Predicate Devices

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The description details a standard RT-PCR process with automated analysis of melt curves and internal controls by the FilmArray software. There is no mention of AI, ML, or any learning algorithms being used for the analysis or interpretation of results. The analysis appears to be based on predefined parameters and thresholds.

Therapeutic

No

Explanation: This device is an in vitro diagnostic test designed to detect the presence of SARS-CoV-2 RNA. It provides diagnostic information but does not actively treat or alleviate a disease, which is the function of a therapeutic device.

Diagnostic

Yes

The "Intended Use / Indications for Use" section explicitly states that "The BioFire COVID-19 Test 2 is a qualitative nested multiplexed RT-PCR in vitro diagnostic test". It further specifies its purpose to "detects nucleic acids from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swabs (NPS) from symptomatic individuals suspected of COVID-19 by their healthcare provider," indicating its use in diagnosing a medical condition.

SaMD (Software as a Medical Device)

No

The device is an in vitro diagnostic test that includes physical components (pouch with reagents, sample buffer) and relies on a specific hardware system (BioFire FilmArray 2.0 or Torch Systems) to perform the test and analysis. While software is involved in guiding the user and analyzing results, it is integral to a larger hardware and reagent-based system, not a standalone software-only device.

IVD (In Vitro Diagnostic)

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

Here's why, based on the provided text:

Explicitly stated in the Intended Use: The very first sentence of the "Intended Use / Indications for Use" section clearly states: "The BioFire COVID-19 Test 2 is a qualitative nested multiplexed RT-PCR in vitro diagnostic test intended for use with the BioFire FilmArray 2.0 and BioFire FilmArray Torch Systems."
Purpose of the test: The test is designed to detect nucleic acids from SARS-CoV-2 in patient specimens (nasopharyngeal swabs) to aid in the diagnosis of COVID-19. This is a core function of an in vitro diagnostic device.
Testing performed outside the body: The test is performed on a specimen collected from the patient (nasopharyngeal swab) in a laboratory setting, not directly on or within the patient's body. This is the definition of "in vitro."

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The BioFire COVID-19 Test 2 is a qualitative nested multiplexed RT-PCR in vitro diagnostic test intended for use with the BioFire FilmArray 2.0 and BioFire FilmArray Torch Systems. The BioFire COVID-19 Test 2 detects nucleic acids from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swabs (NPS) from symptomatic individuals suspected of COVID-19 by their healthcare provider.

Results are for the identification of SARS-CoV-2 RNA. The SARS-CoV-2 RNA is generally detectable in NPS specimens during the acute phase of infection. Positive results are indicative of the presence of SARS-CoV-2 RNA; clinical correlation with patient history and other diagnostic in necessary to determine patient infection status. Positive results do not rule out co-infection with other pathogens.

Results are meant to be used in conjunction with other clinical, epidemiologic, and laboratory data, in accordance with the guidelines provided by the relevant public health authorities. The BioFire COVID-19 Test 2 is intended for use by trained medical and laboratory professionals in a laboratory setting or under the supervision of a trained laboratory professional.

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

OOX, QQX

Device Description

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense, singlestranded RNA betacoronavirus and is the etiological agent of Coronavirus Disease 2019 (COVID-19).12 The disease is primarily characterized by shortness of breath, fever or chills, cough, fatigue, and muscle or body aches.23 Older adults or people with underlying medical conditions may be at higher risk for developing more severe cases which can be fatal.43 The virus is thought to be of zoonotic origin and is highly transmissible through the inhalation of respiratory droplets. 1,3,4

The BioFire COVID-19 Test 2 is a multiplexed nucleic acid-based test designed to be used with BioFire® FilmArray® Systems (BioFire® FilmArray® 2.0 or BioFire® FilmArray® Torch). The BioFire COVID-19 Test 2 consists of the BioFire COVID-19 Test 2 pouch which contains freeze-dried reagents to perform nucleic acid purification and nested, multiplexed polymerase chain reaction (PCR) with DNA melt analysis. The BioFire COVID-19 Test 2 conducts three independent tests for the detection of SARS-CoV-2 RNA in nasopharyngeal swabs (NPS) eluted in transport medium or saline. Results from the BioFire COVID-19 Test 2 are available in about 45 minutes.

A test is initiated by loading Hydration Solution into one port of the pouch and a NPS specimen mixed with the provided Sample Buffer into the other port of the pouch. The pouch contains all the reagents required for specimen testing and analysis in a freeze-dried format; the addition of Hydration Solution and the Sample Buffer rehydrates the reagents. After the pouch is prepared, the FilmArray Software on the FilmArray System guides the user through the steps of placing the pouch into the instrument, scanning the pouch barcode, entering the sample identification, selecting the appropriate protocol, and initiating the run on the FilmArray System.

The FilmArray instruments contain a coordinated system of inflatable bladders and seal points, which act on the pouch to control the movement of liquid between the pouch blisters. When a bladder is inflated over a reagent blister, it forces liquid from the blister into connecting channels. Alternatively, when a seal is placed over a connecting channel it acts as a valve to open or close a channel. In addition, electronically controlled pneumatic pistons are positioned over multiple plungers to deliver the rehydrated reagents into the blisters at the appropriate times. Two Peltier devices control heating and cooling of the pouch to drive the PCR reactions and subsequent melt.

Nucleic acid extraction occurs within the FilmArray pouch using mechanical and chemical lysis followed by purification using standard magnetic bead technology. After extracting and purifying nucleic acids from the unprocessed sample, a nested multiplexed PCR is executed in two stages. During the first stage, a single, large volume, multiplexed reverse transcription PCR (rt-PCR) reaction is performed. The products from first stage PCR are then diluted and combined with a fresh, primer-free master mix and a fluorescent double stranded DNA binding dye (LC Green® Plus, BioFire Defense, LLC). The solution is then distributed to each well of the arrav. Array wells contain sets of primers designed specifically to amplify sequences internal to the PCR products generated during the first stage PCR reaction. The 2nd stage PCR and melt, or nested PCR, is performed in each well of the array. At the conclusion of the 2nd stage PCR, the array is interrogated by melt curve analysis for the detection of signature amplicons denoting the presence of specific targets. A digital camera placed in front of the array captures fluorescent images of the PCR2 reactions The FilmArray software automatically analyzes the results of each DNA melt curve and the results of the internal pouch controls to provide a final test interpretation.

Materials provided in each BioFire COVID-19 Test 2 Kit:

Individually packaged BioFire COVID-19 Test 2 Pouches
Single-use (1.0 mL) Sample Buffer Tubes
· Single-use pre-filled (1.5 mL) Hydration Injection Vials (blue)
Individually packaged Sample Injection Vials (red)
Individually packaged Transfer Pipettes

Materials required but not provided:

10% bleach solution
FilmArray system including:
o FilmArray 2.0 instruments or FilmArray Torch modules and accompanying software
o FilmArray Pouch Loading Station

Mentions image processing

Yes

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

nasopharyngeal swabs (NPS)

Indicated Patient Age Range

Not Found

Intended User / Care Setting

trained medical and laboratory professionals in a laboratory setting or under the supervision of a trained laboratory professional.

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

Not Found

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

The prospective clinical study evaluated the performance of the BioFire COVID-19 Test 2 with NPS specimens in transport medium. Specimens were residual after standard of care (SoC) testing for SARS-CoV-2 using a molecular test with Emergency Use Authorization (EUA). Specimens were de-identified and tested at three study sites in the United States over four months (July-October 2020) during the COVID-19 global pandemic.
A total of 534 specimens were assigned a study code number (SCN) and were enrolled in the study. Eleven (11/534) were excluded for various reasons. Seven (7/11) specimens failed inclusion criteria and were excluded: six did not meet storage requirements, and one failed to obtain a BioFire RP2.1 comparator result (a BioFire COVID-19 Test 2 was never attempted). Thus, 527 specimens met inclusion criteria and were available for testing on the BioFire COVID-19 Test 2. Four specimens (4/527) were later excluded from analyses due to inability to obtain a BioFire COVID-19 Test 2 result and not having sufficient specimen volume for retest.
Specimens were tested on the BioFire COVID-19 Test 2 at clinical study sites. As a comparator for performance evaluation, specimens were also tested at clinical sites on BioFire RP2.1. A BioFire COVID-19 Test 2 result ('Detected' or 'Not Detected') was considered True Positive (TP) or True Negative (TN) only when it agreed with the BioFire RP2.1 comparator result. Positive Percent Agreement (PPA) or Sensitivity for each analyte was calculated as 100% x (TP / (TP + FN)). False Negative (FN) indicates that the BioFire COVID-19 Test 2 result was 'Not Detected', while the BioFire RP2.1 comparator result was positive. Negative Percent Agreement (NPA) or Specificity was calculated as 100%x (TN / (TN + FP)). False Positive (FP) indicates that the BioFire COVID-19 Test 2 result was 'Detected', but the BioFire RP2.1 comparator result was negative. The exact binomial twosided 95% confidence interval was calculated.

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

Clinical Performance
Study Type: Prospective Clinical Study
Sample Size: 523 specimens for analysis after exclusions.
Key Results:

Positive Percent Agreement (PPA): 98.6% (95% CI: 92.2-99.7%) for 68 TP and 1 FN. The FN specimen was negative for SARS-CoV-2 by SoC and Central Reference Lab (CRL) testing.
Negative Percent Agreement (NPA) or Specificity: 99.6% (95% CI: 98.4-99.9%) for 452 TN and 2 FP. Evidence of SARS-CoV-2 was found in one FP by SoC and CRL testing.

Limit of Detection (LoD)
Study Type: Analytical Study
Sample Size: 20 replicates tested at the estimated LoD concentration for each virus type.
Key Results:

SARS-CoV-2 USA-WA1/2020 (infectious culture; WRCEVA): LoD Concentration 3.3E+02 GC/mL (2.2E-02 TCID50/mL), Detection Rate 20/20 (100%).
SARS-CoV-2 USA-WA1/2020 (heat-inactivated: BEI NR-52286): LoD Concentration 3.3E+02 GE/mL (4.3E-02 TCID50/mL), Detection Rate 20/20 (100%).

NPS in Saline Sensitivity Validation
Study Type: Analytical Study
Sample Size: 20 individually contrived samples.
Key Results: Detection Rate 20/20 (100%) for NPS in Saline at 3.3E+02 GE/mL (1x LoD), demonstrating equivalent sensitivity to samples prepared with NPS in transport medium.

FDA SARS-CoV-2 Reference Panel
Study Type: Analytical Evaluation
Key Results:

SARS-CoV-2: Product LoD 5.4E+03 NDU/mL (NPS in transport medium).
MERS-CoV: No cross-reactivity observed.

Inclusivity (Reactivity)
Study Type: Analytical Study
Sample Size: Triplicate testing for each variant.
Key Results: All five SARS-CoV-2 variants (USA-WA1/2020, Chile/Santiago_op4d1/2020, Hong Kong/VM20001061/2020, Italy-INMI1, New York-PV08410/2020) were detected within 3-fold the concentration of the USA-WA1/2020 reference variant. Mean melting temperature (Tm) values were within 0.5 ℃ of the reference variant.

Exclusivity (Specificity)
Study Type: Analytical Study
Sample Size: Panel of 77 viruses and organisms tested.
Key Results: No assay cross-reactivity with any of the tested viruses/organisms was observed. In-silico analysis predicted two non-SARS-CoV-2 accessions (bat coronavirus RaTG13 (MN996532) and pangolin coronavirus isolate MP789 (MT084071)) to be cross-reactive, but neither is expected in human NPS.

Interference
Study Type: Analytical Study
Key Results: None of the tested potentially interfering substances (e.g., toothpaste, tobacco, oral rinse, nasal sprays, blood, mucin, various drugs, transport media components) were determined to be inhibitory to the BioFire COVID-19 Test 2.

Reproducibility
Study Type: Reproducibility Study
Sample Size: 90 replicate test results per sample (6 replicates x 3 locations x 5 days), totaling 270 valid test results.
Key Results:

Moderate Positive (3x LoD) SARS-CoV-2: 100% detection rate (95/95.9-100% Confidence Interval).
Low Positive (1x LoD) SARS-CoV-2: 100% detection rate (95/95.9-100% Confidence Interval).
Negative (No Analyte): 96.7% detection rate (87/90 - 90.7-98.9% Confidence Interval).
Standard deviations for Tm were ≤0.3 ℃, CV ≤0.4%, demonstrating accurate and highly reproducible results.

Specimen Storage
Study Type: Analytical Study
Sample Size: 10 contrived samples per storage condition, tested immediately and then after different storage durations.
Key Results: Data supports claimed storage conditions for NPS specimens: up to 4 hours at 15-30°C, up to 3 days at 2-8°C, and up to 30 days at

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.

Summary

0

Image /page/0/Picture/0 description: The image contains two logos. On the left is the Department of Health & Human Services logo. On the right is the FDA logo, which includes the letters "FDA" in a blue square, followed by the words "U.S. FOOD & DRUG ADMINISTRATION" in blue text.

November 1, 2021

BioFire Defense, LLC Cynthia Phillips VP of Regulatory, Quality, and Clinical Affairs 79 W 4500 S. Suite 14 Salt Lake City, Utah 84107

Re: K211079

Trade/Device Name: BioFire COVID-19 Test 2 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: OOX Dated: April 8, 2021 Received: April 12, 2021

Dear Cynthia Phillips:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. 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 located 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.

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

1

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 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-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 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to 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,

Kristian Roth, Ph.D. Deputy Director Division of Microbiology Devices OHT7: Office of In Vitro Diagnostics and Radiological Health Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

2

Indications for Use

510(k) Number (if known) K211079

Device Name BioFire COVID-19 Test 2

Indications for Use (Describe)

The BioFire COVID-19 Test 2 is a qualitative nested multiplexed RT-PCR in vitro diagnostic test intended for use with the BioFire FilmArray 2.0 and BioFire FilmArray Torch Systems. The BioFire COVID-19 Test 2 detects nucleic acids from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swabs (NPS) from symptomatic individuals suspected of COVID-19 by their healthcare provider.

Results are for the identification of SARS-CoV-2 RNA. The SARS-CoV-2 RNA is generally detectable in NPS specimens during the acute phase of infection. Positive results are indicative of the presence of SARS-CoV-2 RNA; clinical correlation with patient history and other diagnostic in necessary to determine patient infection status. Positive results do not rule out co-infection with other pathogens.

Results are meant to be used in conjunction with other clinical, epidemiologic, and laboratory data, in accordance with the guidelines provided by the relevant public health authorities. The BioFire COVID-19 Test 2 is intended for use by trained medical and laboratory professionals in a laboratory setting or under the supervision of a trained laboratory professional.

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

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

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3

510(k) Summary

I. Submitter

BioFire Defense, LLC Salt Lake City, UT 84107 Phone: (801) 262-3592

Contact Person: Cynthia L. Phillips Date Prepared: 2021-Apr-08

II. Device

Name of Device: BioFire® COVID-19 Test 2 Common or Usual Name: Same Classification Name: Multi-Target Respiratory Specimen Nucleic Acid Test Including SARS-CoV-2 and Other Microbial Agents. Regulatory Class: Class II (Special Controls) Regulation: 21 CFR 866.3981 Panel: Microbiology - 83 Product Code: QQX

III. Predicate Device

BioFire® Respiratory Panel 2.1 (RP2.1) (BioFire Diagnostics, LLC) [DEN200031] This predicate has not been subject to a design-related recall.

Device Description IV.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense, singlestranded RNA betacoronavirus and is the etiological agent of Coronavirus Disease 2019 (COVID-19).12 The disease is primarily characterized by shortness of breath, fever or chills, cough, fatigue, and muscle or body aches.23 Older adults or people with underlying medical conditions may be at higher risk for developing more severe cases which can be fatal.43 The virus is thought to be of zoonotic origin and is highly transmissible through the inhalation of respiratory droplets. 1,3,4

The BioFire COVID-19 Test 2 is a multiplexed nucleic acid-based test designed to be used with BioFire® FilmArray® Systems (BioFire® FilmArray® 2.0 or BioFire® FilmArray® Torch). The BioFire COVID-19 Test 2 consists of the BioFire COVID-19 Test 2 pouch which contains freeze-dried reagents to perform nucleic acid purification and nested, multiplexed polymerase chain reaction (PCR) with DNA melt analysis. The BioFire COVID-19 Test 2 conducts three independent tests for the detection of SARS-CoV-2 RNA in nasopharyngeal swabs (NPS) eluted in transport medium or saline. Results from the BioFire COVID-19 Test 2 are available in about 45 minutes.

4

A test is initiated by loading Hydration Solution into one port of the pouch and a NPS specimen mixed with the provided Sample Buffer into the other port of the pouch. The pouch contains all the reagents required for specimen testing and analysis in a freeze-dried format; the addition of Hydration Solution and the Sample Buffer rehydrates the reagents. After the pouch is prepared, the FilmArray Software on the FilmArray System guides the user through the steps of placing the pouch into the instrument, scanning the pouch barcode, entering the sample identification, selecting the appropriate protocol, and initiating the run on the FilmArray System.

The FilmArray instruments contain a coordinated system of inflatable bladders and seal points, which act on the pouch to control the movement of liquid between the pouch blisters. When a bladder is inflated over a reagent blister, it forces liquid from the blister into connecting channels. Alternatively, when a seal is placed over a connecting channel it acts as a valve to open or close a channel. In addition, electronically controlled pneumatic pistons are positioned over multiple plungers to deliver the rehydrated reagents into the blisters at the appropriate times. Two Peltier devices control heating and cooling of the pouch to drive the PCR reactions and subsequent melt.

Nucleic acid extraction occurs within the FilmArray pouch using mechanical and chemical lysis followed by purification using standard magnetic bead technology. After extracting and purifying nucleic acids from the unprocessed sample, a nested multiplexed PCR is executed in two stages. During the first stage, a single, large volume, multiplexed reverse transcription PCR (rt-PCR) reaction is performed. The products from first stage PCR are then diluted and combined with a fresh, primer-free master mix and a fluorescent double stranded DNA binding dye (LC Green® Plus, BioFire Defense, LLC). The solution is then distributed to each well of the arrav. Array wells contain sets of primers designed specifically to amplify sequences internal to the PCR products generated during the first stage PCR reaction. The 2nd stage PCR and melt, or nested PCR, is performed in each well of the array. At the conclusion of the 2nd stage PCR, the array is interrogated by melt curve analysis for the detection of signature amplicons denoting the presence of specific targets. A digital camera placed in front of the array captures fluorescent images of the PCR2 reactions The FilmArray software automatically analyzes the results of each DNA melt curve and the results of the internal pouch controls to provide a final test interpretation.

Materials provided in each BioFire COVID-19 Test 2 Kit:

Individually packaged BioFire COVID-19 Test 2 Pouches
Single-use (1.0 mL) Sample Buffer Tubes
· Single-use pre-filled (1.5 mL) Hydration Injection Vials (blue)
Individually packaged Sample Injection Vials (red)
Individually packaged Transfer Pipettes

Materials required but not provided:

10% bleach solution
FilmArray system including:

o FilmArray 2.0 instruments or FilmArray Torch modules and accompanying software

o FilmArray Pouch Loading Station
BioFire Defense 510(k) BioFire® COVID-19 Test 2

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V. Intended Use

The BioFire® COVID-19 Test 2 is a qualitative nested multiplexed RT-PCR in vitro diagnostic test intended for use with the BioFire® FilmArray® 2.0 and BioFire® FilmArray® Torch Systems. The BioFire COVID-19 Test 2 detects nucleic acids from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swabs (NPS) from symptomatic individuals suspected of COVID-19 by their healthcare provider.

Results are for the identification of SARS-CoV-2 RNA. The SARS-CoV-2 RNA is generally detectable in NPS specimens during the acute phase of infection. Positive results are indicative of the presence of SARS-CoV-2 RNA; clinical correlation with patient history and other diagnostic information is necessary to determine patient infection status. Positive results do not rule out co-infection with other pathogens.

Results are meant to be used in conjunction with other clinical, epidemiologic, and laboratory data, in accordance with the guidelines provided by the relevant public health authorities. The BioFire COVID-19 Test 2 is intended for use by trained medical and laboratory professionals in a laboratory setting or under the supervision of a trained laboratory professional.

For In Vitro Diagnostic Use.

Substantial Equivalence VI.

The BioFire COVID-19 Test 2 and the BioFire Respiratory Panel 2.1 (RP2.1) are substantially equivalent. The BioFire COVID-19 Test 2 is essentially a BioFire RP2.1 pouch which has been modified to detect only SARS-CoV-2 RNA targets. The instrument platforms, sample type, chemistry, and protocols for the BioFire COVID-19 Test 2 are identical to those of the BioFire RP2.1. The BioFire RP2.1 was granted clearance on March 17, 2021 and determined to be a Class II device [DEN200031].

Table 1 compares the BioFire COVID-19 Test 2 to the BioFire Respiratory Panel 2.1 and outlines the similarities and differences between the two tests.

| Element | New Device:
BioFire COVID-19 Test 2 | Predicate Device:
BioFire Respiratory Panel 2.1 |
|----------------|--------------------------------------------------------------|----------------------------------------------------|
| Specimen Types | Nasopharyngeal swabs eluted in
transport medium or saline | Same |

Table 1. Comparison of the BioFire COVID-19 Test 2 and the BioFire Respiratory Panel 2.1

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| Element | New Device:
BioFire COVID-19 Test 2 | Predicate Device:
BioFire Respiratory Panel 2.1 |
|-------------------------------|------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Viruses/Organisms
Detected | SARS-CoV-2
(multiple targets independent of
BioFire RP2.1) | SARS-CoV-2
AND
adenovirus, coronavirus 229E, coronavirus
HKU1, coronavirus NL63, coronavirus
OC43, human metapneumovirus, human
rhinovirus/enterovirus, influenza A
(including subtypes H1, H3, and H1-2009),
influenza B, parainfluenza virus 1,
parainfluenza virus 2, parainfluenza virus
3, parainfluenza virus 4, respiratory
syncytial virus, Bordetella parapertussis,
Bordetella pertussis, Chlamydia
pneumoniae, Mycoplasma pneumoniae |
| Analyte | RNA | DNA/RNA |
| Technological
Principles | Nested multiplex RT-PCR followed
by high resolution melting analysis
to confirm identity of amplified
product | Same |
| Instrumentation | FilmArray 2.0 or FilmArray Torch | Same |
| Time to Result | About 45 minutes | Same |
| Reagent Storage | Room temperature | Same |
| Test Interpretation | Automated test interpretation and
report generation. User cannot
access raw data. | Same |
| Controls | Two controls are included in each
reagent pouch to control for
sample processing and both stages
of PCR and melt analysis | Same |
| User Complexity | Moderate | Same |

VII. Summary of Performance Data

Clinical Performance

The prospective clinical study evaluated the performance of the BioFire COVID-19 Test 2 with NPS specimens in transport medium. Specimens were residual after standard of care (SoC) testing for SARS-CoV-2 using a molecular test with Emergency Use Authorization (EUA). Specimens were de-identified and tested at three study sites in the United States over four months (July-October 2020) during the COVID-19 global pandemic.

This study was conducted as part of concurrent evaluation of the BioFire Diagnostics Respiratory Panel 2.1 (RP2.1). At the time of the study, BioFire RP2.1 was authorized under

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EUA202392. In March 2021. BioFire RP2.1 was granted de novo classification under DEN200031. BioFire Diagnostics selected clinical sites, sponsored the study, and shared the collected data with BioFire Defense for secondary analysis.

A total of 534 specimens were assigned a study code number (SCN) and were enrolled in the study. Eleven (11/534) were excluded for various reasons. Seven (7/11) specimens failed inclusion criteria and were excluded: six did not meet storage requirements, and one failed to obtain a BioFire RP2.1 comparator result (a BioFire COVID-19 Test 2 was never attempted). Thus, 527 specimens met inclusion criteria and were available for testing on the BioFire COVID-19 Test 2. Four specimens (4/527) were later excluded from analyses due to inability to obtain a BioFire COVID-19 Test 2 result and not having sufficient specimen volume for retest.

The study included approximately equal numbers of females enrolled compared to males (48.4%, 253/523 and 51.1%, 267/523 respectively) (Table 2). Study enrollees were predominantly adults (only 10.5% (55/523) were 0-18 years).

		Overall
Sex	Female	253 (48.4%)
	Male	267 (51.1%)
	Unknown	3 (0.6%)
Age Range	0-18 years	55 (10.5%)
	19-40 years	170 (32.5%)
	41-60 years	146 (27.9%)
	61+ years	152 (29.1%)
	Total	523

Table 2. Overall Enrollment

Specimens were tested on the BioFire COVID-19 Test 2 at clinical study sites. As a comparator for performance evaluation, specimens were also tested at clinical sites on BioFire RP2.1. A BioFire COVID-19 Test 2 result ('Detected' or 'Not Detected') was considered True Positive (TP) or True Negative (TN) only when it agreed with the BioFire RP2.1 comparator result. Positive Percent Agreement (PPA) or Sensitivity for each analyte was calculated as 100% × (TP / (TP + FN)). False Negative (FN) indicates that the BioFire COVID-19 Test 2 result was 'Not Detected', while the BioFire RP2.1 comparator result was positive. Negative Percent Agreement (NPA) or Specificity was calculated as 100%× (TN / (TN + FP)). False Positive (FP) indicates that the BioFire COVID-19 Test 2 result was 'Detected', but the BioFire RP2.1 comparator result was negative. The exact binomial twosided 95% confidence interval was calculated. The prospective clinical study results are summarized in Table 3.

Table 3. BioFire COVID-19 Test 2 Clinical Performance Summary

Positive Agreement (PPA)				Negative Agreement (NPA)
TP	FN	%	95% CI	TN	FP	%	95% CI
68	1a	98.6%	92.2-99.7%	452	2b	99.6%	98.4-99.9%

a The FN specimen was negative for SARS-CoV-2 by SoC and Central Reference Lab (CRL) testing. b Evidence of SARS-CoV-2 was found in one FP by SoC and CRL testing.

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Select Analytical Studies

Limit of Detection

The BioFire COVID-19 Test 2 limit of detection (LoD) was determined using contrived samples containing known concentrations of inactivated or infectious SARS-CoV-2 material. The LoD concentration was first estimated based on results of serial dilutions spanning concentrations bracketing the anticipated LoD concentration. Additional dilutions were tested, if needed, to reach a concentration at which loss of detection could be observed.

The LoD was then confirmed by testing 20 replicates at the estimated LoD concentration. Contrived specimens for LoD confirmation were prepared with SARS-CoV-2 at known concentrations in NPS eluted in transport medium. The required criteria for confirmation of LoD was a detection rate of at least 95% at the LoD concentration (≥ 19/20). Results are shown in Table 4.

Virus	LoD Concentration	Detection Rate
SARS-CoV-2
USA-WA1/2020
(infectious culture; WRCEVA)a	3.3E+02 GC/mL
(2.2E-02 TCID50/mL)	20/20 (100%)
SARS-CoV-2
USA-WA1/2020
(heat-inactivated: BEI NR-52286)b	3.3E+02 GE/mL
(4.3E-02 TCID50/mL)	20/20 (100%)

Table 4. Summary of LoD Results for the BioFire COVID-19 Test 2

a Obtained for culture in a biosafety level 3 laboratory from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA). Concentration determined by quantitative real-time PCR as described on the World Health Organization website: https://www.who.int/docs/default-source/coronaviruse/protocol-v2-1.pdf

ს Concentration determined by digital droplet PCR as indicated on the Certificate of Analysis provided by BEI Resources. TCID50/mL was determined prior to inactivation.

NPS in Saline Sensitivity Validation

Sensitivity of the BioFire COVID-19 Test 2 when testing NPS collected in saline was evaluated by confirming the reliable detection (≥ 95%) of SARS-CoV-2 at 1× the LoD. Twenty (20) samples were individually contrived by spiking inactivated SARS-CoV-2 from the USA-WA1/2020 isolate (BEI/NR-52286) into NPS specimens that had been collected in saline. Each replicate was contrived at a concentration of 3.3E+02 GE/mL (1× LoD). Results, shown in Table 5, demonstrated equivalent sensitivity to samples prepared with NPS in transport medium.

Table 5. Summary of NPS in Saline Sensitivity Testing

Clinical Matrix	Test Concentration	Detection Rate
NPS in Saline	3.3E+02 GE/mL (1× LoD)	20/20 (100%)

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FDA SARS-CoV-2 Reference Panel

SARS-CoV-2 sensitivity and MERS-CoV cross-reactivity were evaluated using the FDA SARS-CoV-2 Reference Panel according to the standard protocol provided by the FDA. The evaluation was performed using the supplied reference material and blinded samples. The study included a range finding study and a confirmatory study for LoD. Blinded sample testing was used to establish specificity and to confirm the LoD. The product LoD when using the FDA Reference Panel is presented in Table 6. No cross-reactivity with MERS-CoV was reported.

| Reference Materials
Provided by FDA | Specimen Type | Product LoD | Cross-
Reactivity |
|----------------------------------------|-------------------------|-----------------|----------------------|
| SARS-CoV-2 | NPS in transport medium | 5.4E+03 NDU/mL1 | N/A2 |
| MERS-CoV | | N/A2 | ND3 |

Table 6. Summary of FDA SARS-CoV-2 Reference Panel Testing

1 NDU: Nucleic acid amplification test (NAAT) Detectable Units

2 N/A: Not applicable

3 ND: Not detected

Inclusivity (Reactivity)

Five variants of infectious SARS-CoV-2 were spiked into pooled NPS specimens in triplicate. Alignments of these SARS-CoV-2 variant sequences did not reveal any mismatches within the assay primer binding regions; therefore, selection of the variants was based on geographic location. Because the quantification method differed from the method used to quantify virus for LoD testing, the evaluation was carried out relative to the SARS-CoV-2 USA-WA1/2020 reference strain. The testing concentration of the reference strain was determined to be ~1× LoD. Results for inclusivity testing are summarized in Table 7. All four SARS-CoV-2 variants were detected within 3-fold the concentration of the USA-WA1/2020 reference variant. Mean melting temperature (Tm) values for each of the SARS-CoV-2 assays when testing the variants were within 0.5 ℃ of values obtained with the USA-WA1/2020 reference variant.

| SARS-CoV-2 Variant
(Source / ID) | SARS-CoV-2
Detection Rate | Concentration
Detected | |
|-----------------------------------------------|------------------------------|---------------------------|---------|
| (GC/mL) | PFU/mL | | |
| USA-WA1/2020
(BEI / NR-52281) | 3/3 | 9.9E+02 | 1.3E-02 |
| Chile/Santiago_op4d1/2020
(BEI / NR-52439) | 3/3 | 9.9E+02 | 6.3E-02 |
| Hong Kong/VM20001061/2020
(BEI / NR-52282) | 3/3 | 9.9E+02 | 1.5E-02 |
| Italy-INMI1
(BEI / NR-52284) | 3/3 | 9.9E+02 | 7.6E-02 |
| New York-PV08410/2020
(BEI / NR-52284) | 3/3 | 3.3E+03 | 2.4E-02 |

Table 7. Summary of Inclusivity Results for the BioFire COVID-19 Test 2

BioFire Defense 510(k) BioFire® COVID-19 Test 2

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| SARS-CoV-2 Variant
(Source / ID) | SARS-CoV-2
Detection Rate | Concentration
Detected | |
|-------------------------------------|------------------------------|---------------------------|--------|
| | | (GC/mL) | PFU/mL |
| (BEI / NR-53514) | | | |

An in-silico analysis was also performed using publicly available SARS-CoV-2 sequences. At this time BioFire Defense predicts all strains of SARS-CoV-2 will be positively identified even if sensitivity to one or two assays is reduced. BioFire Defense will continue monitoring emerging SARS-CoV-2 strains and sequence variants and assessing any predicted changes in BioFire COVID-19 Test 2 performance.

Exclusivity (Specificity)

The potential for non-specific amplification and detection by the BioFire COVID-19 Test 2 was evaluated by challenging the test with a panel of 77 viruses and organisms at high concentrations in sterile saline. The panel of viruses and organisms included normal respiratory flora and pathogens that may be present in NPS specimens, as well as SARS-CoV-2 related viruses. A list of viruses and organisms tested is shown in Table 8. No assay cross-reactivity with any of the tested viruses/organisms was observed.

Organism/Virus
Viruses (SARS-CoV-2 Related)
Human coronavirus 229E	Human coronavirus NL63	Middle East Respiratory Syndrome
coronavirus
(MERS-CoV)
Human coronavirus HKU1	Human coronavirus OC43	Severe Acute Respiratory Syndrome
coronavirus
(SARS-CoV)
Viruses
Adenovirus 1
(species C)	Enterovirus species D
(68)	Mumps virus
Adenovirus 4
(species E)	Epstein-Barr virus
(B95-8)	Parainfluenza virus 1
Adenovirus 7
(species B)	Herpes simplex virus	Parainfluenza virus 2
Bocavirus	Human Metapneumovirus	Parainfluenza virus 3
Cytomegalovirus	Influenza A subtype H1	Parainfluenza virus 4
Enterovirus species A
(EV71)	Influenza A subtype H3	Respiratory syncytial virus
Enterovirus species B
(Echovirus 6)	Influenza B	Rhinovirus
Enterovirus species C
(Coxsackievirus A17)	Measles virus
Bacteria

Table 8. List of Viruses/Organisms Tested for Exclusivity on the BioFire COVID-19 Test 2

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Organism/Virus
Acinetobacter calcoaceticus	Klebsiella aerogenes
(Enterobacter aerogenes)	Neisseria elongate
Bordetella avium	Klebsiella oxytoca	Neisseria gonorrhoeae
Bordetella bronchiseptica	Klebsiella pneumoniae	Neisseria meningitidis
Bordetella hinzii	Lactobacillus acidophilus	Proteus mirabilis
Bordetella holmesii	Lactobacillus plantarum	Pseudomonas aeruginosa
Bordetella parapertussis	Legionella feeleii	Serratia marcescens
Bordetella pertussis	Legionella longbeacheae	Staphylococcus aureus
Chlamydia pneumoniae	Legionella pneumophila	Staphylococcus epidermidis
Chlamydia trachomatis	Moraxella catarrhalis	Stenotrophomonas maltophilia
Corynebacterium diptheriae	Mycobacterium tuberculosis	Streptococcus agalactiae
Escherichia coli	Mycoplasma genitalium	Streptococcus pneumoniae
Fluoribacter bozemanae
(Legionella bozemanii)	Mycoplasma hominis	Streptococcus pyogenes
Fluoribacter dumoffii
(Legionella dumoffii)	Mycoplasma orale	Streptococcus salivarius
Haemophilus influenzae	Mycoplasma pneumoniae	Tatlockia micdadei
(Legionella micdadei)
Ureaplasma urealyticum
Fungi
Aspergillus flavus	Candida albicans	Pneumocystis jirovecii
Aspergillus fumigatus	Cryptococcus neoformans

An in-silico analysis was also performed using an alignment of 1,124 publicly available Coronaviridae sequences. Two non-SARS-CoV-2 accessions were predicted to be crossreactive with the BioFire COVID-19 Test 2: bat coronavirus RaTG13 (MN996532) and pangolin coronavirus isolate MP789 (MT084071). Neither of these viruses are expected to be present within human NPS specimens. However, if present, the cross-reactive product(s) may be detected as SARS-CoV-2.

Interference

For this study, potentially interfering substances were selected based upon FDA recommended interferents specific to the respiratory sample type. Potential interference was evaluated by comparing BioFire COVID-19 Test 2 results from samples containing SARS-CoV-2 analyte at 1× LoD to results from samples with the same analyte composition but including the test substance. Potential effects of the substance on the BioFire COVID-19 Test 2 control assays and analyte detection were evaluated. Table 9 provides a list of the potentially interfering substances evaluated during this study and the observed results of testing in the presence of the substances. None of the substances tested were determined to be inhibitory to the BioFire COVID-19 Test 2.

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| Substance | Specific Active Ingredient | Test
Concentration |
|------------------------------------------------------|----------------------------------------------------------------------------------|-----------------------|
| Toothpaste (Colgate Total) | Stannous fluoride 0.454% | 2% v/v |
| Tobacco (Camel Snus) | Tobacco | 10 mg/mL |
| Oral Rinse (Listerine) | Eucalyptol 0.092%
Menthol 0.042%
Methyl Salicylate 0.060%
Thymol 0.064% | 1% v/v |
| Throat lozenges (Cepacol) | Benzocaine 7.5 mg
Dextromethorphan HBr 5 mg | 2.2 mg/mL |
| Oral anesthetic and analgesic
(Mouth Sore Relief) | Benzocaine 20% | 1% v/v |
| Cough Drops | Menthol 5.4 mg | 2.2 mg/mL |
| Cleanser (Dye-Free Antiseptic
Cleanser) | Chlorhexidine Gluconate 4% | 1% v/v |
| Nicotine | Nicotine | 10 mg/mL |
| Mucin | Bovine submaxillary gland, type I-S Sigma
M3895 | 5 mg/mL |
| Blood (human) | Human DNA | 5% v/v |
| Leukocytes | Human DNA | 1% v/v |
| Nasal spray (Wal-Four Nasal
Spray) | Phenylephrine hydrochloride 1% | 10% v/v |
| Afrin Nasal Spray | Oxymetazoline hydrochloride 0.05% | 10% v/v |
| Saline Nasal Spray | NaCl 0.65% with preservatives (Phenylcarbinol,
Benzalkonium Chloride) | 10% v/v |
| Nasal corticosteroids | Beclomethasone | 2 mg/mL |
| | Dexamethasone | 1.5 mg/mL |
| | Flunisolide | 2 mg/mL |
| | Triamcinolone | 5 mg/mL |
| | Mometasone | 1 mg/mL |
| Budesonide Nasal Spray | Budesonide 32 mcg/ spray | 1% v/v |
| Allergy Nasal Spray | Fluticasone 50 mcg/ spray | 1% v/v |
| Nasal gel
(Zicam) | Luffa opperculata 4x
Galphimia glauca 4x
Sabadilla 4x | 1% v/v |
| Sulfur | Sulfur | 0.17 mg/mL |
| Allergy Relief (RhinAllergy) | Histaminum hydrochloricum 9C HPUS | 10 mg/mL |
| Anti-viral drugs | Zanamivir | 5.5 mg/mL |
| Antibiotic, Nasal ointment | Mupirocin | 3.3 mg/mL |
| Antibacterial, systemic | Tobramycin | 4 µg/mL |
| Transport Media | Remel M4 (R12503) | 100% |
| | Remel M4RT (R12591) | 100% |
| | Copan UTM-RT (UTM 330C) | 100% |
| | PrimeStore MTM (MTM-LH102) | 100% |
| Substance | Specific Active Ingredient | Test
Concentration |
| | Merit Medical Cultura Media | 100% |
| | Neuronics VTM | 100% |
| | Azer UTM | 100% |
| | Bartels FlexTrans | 100% |
| | S2 VTM | 100% |
| Diluents | Ethanol | 10% v/v |
| | DMSO | 10% v/v |
| | Methanol | 10% v/v |
| | Chloroform | 10% v/v |
| | DMF | 10% v/v |

Table 9. Results of Potentially Interfering Substances Testing on the BioFire COVID-19 Test 2

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Reproducibility

Assay reproducibility was evaluated by preparing three samples using pooled negative clinical NPS matrix. Two samples were spiked with SARS-CoV-2 at either moderate positive (3× LoD) or low positive (1× LoD) concentrations; a third sample was not spiked (negative sample). Six replicates of each sample were tested at three locations over five different days, providing a total of 90 replicate test results per sample. Reproducibility was evaluated using both FilmArray 2.0 instruments and FilmArray Torch modules. BioFire COVID-19 Test pouch reagent lots were rotated daily. In total, 270 valid test results were obtained.

The primary assessment of reproducibility was based on a comparison of the observed test results ('Detected') 'Not Detected') to the expected test results. The detection rate and percent agreement between observed and expected test results are shown in Table 10; the expected percent agreement was ≥ 95%. Based on the percent agreement between observed and expected test results and the variability in Tm (standard deviations ≤0.3 ℃, CV ≤0.4%), the reproducibility data demonstrates that the BioFire COVID-19 Test 2 can provide accurate and highly reproducible test results in the context of multiple variables that may be expected in a clinical testing environment.

| Analyte
(Source / ID) | Test
Concentration
×LoD (GE/mL) | Expected
Result | Detection Rate (n/N)
(% Agreement with Expected Result) | | | |
|--------------------------------------------------------|---------------------------------------------|--------------------|------------------------------------------------------------|-----------------|-----------------|--------------------------------------|
| | | | Site 1 | Site 2 | Site 3 | All Sites
[95% Conf.
Interval] |
| SARS-CoV-2
USA-
WA1/2020
(BEI / NR-
52286) | Moderate
Positive
3× LoD
(9.9E+02) | Detected | 30/30
(100%) | 30/30
(100%) | 30/30
(100%) | 90/90
(100%)
[95.9-100%] |
| | Low Positive
1× LoD
(3.3E+02) | Detected | 30/30
(100%) | 30/30
(100%) | 30/30
(100%) | 90/90
(100%)
[95.9-100%] |

		Table 10. Summary of BioFire COVID-19 Test 2 Reproducibility Results

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| Negative
(No Analyte) | Not
Detected | 30/30
(100%) | 28/30
(93.3%) | 29/30
(96.7%) | 87/90a
(96.7%)
[90.7-98.9%] |

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

a Clinical NPS specimens in transport medium were collected during the COVID-19 outbreak and therefore SARS-CoV-2 at levels below LoD may have gone undetected during characterization of the pooled sample matrix.

Specimen Storage

Ten samples were contrived by spiking pooled clinical NPS specimens with SARS-CoV-2 at three concentrations relative to the limit of detection (LoD) (3× LoD, 5× LoD, and 10× LoD). Each contrived specimen was tested immediately as a control, then aliquoted and stored under six different storage conditions. Storage conditions were selected based on the desired claims for specimen storage and included one additional time point beyond the desired claim. A summary of detection rates under each storage condition is shown in Table 11. These data support the claimed storage conditions for NPS specimens as up to 4 hours at 15-30°C, up to 3 days at 2-8°C, and up to 30 days at