available from GISAID EpiCoV database as of November 30, 2023, including sequences from all defined variants of concern or interest, are 100% detectable by NxTAG® Respiratory Pathogen Panel v2 (NxTAG RPP v2) assay.

Influenza A and B inclusivity was assessed with sequences available from the GISAID EpiFlu database between January 1, 2017 and May 5, 2023, as well as between January 1, 2000 and December 31, 2008. The assay oligos for Influenza A, Influenza A H1 (including H1pdm09), Influenza A H3, and Influenza B are predicted to have ~99% inclusivity against the analyzed sequences.

For all targets other than SARS-CoV-2 and the influenza viruses, in silico inclusivity analysis was performed with sequences available from the GenBank" Nucleotide (nt) database as of April 8, 2023. Based on this analysis, ≥96% of sequences of each analyte were predicted to be detected by NxTAG® RPP v2, except for Parainfluenza Virus 2 (~92%) and untyped strains of Parainfluenza Virus 4 (~94%), which exhibited lower homology.

g. Analytical Specificity

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i) Cross-Reactivity

Analytical Specificity (Exclusivity) of the NxTAG® Respiratory Pathogen Panel v2 (NxTAG® RPP v2) assay was assessed with pathogens that cause respiratory infections or those that may be found in respiratory specimens. Sixty-three (63) organisms (82 strains total) were tested, including 41 pathogens that are not detected by NxTAG® RPP v2, and pooled nasal wash (referred to as "Off-panel organisms" – Table 6) and 22 that are detected by the assay (referred to as "On-Panel organisms" – Table 7). Each strain was prepared in negative simulated matrix (NSM) to reach the high positive concentration and tested in triplicate on the NxTAG® RPP v2 assay. None of the off-panel or on-panel organisms tested showed cross-reactivity, with the exception of one strain: Enterovirus (Species D, Type 68, US/IL/14-18952). This strain generated a false positive call for Influenza A H3 when it was tested at ≥ 1.00E+03 TCIDso/mL, although the influenza A matrix gene target was negative. No false positive call was generated when the strain was tested at 1.00E+02 TCID50/mL and five other isolates of Enterovirus D showed no evidence of cross-reaction.

Off-Panel Organisms	Concentration Tested	Cross-ReactivityDetected
Aspergillus flavus	1.00E+06	CFU/mL	None
Aspergillus fumigatus	1.00E+06	CFU/mL	None
Bordetella parapertussis	1.00E+06	CFU/mL	None
Bordetella pertussis	1.00E+06	CFU/mL	None
Candida albicans	1.00E+06	CFU/mL	None
Chlamydia trachomatis	1.00E+06	IFU/mL	None
Corynebacterium diphtheriae	1.00E+06	CFU/mL	None
Corynebacterium striatum	1.00E+06	CFU/mL	None
Cytomegalovirus	1.00E+05	TCID50/mL	None
Epstein Barr Virus	1.00E+07	Copies/mL	None
Escherichia coli	1.00E+06	CFU/mL	None
Fusobacterium necrophorum	1.00E+06	CFU/mL	None
Haemophilus influenzae	1.00E+06	CFU/mL	None
Herpes Simplex virus Type 1	1.00E+05	TCID50/mL	None
Human Bocavirus	1.00E+07	Copies/mL	None
Klebsiella pneumoniae	1.00E+06	CFU/mL	None
Lactobacillus acidophilus	1.00E+06	CFU/mL	None
Lactobacillus plantarum	1.00E+06	CFU/mL	None
Legionella (Tatlockia) micdadei	1.00E+06	CFU/mL	None

Table 6: NxTAG® RPP v2 Analytical Specificity (Off-Panel Organisms)

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Off-Panel Organisms	Concentration Tested	Cross-ReactivityDetected
Legionella pneumophila	1.00E+06 CFU/mL	None
Measles Virus	1.00E+05 TCID50/mL	None
MERS-coronavirus	1.00E+05 TCID50/mL	None
Moraxella catarrhalis	1.00E+06 CFU/mL	None
Mumps Virus	1.00E+05 TCID50/mL	None
Mycobacterium tuberculosis	1.00E+06 CFU/mL	None
Mycoplasma genitalium	1.00E+051 CCU/mL(Approximate)	None
Mycoplasma hominis	1.00E+06 CCU/mL	None
Neisseria elongata	1.00E+06 CFU/mL	None
Neisseria gonorrhoeae	1.00E+06 CFU/mL	None
Neisseria meningitidis	1.00E+06 CFU/mL	None
Pneumocystis carinii	1.00E+06 nuclei/mL	None
Pseudomonas aeruginosa	1.00E+06 CFU/mL	None
Serratia marcescens	1.00E+06 CFU/mL	None
Staphylococcus aureus	1.00E+06 CFU/mL	None
Staphylococcus epidermidis	1.00E+06 CFU/mL	None
Streptococcus agalactiae	1.00E+06 CFU/mL	None
Streptococcus pneumoniae	1.00E+06 CFU/mL	None
Streptococcus pyogenes	1.00E+06 CFU/mL	None
Streptococcus salivarius	1.00E+06 CFU/mL	None
SARS-coronavirus	3.01E+051 Copies/mL	None
Varicella Zoster Virus	1.00E+05 TCID50/mL	None
N/A (Pooled Nasal Wash)	N/A N/A	None

¹Highest concentration based on the available stock.

				Table 7: NxTAG® RPP v2 Analytical Specificity (On-Panel Organisms)
--	--	--	--	--------------------------------------------------------------------

On-Panel Organisms (strain/subtype)	Concentration Tested	Cross-ReactivityDetected
Influenza A H1N1 (Brisbane/59/07)	1.00E+05 TCID50/mL	None
Influenza A 2009 H1N1 (A/NY/02/09)	1.00E+05 TCID50/mL	None
Influenza A H3N2 (Wisconsin/67/05)	1.00E+05 TCID50/mL	None
Influenza B (Florida/02/06) (LN: 325286)	1.00E+05 TCID50/mL	None
Influenza B (Florida/02/06) (LN: 325345)	1.00E+05 TCID50/mL	None
Influenza B (Florida/02/06) (LN: 307551)	1.00E+05 TCID50/mL	None
Influenza B (B/Brisbane/33/2008)	1.00E+05 TCID50/mL	None
Influenza B (B/Massachusetts/2/12)	1.00E+05 TCID50/mL	None
Influenza B (B/Wisconsin/1/2010)	1.00E+05 TCID50/mL	None
On-Panel Organisms (strain/subtype)	Concentration Tested	Cross-ReactivityDetected
Influenza B (B/Brigit (B/Russia/69))	1.00E+05	CEID50/mL	None
Influenza B (B/Hong Kong/5/72)	1.00E+05	CEID50/mL	None
Influenza B (B/Russia/69)	1.00E+05	CEID50/mL	None
Influenza B (B/GreLakes/1739/1954)	1.00E+05	CEID50/mL	None
Influenza B (B/Bangladesh/5972/2007)	1.00E+05	TCID50/mL	None
Influenza B (B/Hong Kong/259/2010)	1.00E+05	CEID50/mL	None
Influenza B (B/Texas/02/2013)	1.00E+05	TCID50/mL	None
Influenza B (B/Hubei-Wujiagang/158/2009)	1.00E+05	CEID50/mL	None
Influenza B (B/New Jersey/1/2012)	1.00E+05	TCID50/mL	None
Influenza B (B/Brisbane/3/2007)	1.00E+05	CEID50/mL	None
Human Respiratory Syncytial Virus A (A2)	1.00E+05	PFU/mL	None
Human Respiratory Syncytial Virus B (18537)	7.00E+04a	PFU/mL	None
SARS-CoV-2 (USA-WA-1/2020)	1.00E+07	Copies/mL	None
Coronavirus 229E	1.00E+05	TCID50/mL	None
Coronavirus OC43	1.00E+05	TCID50/mL	None
Coronavirus NL63	1.00E+05	TCID50/mL	None
Coronavirus HKU1	1.00E+06a	Copies/mL	None
Human Metapneumovirus (hMPV-16, Type A1,IA10-2003)	1.00E+05	TCID50/mL	None
Human Metapneumovirus (hMPV-2, Type B1,Peru2-2002)	3.89E+04a	TCID50/mL	None
Rhinovirus (Type 85, 50-525-CV54 [V-192-001-021])	1.00E+05	TCID50/mL	None
Enterovirus (Species D, Type 68, 2007 Isolate)	1.00E+05	TCID50/mL	None
Enterovirus (Species D, Type 68, US/MO/14-18947)	1.00E+05	TCID50/mL	None
Enterovirus (Species D, Type 68, US/IL/14-18952)	1.00E+03	TCID50/mL	Influenza AH3
Enterovirus (Species D, Type 68, US/KY/14-18953)	1.00E+05	TCID50/mL	None
Enterovirus (Species D, Type 68, Fermon)	1.00E+05	TCID50/mL	None
Enterovirus (68, 2014 Isolate 1)	1.00E+05	TCID50/mL	None
Adenovirus (Species B; Type 14, 2006 isolate)	1.00E+05	TCID50/mL	None
Parainfluenza virus 1 (Type 1)	1.00E+05	TCID50/mL	None
Parainfluenza virus 2 (Greer)	1.00E+05	TCID50/mL	None
Parainfluenza virus 3 (C243)	1.00E+05	TCID50/mL	None
Parainfluenza virus 4 (Subtype A)	1.00E+05	TCID50/mL	None
Parainfluenza virus 4 (Subtype B; CH 19503)	1.00E+05	TCID50/mL	None
On-Panel Organisms (strain/subtype)	Concentration Tested	Cross-ReactivityDetected
Chlamydia pneumoniae (TW-183)	1.00E+06 IFU/mL	None
Mycoplasma pneumoniae (M129)	1.00E+06 CCU/mL	None

510(k) Summary

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Confidential & Restricted

510(k) Summary

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ªHighest concentration possible based on the available stock.

Analytical Cross-Reactivity In Silico Analysis

For in silico exclusivity assessment of the assay oligos against on-panel and off-panel organisms listed in Table 8 below, based on analysis of sequences available in the GenBank Nucleotide (nt) database as of April 9, 2023, the following potential cross-reactivity is predicted:

. SARS-CoV-2 oligos are likely to detect some SARS-related coronavirus strains, as well as some bat coronavirus and bat SARS-like coronavirus strains.
. One bat 229E-like coronavirus sequence (KT253270) is likely to be detected by the Coronavirus 229E oligos at high viral titer.

On-Panel Organisms	Off-Panel Organisms	Off-Panel Organisms
Adenovirus	Bat SARS-like Coronavirus	Chlamydia psittaci
Enterovirus	Bat SARS-like CoronavirusHKU5	Coxiella burnetii
Human coronavirus 229E	Herpes Simplex Virus 2 (HSV2)	Cryptococcus neoformans
Human coronavirus HKU1	Human Bocavirus	Fusobacterium necrophorum
Human coronavirus NL63	Human Herpes Virus 6 (HHV6)	Haemophilus influenza
Human coronavirus OC43	Human Parechovirus (HPeV)	Histoplasma capsulatum
Human metapneumovirus (hMPV)	Influenza C	Klebsiella (Enterobacter) aerogenes
Influenza A	MERS-coronavirus	Klebsiella oxytoca
Influenza B	SARS-coronavirus	Legionella pneumophila
Parainfluenza virus 1	Acinetobacter calcoaceticus	Leptospira interrogans
Parainfluenza virus 2	Arcanobacterium haemolyticum	Mycobacterium tuberculosis
Parainfluenza virus 3	Aspergillus fumigatus	Mycoplasma orale
Parainfluenza virus 4	Aspergillus flavus	Pneumocystis jirovecii (PJP)
Respiratory syncytial virus A	Bacillus anthracis	Proteus mirabilis
Respiratory syncytial virus B	Blastomyces dennatitidis	Pseudomonas aeruginosa
Rhinovirus	Bordetella avium	Staphylococcus epidermidis
SARS-CoV-2	Bordetella bronchiseptica	Stenotrophomonas maltophilia
Chlamydia pneumoniae	Bordetella hinzii	Streptococcus dysgalactiae
Mycoplasma pneumoniae	Bordetella holmesii	Streptococcus pneumonia
	Bordetella pertussis	Streptococcus pyogenes
	Burkholderia cepacia	Streptococcus salivarius

Table 8. Potential Cross-Reactive Organisms assessed in the In Silico Exclusivity Analysis

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On-Panel Organisms	Off-Panel Organisms
	Candida albicansUreaplasma urealyticum

ii) Microbial Interference

The performance of NxTAG® RPP v2 in the presence of potentially interfering offpanel organisms (i.e., pathogens that are not detected by NxTAG® RPP v2) was evaluated by testing 11 off-panel organisms against 22 on-panel organisms detected by the assay. The samples were prepared in negative simulated matrix (NSM), with on-panel targets in multi-analyte samples at Low-Moderate Positive concentration (3x-6x LoD) and pools of off-panel organisms at the high positive concentration. Each combination was tested in triplicate on the NxTAG® RPP v2 assay. No off-panel organism present at high positive concentration interfered with the detection of any on-panel organism present at low positive concentration. The summary of results is shown in Table 9.

Off-Panel Organisms	Strain	Concentration Tested	MicrobialInterferenceDetected
Bordetella pertussis	A639	1.00E+06 CFU/mL	None
Cytomegalovirus	Merlin	1.00E+05 TCID50/mL	None
Corynebacterium diphtheriae	Z116	1.00E+06 CFU/mL	None
Haemophilus influenzae	Type b; MinnA	1.00E+06 CFU/mL	None
Measles Virus	N/A	1.00E+05 TCID50/mL	None
Moraxella catarrhalis	Strain NE 11	1.00E+06 CFU/mL	None
Mumps Virus	N/A	1.00E+05 TCID50/mL	None
Neisseria meningitides	Serotype A	1.00E+06 CFU/mL	None
Pseudomonas aeruginosa	Clinical isolate	1.00E+06 CFU/mL	None
Staphylococcus aureus	102-04	1.00E+06 CFU/mL	None
Streptococcus pneumoniae	Z022	1.00E+06 CFU/mL	None

Table 9: NxTAG® RPP v2 Microbial Interference

iii) Competitive Interference (Co-infection)

The performance of NxTAG® RPP v2 assay in the presence of potentially interfering on-panel organisms (i.e., pathogens that are detected by NxTAG® RPP v2) was

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

evaluated by testing samples that contain multiple on-panel targets, which included clinically relevant co-infections that occur in respiratory samples. The samples were prepared in negative simulated matrix (NSM), with on-panel organisms (in either single-analyte or multi-analyte samples) at Low-Moderate Positive concentration (3x-6x LoD), and the potentially interfering on-panel organisms at high positive concentration. Each combination was tested in triplicate on the NxTAG® RPP v2 assay. No on-panel organism present at high positive concentration interfered with the detection of any other on-panel organism present at low-moderate positive concentration. The summary of results is shown in Table 10.

High Positive		Low-Moderate Positive	CompetitiveInterferenceDetected
Target	Concentration	Target
Influenza A H1pdm09(strain: A/NY/02/09)	1.00E+05TCID50/mL	Respiratory Syncytial Virus A	None
		Rhinovirus	None
		Respiratory Syncytial Virus B	None
		Influenza A H3	None
		Influenza B	None
		Parainfluenza virus 3	None
		Mycoplasma pneumoniae	None
		SARS-CoV-2	None
		Human Metapneumovirus	None
		Adenovirus	None
Influenza A H3(Strain:Wisconsin/67/05)	1.00E+05TCID50/mL	Influenza A H1pdm09	None
		Respiratory Syncytial Virus A	None
		Rhinovirus	None
		Respiratory Syncytial Virus B	None
		Influenza B	None
		Parainfluenza virus 3	None
		Mycoplasma pneumoniae	None
		SARS-CoV-2	None
		Human Metapneumovirus	None
		Adenovirus	None
Influenza B	1.00E+05TCID50/mL	Influenza A H1pdm09	None
High Positive		Low-Moderate Positive	CompetitiveInterferenceDetected
Target	Concentration	Target
(Strain: Florida/02/06)	TCID50/mL	Respiratory Syncytial Virus A	None
		Rhinovirus	None
		Respiratory Syncytial Virus B	None
		Influenza A H3	None
		Parainfluenza virus 3	None
		Mycoplasma pneumoniae	None
		SARS-CoV-2	None
		Human Metapneumovirus	None
		Adenovirus	None
		Influenza A H1pdm09	None
		Respiratory Syncytial Virus A	None
Respiratory SyncytialVirus B(Strain: B/18537)		Rhinovirus	None
		Influenza A H3	None
		Influenza B	None
		Parainfluenza virus 3	None
		Mycoplasma pneumoniae	None
		SARS-CoV-2	None
		Human Metapneumovirus	None
		Adenovirus	None
		Influenza A H1pdm09	None
	$6.30E+04^a$PFU/mL	Respiratory Syncytial Virus A	None
Respiratory SyncytialVirus B(Strain:B/WV/14617/85)		Rhinovirus	None
		Influenza A H3	None
		Influenza B	None
		Parainfluenza virus 3	None
		Mycoplasma pneumoniae	None
		SARS-CoV-2	None
		Human Metapneumovirus	None
		Adenovirus	None
SARS-CoV-2(Strain: USA-WA-	1.00E+07Copies/mL	Influenza A H1pdm09	None
		Respiratory Syncytial Virus A	None
High Positive		Low-Moderate Positive	CompetitiveInterferenceDetected
Target	Concentration	Target
1/2020)		Rhinovirus	None
		Respiratory Syncytial Virus B	None
		Influenza A H3	None
		Influenza B	None
		Parainfluenza virus 3	None
		Mycoplasma pneumoniae	None
		Human Metapneumovirus	None
		Adenovirus	None
		Influenza A H1pdm09	None
		Respiratory Syncytial Virus A	None
		Rhinovirus	None
		Respiratory Syncytial Virus B	None
Coronavirus NL63(Strain: NL63)	1.00E+05TCID50/mL	Influenza A H3	None
		Influenza B	None
		Parainfluenza virus 3	None
		Mycoplasma pneumoniae	None
		SARS-CoV-2	None
		Human Metapneumovirus	None
		Adenovirus	None
		Influenza A H1pdm09	None
HumanMetapneumovirus(Strain: hMPV-16, TypeA1, IA10-2003)	1.00E+05TCID50/mL	Respiratory Syncytial Virus A	None
		Rhinovirus	None
		Respiratory Syncytial Virus B	None
		Influenza A H3	None
		Influenza B	None
		Parainfluenza virus 3	None
		Mycoplasma pneumoniae	None
		SARS-CoV-2	None
		Human Metapneumovirus	None
		Adenovirus	None
Human	3.50E+04a	Influenza A H1pdm09	None
High Positive		Low-Moderate Positive	CompetitiveInterferenceDetected
Target	Concentration	Target
Metapneumovirus(Strain: hMPV-3, TypeB1, Peru2-2002)	TCID50/mL	Respiratory Syncytial Virus A	None
		Rhinovirus	None
		Respiratory Syncytial Virus B	None
		Influenza A H3	None
		Influenza B	None
		Parainfluenza virus 3	None
		Mycoplasma pneumoniae	None
		SARS-CoV-2	None
		Adenovirus	None
		Influenza A H1pdm09	None
HumanMetapneumovirus(Strain: hMPV-5, TypeB1, Peru3-2003 G gene)	1.00E+05TCID50/mL	Respiratory Syncytial Virus A	None
		Rhinovirus	None
		Respiratory Syncytial Virus B	None
		Influenza A H3	None
		Influenza B	None
		Parainfluenza virus 3	None
		Mycoplasma pneumoniae	None
		SARS-CoV-2	None
		Adenovirus	None
		Influenza A H1pdm09	None
Rhinovirus(Strain: Type 85, 50-525-CV54 [V-192-001-021])	1.00E+05TCID50/mL	Respiratory Syncytial Virus A	None
		Respiratory Syncytial Virus B	None
		Influenza A H3	None
		Influenza B	None
		Parainfluenza virus 3	None
		Mycoplasma pneumoniae	None
		SARS-CoV-2	None
		Human Metapneumovirus	None
		Adenovirus	None
Adenovirus B		Influenza A H1pdm09	None
High Positive	Low-Moderate Positive	CompetitiveInterferenceDetected
Target	Concentration	Target
(Strain: Type 14, 2006isolate)	$1.00E+05$TCID50/mL	Respiratory Syncytial Virus A	None
		Rhinovirus	None
		Respiratory Syncytial Virus B	None
		Influenza A H3	None
		Influenza B	None
		Parainfluenza virus 3	None
		Mycoplasma pneumoniae	None
		SARS-CoV-2	None
		Human Metapneumovirus	None

Table 10: NxTAG® RPP v2 Competitive Interference

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510(k) Summary

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ª The highest possible concentration was tested.

Additional competitive interference was evaluated for SARS-CoV-2. Low Positive SARS-CoV-2 (3x LoD) was tested against the remaining 13 NxTAG® RPP v2 assay targets that were not tested with the SARS-CoV-2 containing multi-analyte combinations. High Positive targets were tested at 1.00E+06 CCU/mL or IFU/mL for bacteria; 1.00E+05 TCIDso/mL, PFU/mL, or 1.00E+07 Copies/mL, or highest possible concentration for virus. Samples were prepared in negative simulated matrix and each combination was tested in triplicate with the assay. No interference of SARS-CoV-2 low positive detection was observed (Table 11).

Table 11: NxTAG® RPP v2 Competitive Interference Study – Additional Tests Performed with SARS-CoV-2

High Positive		Low Positive	CompetitiveInterferenceDetected
Target	Concentration	Target
Influenza A H1	1.00E+05 TCID50/mL	SARS-CoV-2	None
Respiratory Syncytial Virus A	1.00E+05 PFU/mL		None
Parainfluenza virus 1	1.00E+05 TCID50/mL		None
Parainfluenza virus 2	1.00E+05 TCID50/mL		None
Parainfluenza virus 3	1.00E+05 TCID50/mL		None

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Parainfluenza virus 4Aa	1.00E+05 TCID50/mL
Parainfluenza virus 4Ba	1.00E+05 TCID50/mL
Coronavirus 229E	1.00E+05 TCID50/mL
Coronavirus OC43	1.00E+05 TCID50/mL
Coronavirus HKU1	1.00E+06b Copies/mL
Enterovirusc	1.00E+05 TCID50/mL
Chlamydia pneumoniae	1.00E+06 IFU/mL
Mycoplasma pneumoniae	1.00E+06 CCU/mL

ª Reported by NxTAG RPP as Parainfluenza virus 4

b The highest possible concentration was tested

¢ Reported by NxTAG RPP v2 as Rhinovirus/Enterovirus

iv) Interfering Substances:

The performance of NxTAG® RPP v2 in the presence of potentially interfering substances was assessed. Twenty non-microbial substances commonly found in respiratory specimens were tested on the assay alone or in the presence of pathogens detected by the assay in multi-analyte samples. The samples were prepared in negative simulated matrix (NSM), with the on-panel organisms at Low-Moderate Positive concentration (3x-6x LoD) and the potentially interfering substance at the concentration listed in Table 14. All samples were tested in triplicate on the NxTAG® RPP v2 assay. None of the substances tested interfered with the detection of on-panel organisms present in the sample, with the exception of menthol and FluMist®. Menthol interfered with the detection of Coronavirus OC43 at 1% w/v; no interference was observed when menthol was tested at 0.5% w/v. FluMist® generated positive calls for Influenza A (matrix), Influenza A H1pdm09, Influenza A H3, and Influenza B for all replicates it was present in. These positive calls are expected as FluMist® contains attenuated Influenza A H1N1, Influenza A H3N2, and Influenza B strains. Positive influenza results obtained in a patient who received FluMist® prior to sample collection may be due to detection of vaccine virus and may mask a true positive result due to infection by one or more of these analytes. The list of potentially interfering substances and the concentrations tested are shown in Table 12.

Potential Interferent	1991 - 1971 - 111 1 - 11 1 Vá 1116 - 111 1 Vá 1116 - 111 - 111 - Vá 1116 - 111 - 11 - 111 - 111 - 111 - 111 - 111 - 111 - 111 - 111 - 111 - 111 - 111 - 111 - 111 - 111 -Active Ingredient	Concentration Tested
Human Whole Blood	N/A	5% (v/v)
Potential Interferent	Active Ingredient	Concentration Tested
Human Genomic DNA	N/A	20 ng/µL
Mucin	Mucin	100 µg/mL
Phenylephrine	Phenylephrine	0.03 µg/mL
Beclomethasonedipropionate	Beclomethasonedipropionate	25 µg/mL
Dexamethasone	Dexamethasone	12 µg/mL
Flunisolide	Flunisolide	5 µg/mL
Triamcinolone acetonide	Triamcinolone acetonide	22 µg/mL
Budesonide	Budesonide	$6.30E-03$ µg/mL
Mometasone furoate	Mometasone furoate	$4.50E-04$ µg/mL
Fluticasone proprionate	Fluticasone	$1.26E-03$ µg/mL
Drixoral®	Oxymetaxoline	10% (v/v)
		15% (v/v)
ZICAM® Allergy Relief	Galphimia GlaucaHistaminumHydrochloricumLuffa operculataSulfur	1% (v/v)
		5% (v/v)
Salinex®	Sodium Chloride	1% (v/v)
		15% (v/v)
Mupirocin	Mupirocin	1.5 µg/mL
Tobramycin	Tobramycin	33 µg/mL
		600 µg/mL
Zanamivir	Zanamivir	100 µg/mL
FluMist®	Influenza A H1N1,Influenza A H3N2,Influenza B Yamagatalineage, Influenza B VictoriaLineage	0.5% (v/v) a
Benzocaine	Benzocaine	10% (w/v)
		1% (w/v) b
Menthol	Menthol	0.5% (w/v)
Leukocyte	Leukocyte	$1.00E+03$ cells/µL
Early Defence Nasal Spray®	Zinc	5% (v/v)

Table 12: NxTAG® RPP v2 Interfering Substances

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ª FluMist® demonstrated interference with the detection of Influenza A (matrix), Influenza A H1pdm09, Influenza A H3, and Influenza B at 0.5% v/v.

₪ Menthol demonstrated interference with the detection of Coronavirus OC43 at 1% w/v; no interference was observed at 0.5% w/v.

ν) Carry-Over/Cross-Contamination:

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The risk of carry-over and cross-contamination events for the NxTAG® RPP v2 assay was assessed by testing samples at high titer in alternating pattern with negative samples. Two representative targets, SARS-CoV-2 (viral) and Mycoplasma pneumoniae (bacterial), were each extracted in an alternating arrangement with the negative sample across two easyMAG instruments. The extracts were then tested in an alternating arrangement with the NxTAG® RPP v2 assay. Two false positives were observed for the SARS-CoV-2 target; one after a negative sample and the other after a high titer samples were re-run to determine whether the extracts were contaminated, and the re-run results confirmed the contamination of the Following this, the samples were re-prepared and re-tested, generating extracts. the expected results.

h. Assay cut-off

Thresholds for the NxTAG® RPP v2 assay are provided in Table 13.

Analyte	MFI Threshold	MDD Threshold
Influenza A	45	35
Influenza A H1 (H1-A)	90	75
Influenza A H1pdm09 (H1-B)	55	45
Influenza A H3	80	50
Influenza B	60	40
RSV A	50	45
RSV B	45	35
Parainfluenza 1	65	50
Parainfluenza 2	70	55
Parainfluenza 3	60	50
Parainfluenza 4A a	70	55
Parainfluenza 4B a	60	45
SARS-CoV-2 (ORF1ab) b	35	30
SARS-CoV-2 (M) b	35	30
Coronavirus 229E	60	50
Coronavirus NL63	75	60
Coronavirus OC43	70	55
Coronavirus HKU1	60	40
Metapneumovirus	55	40
Rhinovirus/Enterovirus	50	40
Adenovirus	75	65
Chlamydia pneumoniae	45	40

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Analyte	MFI Threshold	MDD Threshold
Mycoplasma pneumoniae	40	30
Internal Control	120	100

ª NxTAG RPP v2 does not differentiate Parainfluenza virus 4A and 4B, both of which are reported as Parainfluenza virus 4

₪ NxTAG RPP v2 reports a single combined result for the SARS-CoV-2 ORF1ab and M gene targets

2. Comparison Studies:

Method comparison with predicate device: a.
Not applicable.
b. Matrix and Multi-Analyte Sample Comparison:
Equivalency between multi-analyte (MA) and single target samples, and NCM and negative simulated matrix (NSM) were assessed to demonstrate the validity of using MA samples and/or NSM in applicable analytical studies. Eight (8) MA samples, covering all targets probed by NxTAG® RPP v2, were evaluated. Each MA sample consisted of 2-3 targets and was prepared in both NCM and NSM at or near the LoD concentration (1x – 2x LoD) confirmed with single target samples in NCM. Twenty (20) replicates of each MA were tested, and all targets in MA samples generated ≥ 95% positivity. The results demonstrate equivalency between single-analyte and multi-analyte samples as well as between NCM and NSM.

The multi-analyte sample composition and the equivalency test results are shown in Table 14.

Multi-analyteSample	Organism	Confirmed LoD(Copies/mL)	Positivity (%)
			MA Sample inNCM	MA Sample inNSM
MA-1	Influenza A 2009 H1N1 (subtype)	9.84E+02	20/20 (100%)	20/20 (100%)
	Respiratory Syncytial Virus A	4.97E+03	19/20 (95%)	20/20 (100%)
	Rhinovirus	1.54E+03	20/20 (100%)	20/20 (100%)
MA-2	Influenza A H3 (subtype)	5.60E+01	20/20 (100%)	20/20 (100%)
	Respiratory Syncytial Virus B	7.21E+03	20/20 (100%)	20/20 (100%)

Table 14: NxTAG® RPP v2 Multi-Analyte Vs. Single Analyte, and Negative Clinical Matrix Vs. Negative Simulated Matrix Equivalency

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Multi-analyteSample	Organism	Confirmed LoD(Copies/mL)	Positivity (%)
			MA Sample inNCM	MA Sample inNSM
MA-3	Influenza B	6.33E+01	20/20 (100%)	19/20 (95%)
MA-3	Parainfluenza virus 3	1.01E+03	20/20 (100%)	20/20 (100%)
MA-3	Mycoplasma pneumoniae	3.23E+03	20/20 (100%)	20/20 (100%)
MA-4	SARS-CoV-2	5.00E+02	20/20 (100%)	20/20 (100%)
MA-4	Human Metapneumovirus	2.62E+02	20/20 (100%)	19/20 (95%)
MA-4	Adenovirus	1.42E+03	20/20 (100%)	20/20 (100%)
MA-5	Influenza A H3 (Matrix)	1.68E+02	20/20 (100%)	20/20 (100%)
MA-5	Coronavirus NL63	1.00E+02	20/20 (100%)	20/20 (100%)
MA-5	Coronavirus HKU1	4.18E+03	20/20 (100%)	19/20 (95%)
MA-6	Influenza H1 (subtype)	1.60E+03	20/20 (100%)	19/20 (95%)
	Parainfluenza virus 1	6.92E+02	20/20 (100%)	20/20 (100%)
	Chlamydia pneumoniae	2.38E+02	20/20 (100%)	20/20 (100%)
MA-7	Parainfluenza virus 2	3.45E+02	19/20 (95%)	19/20 (95%)
	Parainfluenza virus 4B	7.15E+03	20/20 (100%)	20/20 (100%)
	Coronavirus 229E	3.81E+02	20/20 (100%)	19/20 (95%)
MA-8	Parainfluenza virus 4A	1.69E+04	20/20 (100%)	20/20 (100%)
	Coronavirus OC43	4.55E+03	20/20 (100%)	20/20 (100%)

Specimen Collection Device Comparison: C.

The equivalency between three swab types (swabs with a nylon flocked tip, polyester tip, and rayon tip) for the collection of samples for testing on the NxTAG® RPP v2 was evaluated. The study assessed the transfer of samples from the swab to the collection media. Four multi-analyte (MA) samples consisting of targets detected by the NxTAG® RPP v2 assay were prepared in negative simulated matrix (NSM) and tested at a final concentration of 3x-6x LoD, along with a negative sample.

Samples prepared using swabs with a nylon flocked tip and a polyester tip generated 100% positivity for all targets present in the respective multi-analyte samples and 0% positivity for all targets for the negative sample.

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Samples prepared using swabs with a rayon tip only generated 100% positivity for all targets present in the MA1 and MA2 samples, and 0% positivity for all targets for the negative sample. For the MA3 sample, two of three replicates generated negative results for all targets (Influenza B, Parainfluenza virus 3, and M. pneumoniae) and for the MA4 sample, one of three replicates generated a negative result for Adenovirus.

The results demonstrate equivalency between swabs with nylon flocked tip and polyester tip (Table 15). Based on the results of this study, rayon-tipped swabs should not be used to collect specimens for use with the NxTAG RPP v2 assay.

Sample	Organism	Target Positivity
		Nylon Flocked	Polyester	Rayon
MA1	Influenza A 2009 H1N1 (subtype)	100% (3/3)	100% (3/3)	100% (3/3)
MA1	Respiratory Syncytial Virus A	100% (3/3)	100% (3/3)	100% (3/3)
MA1	Rhinovirus	100% (3/3)	100% (3/3)	100% (3/3)
MA2	Influenza A H3 (subtype)	100% (3/3)	100% (3/3)	100% (3/3)
MA2	Respiratory Syncytial Virus B	100% (3/3)	100% (3/3)	100% (3/3)
MA3	Influenza B	100% (3/3)	100% (3/3)	33% (1/3)
MA3	Parainfluenza virus 3	100% (3/3)	100% (3/3)	33% (1/3)
MA3	Mycoplasma pneumoniae	100% (3/3)	100% (3/3)	33% (1/3)
MA4	SARS-CoV-2	100% (3/3)	100% (3/3)	100% (3/3)
MA4	Human Metapneumovirus	100% (3/3)	100% (3/3)	100% (3/3)
MA4	Adenovirus	100% (3/3)	100% (3/3)	67% (2/3)
NEG	N/A	0% (0/3)	0% (0/3)	0% (0/3)

Table 15: NxTAG® RPP v2 Assay Swab Equivalency

d. Specimen Collection Media and Extraction Comparison:

Equivalency between collection media; Universal Transport Media (UTM), and Remel MicroTest™ M4RT (M4RT), and between two extraction systems (bioMérieux's NucliSENS® easyMAG® and EMAG®) was assessed to verify their compatibility with the NxTAG® RPP v2 assay.

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To assess collection media equivalency, four multi-analyte samples consisting of representative targets were contrived in pooled negative nasopharyngeal swabs (NPS) collected in UTM (NCM), and in pooled negative NPS collected in M4RT (NCM-M4RT). The samples, prepared at 3 dilution levels: Above LoD (5x -9x LoD), At/Near LoD (1x-2x LoD), and Below LoD (1/3x-2/3x LoD), along with a negative sample (negative clinical matrix alone), were extracted using the EMAG system and tested on the NxTAG® RPP v2 assay.

All samples prepared in NCM and extracted using easyMAG generated ≥ 95% positivity for all targets at concentrations that were ≤ 2x LoD concentration confirmed in using the EMAG in the LoD study. The results demonstrate equivalency between extractions by EMAG and easyMAG for use with NxTAG® RPP v2. Results are summarized in Table 16.

Organism	Strain information	Concentration(Copies/mL)	SampleType(LoD)	Target Positivity (%)
Influenza A 2009H1N1	A/NY/02/09	8.52E+03	Above	10/10(100%)	10/10 (100%)
		1.70E+03	At/Near	30/30 (100%)	30/30 (100%)
		5.68E+02	Below	10/10 (100%)	7/10 (70%)
RespiratorySyncytial Virus A	A2	2.48E+04	Above	10/10 (100%)	10/10 (100%)
		4.97E+03	At/Near	30/30 (100%)	30/30 (100%)
		1.66E+03	Below	6/10 (60%)	5/10 (50%)
Rhinovirus	50-525-CV54	7.68E+03	Above	10/10 (100%)	10/10 (100%)
		1.54E+03	At/Near	30/30 (100%)	30/30 (100%)
		5.12E+02	Below	10/10 (100%)	9/10 (90%)
Influenza A H3	A/Wisconsin/67/05	2.80E+02	Above	10/10 (100%)	10/10 (100%)
		5.60E+01	At/Near	30/30 (100%)	30/30 (100%)
		1.87E+01	Below	4/10 (40%)	4/10 (40%)
RespiratorySyncytial Virus B	18537	3.60E+04	Above	10/10 (100%)	10/10 (100%)
		7.21E+03	At/Near	30/30 (100%)	30/30 (100%)
		2.40E+03	Below	6/10 (60%)	3/10 (30%)
Influenza B	B/Florida/02/06	3.16E+02	Above	10/10 (100%)	10/10 (100%)
		6.33E+01	At/Near	29/30 (97%)	29/30 (97%)
		2.11E+01	Below	5/10 (50%)	4/10 (40%)
Parainfluenza virus	C 243	5.07E+03	Above	10/10 (100%)	10/10 (100%)

Table 16: NxTAG® RPP v2 Collection Media Equivalency

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Organism	Strain information	Concentration(Copies/mL)	SampleType(LoD)	Target Positivity (%)
				NCM (UTM)	NCM-M4RT
3		2.03E+03	At/Near	30/30 (100%)	29/30 (97%)
		1.01E+03	At/Near	26/30 (87%)	22/30 (73%)
		3.38E+02	Below	0/10 (0%)	2/10 (20%)
Mycoplasmapneumoniae	M129	2.15E+04	Above	10/10 (100%)	10/10 (100%)
		4.30E+03	At/Near	30/30 (100%)	29/30 (97%)
		1.43E+03	Below	3/10 (30%)	4/10 (40%)
SARS-CoV-2	USA-WA1/2020	2.50E+03	Above	10/10 (100%)	10/10 (100%)
		5.00E+02	At/Near	30/30 (100%)	30/30 (100%)
		1.67E+02	Below	10/10 (100%)	7/10 (70%)
HumanMetapneumovirus	hMPV-3, Type B1,Peru2-2002	1.31E+03	Above	10/10 (100%)	10/10 (100%)
		2.62E+02	At/Near	29/30 (97%)	30/30 (100%)
		8.73E+01	Below	5/10 (50%)	7/10 (70%)
Adenovirus	Type 14	1.23E+04	Above	10/10 (100%)	10/10 (100%)
		3.69E+03	At/Near	30/30 (100%)
		2.46E+03	At/Near	28/30 (93%)	30/30 (100%)
		8.20E+02	Below	4/10 (40%)	0/10 (0%)
N/A(Negative Sample)	N/A	N/A	N/A	0/10(0%)	0/10 (0%)

To demonstrate extractor equivalency, eight multi-analyte samples covering all targets probed by NxTAG® RPP v2 were tested. Multi-analyte samples were contrived in NCM, extracted on the easyMAG system, and tested on the NxTAG® RPP v2 assay.

All samples prepared in NCM and extracted using easyMAG generated ≥ 95% positivity for all targets at concentrations that were within 2x LoD of the confirmed concentration in multi-analyte samples prepared in NCM and extracted using the EMAG. The results demonstrate equivalency between extractions by EMAG and easyMAG for use with NxTAG® RPP v2. Results are summarized in Table 17.

Table 17: NxTAG® RPP v2 Extractor Equivalency

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Multi-analyteSample	Organism	Strain information	Concentration(Copies/mL)	Sample Type(LoD)	Positivity (%)
easyMAG (in NCM)
MA1	Influenza A 2009 H1N1	A/NY/02/09	1.70E+03	At/Near	20/20 (100%)
MA1	Respiratory Syncytial Virus A	A2	4.97E+03	At/Near	20/20 (100%)
MA1	Rhinovirus	50-525-CV54	5.12E+02	Below	20/20 (100%)
MA2	Influenza A H3	A/Wisconsin/67/05	5.60E+01	At/Near	20/20 (100%)
MA2	Respiratory Syncytial Virus B	18537	7.21E+03	At/Near	20/20 (100%)
MA3	Influenza B	B/Florida/02/06	6.33E+01	At/Near	20/20 (100%)
MA3	Parainfluenza virus 3	C 243	1.01E+03	At/Near	20/20 (100%)
MA3	Mycoplasma pneumoniae	M129	4.30E+03	At/Near	20/20 (100%)
MA4	SARS-CoV-2	USA-WA1/2020	5.00E+02	At/Near	20/20 (100%)
MA4	Human Metapneumovirus	hMPV-3, Type B1,Peru2-2002	2.62E+02	At/Near	20/20 (100%)
MA4	Adenovirus	Type 14	2.46E+03	At/Near	20/20 (100%)
MA5	Influenza A Matrix	A/Wisconsin/67/05	1.68E+02	At/Near	20/20 (100%)
MA5	Coronavirus NL63	N/A	1.00E+02	At/Near	20/20 (100%)
MA5	Coronavirus HKU1	Type B	4.18E+03	At/Near	20/20 (100%)
MA6	Influenza H1	A/Brisbane/59/07	5.35E+02	Below	19/20 (95%)
MA6	Parainfluenza virus 1	N/A	6.92E+02	Below	20/20 (100%)
MA6	Chlamydia pneumoniae	TW-183	7.93E+01	Below	20/20 (100%)
MA7	Parainfluenza virus 2	Greer	3.45E+02	At/Near	19/20 (95%)
MA7	Parainfluenza virus 4B	CH 19503	7.15E+03	At/Near	20/20 (100%)
MA7	Coronavirus 229E	N/A	3.81E+02	At/Near	20/20 (100%)
MA8	Parainfluenza virus 4A	N/A	5.65E+03	Below	19/20 (95%)
MA8	Coronavirus OC43	Betacoronavirus 1	4.55E+03	At/Near	20/20 (100%)

3. Clinical Performance:

A multi-site clinical study established the clinical performance of the NxTAG® RPP v2 assay for the detection and identification of nucleic acids from multiple respiratory viruses and bacteria extracted from upper respiratory tract specimens collected from individuals with clinical signs and symptoms of a respiratory tract infection. The clinical performance of the NxTAG® RPP v2 assay was evaluated using clinical specimens prospectively collected between October 2022 and April 2023 from five geographically diverse clinical sites within the United States. The clinical study utilized leftover, de-

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identified specimens collected from pediatric and adult patients exhibiting clinical signs and symptoms of a respiratory tract infection.

The NxTAG® RPP v2 results were compared to those obtained with an FDA-cleared molecular assay and PCR/bi-directional sequencing for influenza A subtyping. The twostep PCR analysis followed by BDS assays employed two independent sets of validated PCR assays. The primers used for PCR analysis and sequencing assays, where possible, were designed to amplify distinct regions from the investigational device. PCR analysis composite positive specimens were confirmed by BDS assays. See Table 18 for comparator method testing by target.

NxTAG® RPP v2 Target	Comparator Method
Adenovirus
Enterovirus/Rhinovirus
Influenza A
Influenza A H3
Influenza B
Respiratory Syncytial Virus A
Respiratory Syncytial Virus B
Parainfluenza 1
Parainfluenza 2
Parainfluenza 3	FDA cleared molecular assay
Parainfluenza 4
Coronavirus 229E
Coronavirus NL63
Coronavirus OC43
Coronavirus HKU1
Human Metapneumovirus
Chlamydia pneumoniae
Mycoplasma pneumoniae
Influenza A H1	FDA cleared molecular assay followed by
Influenza A H1pdm09	composite of PCR followed by BDS NAATs*
SARS-CoV-2	FDA cleared molecular assay

Table 18: Prospective Comparator Method Algorithm

NAAT – Nucleic Acid Amplification Test

A total of 1844 prospective specimens, collected from five geographically diverse US sites were initially enrolled in the study, of which 19 were excluded from the analysis of performance (duplicate specimen, not from a unique subject (11), absence of signs and symptoms (5), improper labeling (2), operator error (1)). Nineteen of the remaining 1825 specimens initially produced invalid NxTAG RPP v2 results, of which 14 resolved upon repeat testing. Therefore, a total of 1820 prospectively collected specimens

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generated valid NxTAG RPP v2 results after allowing for a single retest. Certain additional specimens were excluded from performance calculations for specific analytes based on the availability of valid results for the applicable comparator method. Clinical runs and re-runs using the NxTAG® RPP v2 assay were tested on the MAGPIX System by trained operators at three sites. Prospective specimen (Arm 1) testing occurred between March 2023 and April 2023.

For targets that exhibited low prevalence rates in the prospective study cohort, the prospective specimen set was supplemented with 320 pre-selected left-over, deidentified specimens (Arm 2) sourced from six sites in the United States. Pre-selected specimens were identified by Standard of Care (SoC) results and confirmed by BDS testing prior to enrollment in the study. Of the preselected specimens, 11 were excluded from the analysis of performance because confirmatory testing could not be completed. In addition, 3 specimens initially produced invalid NxTAG RPP v2 results, of which 2 resolved upon repeat testing. Therefore, a total of 308 preselected specimens were included in the analysis of performance. To minimize bias, pre-selected specimens were tested in a randomized, blinded manner with negative specimens at four sites. Pre-selected specimen (Arm 2) testing occurred between December 2021 and May 2023.

To supplement the number of clinical specimens positive for Chlamydia pneumoniae, RSV B, pre-2009 pandemic Influenza A H1N1, and Coronavirus 229E in the prospective and pre-selected arms of the study, additional testing was performed using contrived specimens. Contrived specimens were prepared by spiking representative strains into unique negative human nasopharyngeal specimens at 2x Limit of Detection (LoD), 10x LoD, and 100x LoD, for all strains except influenza A H1N1 which was prepared at 2x and 10x LoD. A total of 199 specimens were contrived and tested as part of Arm 3.

To minimize bias, contrived specimens were blinded, randomized, and tested along with Arm 2 positive and negative clinical specimens at two testing sites between December 2021 and January 2022. Results from contrived specimens were analyzed separately from the prospective and pre-selected data sets.

Out of the 199 specimens included in the contrived study analysis, 198 (99.50%) generated valid NxTAG® RPP v2 Assay results (i.e., Positive or Negative) on the first attempt. There was one specimen (0.50%) with an invalid result on the initial run. This specimen generated a valid result after a single retest for a final success rate of 100%.

The invalid rate for prospective, pre-selected, and contrived specimens combined was 0.98% (23/2344) after the initial run. Of the 23 specimens with initial invalid results, 17 (0.73%) specimens generated valid NxTAG® RPP v2 results after a single retest, three (0.13%) specimens remained invalid on repeat, and three (0.13%) specimens were not retested due to volume limitations.

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Lumi

For each target in the NxTAG® RPP v2 Assay, the performance (Positive Percent Agreement, Negative Percent Agreement, and 95% confidence interval) of the NxTAG® RPP v2 Assay as compared to the reference method are summarized in Tables 19 and 20 for prospective and pre-selected specimen analysis, respectively. The performance of the NxTAG® RPP v2 Assay for contrived specimens is presented separately in Table 21. For each of the contrived specimens (n = 199), negative results were obtained for all other analytes included on the panel that are not listed in Table 21.

	Pathogen Target		Positive Percent Agreement			Negative Percent Agreement
		TP / (TP+FN)	PPA (%)	95% CI	TN / (TN+FP)	NPA (%)	95% CI
	Viruses
	Fresh	39/39	100%	91%-100%	733/736	99.6%	99%-100%
Adenovirus	Frozen	55/59	93.2%	84%-97%	976/980	99.6%	99%-100%
	Overall	94/98a	95.9%	90%-98%	1709/1716b	99.6%	99%-100%
	Fresh	2/2	100%	34%-100%	773/773	100%	100%-100%
Coronavirus 229E	Frozen	5/5	100%	57%-100%	1033/1034	99.9%	99%-100%
	Overall	7/7	100%	65%-100%	1806/1807	99.9%	100%-100%
	Fresh	8/8	100%	68%-100%	767/767	100%	100%-100%
Coronavirus HKU1	Frozen	12/13	92.3%	67%-99%	1026/1026	100%	100%-100%
	Overall	20/21c	95.2%	77%-99%	1793/1793	100%	100%-100%
	Fresh	25/27	92.6%	77%-98%	748/748	100%	99%-100%
Coronavirus NL63	Frozen	23/25	92%	75%-98%	1014/1014	100%	100%-100%
	Overall	48/52	92.3%	82%-97%	1762/1762	100%	100%-100%
	Fresh	10/10	100%	72%-100%	765/765	100%	100%-100%
Coronavirus OC43	Frozen	29/29	100%	88%-100%	1010/1010	100%	100%-100%
	Overall	39/39	100%	91%-100%	1775/1775	100%	100%-100%
	Fresh	87/87	100%	96%-100%	680/688	98.8%	98%-99%
Human Metapneumovirus	Frozen	70/70	100%	95%-100%	963/969	99.4%	99%-100%
	Overall	157/157	100%	98%-100%	1643/1657d	99.2%	99%-99%
	Fresh	20/20	100%	84%-100%	753/755	99.7%	99%-100%
Influenza A	Frozen	54/54	100%	93%-100%	984/985	99.9%	99%-100%
	Overall	74/74	100%	95%-100%	1737/1740e	99.8%	99%-100%
	Fresh	9/9	100%	70%-100%	765/765	100%	100%-100%
Influenza A H1pdm09	Frozen	22/22	100%	85%-100%	1017/1017	100%	100%-100%
	Overall	31/31	100%	89%-100%	1782/1782	100%	100%-100%
	Fresh	0/0	N/A	N/A	774/774	100%	100%-100%
Influenza A H1	Frozen	0/0	N/A	N/A	1039/1039	100%	100%-100%
	Overall	0/0	N/A	N/A	1813/1813	100%	100%-100%
Influenza A H3	Fresh	11/11	100%	74%-100%	764/764	100%	99%-100%
	Frozen	34/36	94.4%	82%-98%	1002/1003	99.9%	99%-100%
Pathogen Target		Positive Percent Agreement			Negative Percent Agreement
		TP / (TP+FN)	PPA (%)	95% CI	TN /(TN+FP)	NPA (%)	95% CI
Influenza B	Overall	45/47f	95.7%	86%-99%	1766/1767	99.9%	100%-100%
	Fresh	5/5	100%	57%-100%	770/770	100%	100%-100%
	Frozen	6/6	100%	61%-100%	1033/1033	100%	100%-100%
Parainfluenza 1	Overall	11/11	100%	74%-100%	1803/1803	100%	100%-100%
	Fresh	7/7	100%	65%-100%	768/768	100%	100%-100%
	Frozen	11/11	100%	74%-100%	1028/1028	100%	100%-100%
Parainfluenza 2	Overall	18/18	100%	82%-100%	1796/1796	100%	100%-100%
	Fresh	4/5	80%	38%-96%	770/770	100%	100%-100%
	Frozen	5/5	100%	57%-100%	1034/1034	100%	100%-100%
Parainfluenza 3	Overall	9/10	90%	60%-98%	1804/1804	100%	100%-100%
	Fresh	19/19	100%	83%-100%	756/756	100%	99%-100%
	Frozen	23/23	100%	86%-100%	1015/1016	99.9%	99%-100%
Parainfluenza 4	Overall	42/42	100%	92%-100%	1771/1772	99.9%	100%-100%
	Fresh	2/3	66.7%	21%-94%	770/772	99.7%	99%-100%
	Frozen	11/12	91.7%	65%-99%	1026/1027	99.9%	99%-100%
RSV A	Overall	13/15g	86.7%	62%-96%	1796/1799h	99.8%	100%-100%
	Fresh	10/10	100%	72%-100%	764/765	99.9%	99%-100%
	Frozen	45/45	100%	92%-100%	992/994	99.8%	99%-100%
RSV B	Overall	55/55	100%	93%-100%	1756/1759i	99.8%	99%-100%
	Fresh	3/3	100%	44%-100%	772/772	100%	100%-100%
	Frozen	17/17	100%	82%-100%	1022/1022	100%	100%-100%
Rhinovirus /Enterovirus	Overall	20/20	100%	84%-100%	1794/1794	100%	100%-100%
	Fresh	123/132	93.2%	88%-96%	643/643	100%	99%-100%
	Frozen	228/237	96.2%	93%-98%	801/802	99.9%	99%-100%
SARS-CoV-2	Overall	351/369j	95.1%	92%-97%	1444/1445	99.9%	100%-100%
	Fresh	103/106	97.2%	92%-99%	656/660	99.4%	98%-100%
	Frozen	126/128	98.4%	94%-100%	902/909	99.2%	98%-100%
	Overall	229/234k	97.9%	95%-99%	1558/1569l	99.3%	99%-100%
Bacteria
Chlamydiapneumoniae	Fresh	0/0	N/A	N/A	775/775	100%	100%-100%
	Frozen	0/0	N/A	N/A	1039/1039	100%	100%-100%
	Overall	0/0	N/A	N/A	1814/1814	100%	100%-100%
Mycoplasmapneumoniae	Fresh	0/0	N/A	N/A	775/775	100%	100%-100%
	Frozen	0/0	N/A	N/A	1039/1039	100%	100%-100%
	Overall	0/0	N/A	N/A	1814/1814	100%	100%-100%

Table 19: NxTAG® RPP v2 Performance for the Prospective Data Set

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ªTwo of the four Adenovirus False Negative by BDS and two were not tested due to volume limitations.

bFour of the seven Adenovirus False Positives were positive by the molecular SoC assay.

'The one prospective Coronavirus HKU1 False Negative by the molecular SoC assay.

ণ Two of the fourteen Human Metapneumovirus False Positive by the molecular SoC assay. Seven samples could not be tested due to volume limitations.

€The Influenza A subtype was detected by the reference method for three Influenza A False Positives.

'One of the two Influenza A H3 False Negatives was negative by the molecular SoC assay.

80ne of the two Parainfluenza 4 False Negatives was negative by the molecular SoC assay.

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1Two of the three Parainfluenza 4 False Positive by the molecular SoC assay.

1One of the three RSV A False Positives was positive by the molecular SoC assay.

Eight of the eighteen Rhinovirus False Negatives were negative by BDS, and three False Negatives were negative by the

molecular SoC assay. Five samples could not be tested due to volume limitations.
*Two of the five SARS-CoV-2 False Negatives were negative by the molecular SoC assay.

'Six of the eleven SARS-CoV-2 False Positives were positive by the molecular SoC assay.

Positive Percent Agreement Negative Percent Agreement Pathogen Target TP / NPA TN / PPA (%) 95% Cl 95% Cl (TP+FN) (TN+FP) (%) Viruses Adenovirus Pre-Selected 1/1 100% 21%-100% 307/307 100% 99%-100% Coronavirus 229E Pre-Selected 11/11 100% 74%-100% 297/297 100% 99%-100% 30/32 80%-98% 276/276 Coronavirus HKU1 Pre-Selected 93.8% 100% 99%-100% 0/0 Coronavirus NL63 Pre-Selected N/A N/A 308/308 100% 99%-100% Coronavirus OC43 308/308 100% 99%-100% Pre-Selected 0/0 N/A N/A Human Pre-Selected 0/0 N/A N/A 308/308 100% 99%-100% Metapneumovirus Influenza A Pre-Selected 30/30 100% 89%-100% 277/278 99.6% 98%-100% Influenza A 29/30 96.7% 83%-99% 278/278 99%-100% Pre-Selected 100% H1pdm09 Influenza A H1 Pre-Selected 0/0 N/A N/A 277/278 99.6% 98%-100% Influenza A H3 Pre-Selected 0/0 N/A N/A 278/278 100% 99%-100% Influenza B 30/30 89%-100% 278/278 100% 99%-100% Pre-Selected 100% 29/29 Parainfluenza 1 Pre-Selected 100% 88%-100% 279/279 100% 99%-100% Parainfluenza 2 Pre-Selected 30/30 100% 89%-100% 278/278 100% 99%-100% Parainfluenza 3 Pre-Selected 0/0 N/A N/A 307/307 100% 99%-100% Parainfluenza 4 Pre-Selected 15/16 93.8% 72%-99% 292/292 100% 99%-100% RSV A Pre-Selected 0/0 N/A N/A 305/307 99.3% 98%-100% RSV B Pre-Selected 0/0 N/A N/A 306/307 99.7% 98%-100% Rhinovirus / Pre-Selected 1/1 100% 21%-100% 302/306 98.7% 97%-99% Enterovirus SARS-CoV-2 0/0 N/A 0/0 N/A N/A Pre-Selected N/A Bacteria Chlamydia Pre-Selected 14/14 100% 78%-100% 293/294 99.7% 98%-100% pneumoniae

Table 20: NxTAG® RPP v2 Performance for the Pre-Selected Data Set

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Pathogen Target	Positive Percent Agreement		Negative Percent Agreement
	TP /(TP+FN)	PPA (%)	95% CI	TN /(TN+FP)	NPA (%)	95% CI
Viruses
Mycoplasma pneumoniae	Pre-Selected	48/52	92.3%	82%-97%	256/256	100%	99%-100%

Pathogen Target			Positive Percent Agreement		Negative Percent Agreement
		TP /(TP+FN)	PPA (%)	95% CI	TN /(TN+FP)	NPA (%)	95% CI
Analyte	LoD	Viruses
	2x	25/25	100%	87%-100%	N/A	N/A	N/A
Coronavirus	10x	12/12	100%	76%-100%	N/A	N/A	N/A
229E	100x	12/12	100%	76%-100%	N/A	N/A	N/A
	Combined	49/49	100%	93%-100%	150/150	100%	98%-100%
	2x	26/26	100%	87%-100%	N/A	N/A	N/A
Influenza A	10x	24/24	100%	86%-100%	N/A	N/A	N/A
(matrix)	100x	0/0	N/A	N/A	N/A	N/A	N/A
	Combined	50/50	100%	93%-100%	149/149	100%	97%-100%
	2x	26/26	100%	87%-100%	N/A	N/A	N/A
Influenza A H1	10x	24/24	100%	86%-100%	N/A	N/A	N/A
(subtype)	100x	0/0	N/A	N/A	N/A	N/A	N/A
	Combined	50/50	100%	93%-100%	149/149	100%	97%-100%
	2x	24/25	96.0%	80%-99%	N/A	N/A	N/A
	10x	13/13	100%	77%-100%	N/A	N/A	N/A
RSV B	100x	12/12	100%	76%-100%	N/A	N/A	N/A
	Combined	49/50	98.0%	90%-100%	148/149	99.3%	96%-100%
Analyte	LoD	Bacteria
	2x	25/25	100%	87%-100%	N/A	N/A	N/A
Chlamydia	10x	12/13	92.3%	67%-99%	N/A	N/A	N/A
pneumoniae	100x	12/12	100%	76%-100%	N/A	N/A	N/A
	Combined	49/50	98.0%	90%-100%	149/149	100%	97%-100%

Table 21: NxTAG® RPP v2 Performance for the Contrived Data Set

The study results demonstrate that the diagnostic accuracy of the NxTAG® RPP v2 assay is acceptable for the detection and identification of respiratory bacteria and viruses from NPS specimens collected from patients exhibiting clinical signs and symptoms of RTI.

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1. Expected values/Reference range:

Table 22: NxTAG® RPP v2 Expected Values for Prospective Specimens by Age

	0-1 years		>1-5 years		>5-21 years		>21-65 years		> 65 years		Unknown		Overall
Target (Analyte)	#Pos	(%)	#Pos	(%)	#Pos	(%)	#Pos	(%)	#Pos	(%)	#Pos	(%)	#Pos	(%)
Adenovirus	34	10.1%(34/337)	40	15.8%(40/253)	22	6.7%(22/329)	5	0.8% (5/641)	0	0.0%(0/242)	0	0.0%(0/12)	101	5.6%(101/1814)
Chlamydia pneumoniae	0	0.0% (0/337)	0	0.0%(0/253)	0	0.0%(0/329)	0	0.0% (0/641)	0	0.0%(0/242)	0	0.0%(0/12)	0	0.0% (0/1814)
Coronavirus 229E	1	0.3% (1/337)	1	0.4%(1/253)	0	0.0%(0/329)	5	0.8% (5/641)	1	0.4%(1/242)	0	0.0%(0/12)	8	0.4% (8/1814)
Coronavirus HKU1	3	0.9% (3/337)	5	2.0%(5/253)	4	1.2%(4/329)	8	1.2% (8/641)	0	0.0%(0/242)	0	0.0%(0/12)	20	1.1%(20/1814)
Coronavirus NL63	17	5.0% (17/337)	12	4.7%(12/253)	8	2.4%(8/329)	9	1.4% (9/641)	2	0.8%(2/242)	0	0.0%(0/12)	48	2.6%(48/1814)
Coronavirus OC43	16	4.7% (16/337)	8	3.2%(8/253)	7	2.1%(7/329)	6	0.9% (6/641)	2	0.8%(2/242)	0	0.0%(0/12)	39	2.1%(39/1814)
HumanMetapneumovirus	45	13.4%(45/337)	41	16.2%(41/253)	29	8.8%(29/329)	39	6.1% (39/641)	14	5.8%(14/242)	3	25%(3/12)	171	9.4%(171/1814)
Influenza A	7	2.1% (7/337)	8	3.2%(8/253)	25	7.6%(25/329)	27	4.2% (27/641)	10	4.1%(10/242)	0	0.0%(0/12)	77	4.2%(77/1814)
Influenza A H1pdm09	3	0.9% (3/337)	3	1.2%(3/253)	8	2.4%(8/329)	14	2.2% (14/641)	3	1.2%(3/242)	0	0.0%(0/12)	31	1.7%(31/1813)
Influenza A H1	0	0.0% (0/337)	0	0.0%(0/253)	0	0.0%(0/328)	0	0.0% (0/641)	0	0.0%(0/242)	0	0.0%(0/12)	0	0.0% (0/1813)
Influenza A H3	4	1.2% (4/337)	6	2.4%(6/253)	18	5.5%(18/329)	11	1.7% (11/641)	7	2.9%(7/242)	0	0.0%(0/12)	46	2.5%(46/1814)
Influenza B	0	0.0% (0/337)	3	1.2%(3/253)	3	0.9%(3/329)	5	0.8% (5/641)	0	0.0%(0/242)	0	0.0%(0/12)	11	0.6%(11/1814)

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	0-1 years		>1-5 years		>5-21 years		>21-65 years		> 65 years		Unknown		Overall
Target (Analyte)	#Pos	(%)	#Pos	(%)	#Pos	(%)	#Pos	(%)	#Pos	(%)	#Pos	(%)	#Pos	(%)
Mycoplasmapneumoniae	0	0.0% (0/337)	0	0.0%(0/253)	0	0.0%(0/329)	0	0.0% (0/641)	0	0.0%(0/242)	0	0.0%(0/12)	0	0.0% (0/1814)
Parainfluenza 1	6	1.8% (6/337)	3	1.2%(3/253)	1	0.3%(1/329)	7	1.1% (7/641)	1	0.4%(1/242)	0	0.0%(0/12)	18	1.0%(18/1814)
Parainfluenza 2	1	0.3% (1/337)	1	0.4%(1/253)	3	0.9%(3/329)	1	0.2% (1/641)	3	1.2%(3/242)	0	0.0%(0/12)	9	0.5% (9/1814)
Parainfluenza 3	15	4.5% (15/337)	16	6.3%(16/253)	5	1.5%(5/329)	7	1.1% (7/641)	0	0.0%(0/242)	0	0.0%(0/12)	43	2.4%(43/1814)
Parainfluenza 4	7	2.1% (7/337)	1	0.4%(1/253)	4	1.2%(4/329)	4	0.6% (4/641)	0	0.0%(0/242)	0	0.0%(0/12)	16	0.9%(16/1814)
RSV A	28	8.3% (28/337)	9	3.6%(9/253)	2	0.6%(2/329)	11	1.7% (11/641)	8	3.3%(8/242)	0	0.0%(0/12)	58	3.2%(58/1814)
RSV B	10	3.0% (10/337)	4	1.6%(4/253)	1	0.3%(1/329)	4	0.6% (4/641)	1	0.4%(1/242)	0	0.0%(0/12)	20	1.1%(20/1814)
Rhinovirus/ Enterovirus	113	33.5%(113/337)	75	29.6%(75/253)	75	22.8%(75/329)	65	10.1%(65/641)	20	8.3%(20/242)	4	33.3%(4/12)	352	19.4%(352/1814)
SARS-CoV-2	32	9.5% (32/336)	12	4.8%(12/251)	21	6.5%(21/325)	118	18.4%(118/640)	56	23.3%(56/240)	1	9.1%(1/11)	240	13.3%(240/1803)

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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.