BIOFIRE SPOTFIRE Respiratory (R) Panel

K213954 · Biofire Diagnostics · QOF · Feb 3, 2023 · Microbiology

Device Facts

• Record ID: K213954
• Device Name: BIOFIRE SPOTFIRE Respiratory (R) Panel
• Applicant: Biofire Diagnostics
• Product Code: QOF · Microbiology
• Decision Date: Feb 3, 2023
• Decision: SESE
• Submission Type: Dual Track
• Regulation: 21 CFR 866.3981
• Device Class: Class 2

Intended Use

The BIOFIRE® SPOTFIRE® Respiratory (R) Panel (SPOTFIRE R Panel) is a multiplexed polymerase chain reaction (PCR) test intended for use with the BIOFIRE® System for the simultaneous, qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swab (NPS) specimens obtained from individuals with signs and symptoms of respiratory tract infection, including COVID-19. The following organism types are identified and differentiated using the SPOTFIRE R Panel: Viruses: Adenovirus Coronavirus (seasonal) Coronavirus SARS-CoV-2 Human metapneumovirus Human rhinovirus/enterovirus Influenza A virus Influenza A virus A/H1-2009 Influenza A virus A/H3 Influenza B virus Parainfluenza virus Respiratory syncytial virus Bacteria: Bordetella parapertussis Bordetella pertussis Chlamydia pneumoniae Mycoplasma pneumoniae Nucleic acids from the viral and bacterial organisms identified by this test are generally detectable in NPS specimens during the acute phase of infection and identification 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 an NPS specimen. Positive results do not rule out coinfection with other organisms. The agent(s) detected by the SPOTFIRE R Panel 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.

Device Story

The BIOFIRE SPOTFIRE R Panel is a multiplexed PCR-based diagnostic test for identifying 15 respiratory pathogens in nasopharyngeal swab (NPS) specimens. Designed for CLIA-waived environments, it is operated by minimally trained personnel in urgent care or emergency departments. The process involves loading a hydrated NPS specimen into a reagent-filled pouch, which is then inserted into the SPOTFIRE System. The system uses pneumatic bladders and pistons to automate reagent movement, nucleic acid extraction via mechanical lysis and magnetic beads, and a two-stage nested multiplex PCR. The first stage is a large-volume RT-PCR; the second stage is a singleplex nested PCR in an array. Detection is achieved via melt curve analysis using a fluorescent DNA-binding dye. A digital camera captures fluorescent images, and the system software automatically interprets results. The output provides a qualitative report for each organism, aiding clinicians in diagnosis and patient management. The device benefits patients by providing rapid (approx. 15-minute) results, facilitating timely clinical decisions.

Clinical Evidence

Clinical performance was established via a prospective multi-center study (1120 valid NPS specimens) and 542 archived specimens. Performance was compared to FDA-cleared multiplexed panels. PPA for analytes ranged from 96.0% to 100% (e.g., SARS-CoV-2 97.3%, HRV/EV 99.1%). NPA ranged from 90.6% to 100%. Instrument success rate was 99.3%. Reproducibility was confirmed across 150 replicates per analyte using minimally trained operators, showing 94-100% agreement with expected results.

Technological Characteristics

Multiplexed nested nucleic acid amplification test with melt analysis. Reagents are freeze-dried in a single-use pouch. Instrumentation includes pneumatic bladders, seal points, and Peltier devices for thermal cycling. Connectivity: Standalone system. Software: Automated interpretation of melt curve data. Materials: Standard plastic pouch components; magnetic beads for extraction. Sterilization: Not specified.

Indications for Use

Indicated for individuals of all ages exhibiting signs/symptoms of respiratory tract infection, including COVID-19. Used for simultaneous, qualitative detection of 15 viral and bacterial respiratory pathogens in nasopharyngeal swab (NPS) specimens. Not for sole diagnostic use; results must be interpreted with clinical/epidemiological data.

Regulatory Classification

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.

Special Controls

*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.

Predicate Devices

• BIOFIRE Respiratory Panel 2.1 (RP2.1) (DEN200031)

Submission Summary (Full Text)

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February 3, 2023

BioFire Diagnostics Kevin Bourzac Vice President, Regulatory & Clinical Affairs 515 Colorow Drive Salt Lake City, Utah 84108

Re: K213954

Trade/Device Name: BIOFIRE SPOTFIRE Respiratory (R) Panel Regulation Number: 21 CFR 866.3981 Regulation Name: Device to detect and identify nucleic acid targets in respiratory specimens from microbial agents that cause the SARS-CoV-2 respiratory infection and other microbial agents when in a multi-target test Regulatory Class: Class II Product Code: QOF, OEM, OOU, OTG, OZE, OZX, OZY, OZZ, OCC, NSU Dated: December 16, 2021 Received: December 17, 2021

Dear Kevin Bourzac:

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.

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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 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 ([e-mail]) or phone [phone] or 301-796-7100).

Sincerely.

Image /page/1/Picture/5 description: The image shows the text "Joseph Briggs-S" in a large, sans-serif font. The name "Joseph" is on the top line, followed by "Briggs-S" on the second line. The text is black, and the background is a light blue color.

Digitally signed by Joseph Briggs -S Date: 2023.02.03 13:34:47 -05'00'

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

Enclosure

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## Indications for Use

510(k) Number (if known) K213954

Device Name BIOFIRE® SPOTFIRE® Respiratory (R) Panel

#### Indications for Use (Describe)

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

The following organism types are identified and differentiated using the SPOTFIRE R Panel:

Viruses: Adenovirus Coronavirus (seasonal) Coronavirus SARS-CoV-2 Human metapneumovirus Human rhinovirus/enterovirus Influenza A virus Influenza A virus A/H1-2009 Influenza A virus A/H3 Influenza B virus Parainfluenza virus Respiratory syncytial virus

Bacteria: Bordetella parapertussis Bordetella pertussis Chlamydia pneumoniae Mycoplasma pneumoniae

Nucleic acids from the viral and bacterial organisms identified by this test are generally detectable in NPS specimens during the acute phase of infection and identification 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 an NPS specimen. Positive results do not rule out coinfection with other organisms. The agent(s) detected by the SPOTFIRE R Panel 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.

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

区 Prescription Use (Part 21 CFR 801 Subpart D)

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

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# BIOFIRE® SPOTFIRE® Respiratory (R) Panel 510(k) Summary BioFire Diagnostics, LLC

## Introduction:

According to the requirements of 21 CFR 807.92, the following information provides sufficient detail to understand the basis for a determination of substantial equivalence.

## Submitted by:

BioFire Diagnostics, LLC 515 Colorow Drive Salt Lake City, UT 84108

Telephone: [phone]

Facsimile: 801-588-0507

Contact: Kevin Bourzac, Ph.D ext. 1358

Date Submitted: December 16, 2021

## Device Name and Classification:

Trade Name: BIOFIRE® SPOTFIRE® Respiratory (R) Panel

Requlation Number: 21 CFR 866.3981

Classification Name: Multi-target Respiratory Specimen Nucleic Acid Test Including SARS-CoV-2 and Other Microbial Agents

## Predicate Device:

DEN200031 - BIOFIRE® Respiratory Panel 2.1 (RP2.1)

## Intended Use:

The BIOFIRE® SPOTFIRE® Respiratory (R) Panel (SPOTFIRE R Panel) is a multiplexed polymerase chain reaction (PCR) test intended for use with the BIOFIRE® System for the simultaneous, qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swab (NPS) 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 SPOTFIRE R Panel:

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| Viruses | Bacteria |
|------------------------------|--------------------------|
| Adenovirus | Bordetella parapertussis |
| Coronavirus (seasonal) | Bordetella pertussis |
| Coronavirus SARS-CoV-2 | Chlamydia pneumoniae |
| Human metapneumovirus | Mycoplasma pneumoniae |
| Human rhinovirus/enterovirus | |
| Influenza A virus | |
| Influenza A virus A/H1-2009 | |
| Influenza A virus A/H3 | |
| Influenza B virus | |
| Parainfluenza virus | |
| Respiratory syncytial virus | |

Nucleic acids from the viral and bacterial organisms identified by this test are generally detectable in NPS 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 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.

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 an NPS specimen. Positive results do not rule out coinfection with other organisms. The agent(s) detected by the SPOTFIRE R Panel 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.

## Device Description:

The BIOFIRE® SPOTFIRE® Respiratory (R) Panel (SPOTFIRE R Panel) simultaneously identifies 15 different respiratory viral and bacterial pathogens in nasopharyngeal swabs (NPS) from individuals with signs and symptoms of respiratory tract infection (see Table 1) . The SPOTFIRE R Panel is compatible with the BIOFIRE® System, a polymerase chain reaction (PCR)-based in vitro diagnostic system for infectious disease testing. The SPOTFIRE System Software executes the SPOTFIRE R Panel test and reports the test results. The SPOTFIRE R Panel was designed to be used in CLIA-waived environments.

| Viruses | Bacteria |
|------------------------------|--------------------------|
| Adenovirus | Bordetella parapertussis |
| Coronavirus (seasonal) | Bordetella pertussis |
| Coronavirus SARS-CoV-2 | Chlamydia pneumoniae |
| Human metapneumovirus | Mycoplasma pneumoniae |
| Human rhinovirus/enterovirus | |
| Influenza A virus | |
| Influenza A virus A/H1-2009 | |
| Influenza A virus A/H3 | |
| Influenza B virus | |
| Parainfluenza virus | |
| Respiratory syncytial virus | |

| Table 1. Analytes Detected by the SPOTFIRE (R) Panel | | |
|------------------------------------------------------|--|--|
| | | |

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A test is initiated by loading Hydration into one port of the SPOTFIRE R Panel pouch and a NPS specimen mixed with the provided Sample Buffer into the other port of the SPOTFIRE R Panel pouch and placing it in the SPOTFIRE System. The pouch contains all of the reagents required for specimen testing and analysis in a freezedried format; the addition of Hydration and Sample/Buffer Mix rehydrates the reagents. After the pouch is prepared, the SPOTFIRE System Software guides the user through the pouch into the instrument, scanning the pouch barcode, entering the sample identification, and initiating the run.

The SPOTFIRE System contains coordinated systems 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 in order to deliver the rehydrated reagents into the appropriate times. Two Pettier devices control heating and cooling of the PCR reactions and the melt curve analysis.

Nucleic acid extraction occurs within the SPOTFIRE R Panel pouch using mechanical lysis followed by purification using standard magnetic bead technology. After extracting and purifying nucleic acids from the unprocessed sample, the SPOTFIRE system performs a nested multiplex PCR that is executed in two stages. During the first stage, the SpotFire system s a single, large volume, highly multiplexed reverse transcription PCR (tt-PCR) reaction. 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 Diagnostics). The solution is then distributed to each well of the array. 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, or nested PCR, is performed in singleplex fashion in each well of 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 2nd stage PCR captures fluorescent images of the PCR reactions and software interprets the data.

The SPOTFIRE System Software automatically interprets the results of each DNA melt curve analysis and combines the data with the results of the internal pouch controls to provide a test result for each organism on the panel.

## Substantial Equivalence:

The SPOTFIRE R Panel is substantially equivalent to the BioFire Respiratory Panel 2.1 (BioFire RP2.1) (DEN200031), which was cleared on March 17, 2021 and determined to be a Class II device under the classification code 21 CFR 866.3981.

| Element | Predicate: BioFire RP2.1 (DEN200031) | New Device: SPOTFIRE R Panel |
|--------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Intended Use | The BioFire Respiratory Panel 2.1 (RP2.1)<br>is a PCR-based multiplexed nucleic acid<br>test intended for use with the BioFire®<br>FilmArray® 2.0 or BioFire® FilmArray®<br>Torch Systems for the simultaneous<br>qualitative detection and identification of<br>multiple respiratory viral and bacterial<br>nucleic acids in nasopharyngeal swabs<br>(NPS) obtained from individuals suspected<br>of respiratory tract infections, including<br>COVID-19 | The SPOTFIRE R Panel is a PCR-based<br>multiplexed nucleic acid test intended for<br>use with the SPOTFIRE System for the<br>simultaneous, qualitative detection and<br>identification of multiple respiratory viral and<br>bacterial nucleic acids in nasopharyngeal<br>swab (NPS) specimens obtained from<br>individuals with signs and symptoms of<br>respiratory tract infection, including COVID-<br>19. |
| Specimen Types | Nasopharyngeal swab in transport media or<br>saline | Nasopharyngeal swab in transport media |
| Organisms detected | Viruses<br>Adenovirus<br>Coronavirus 229E<br>Coronavirus HKU1<br>Coronavirus NL63<br>Coronavirus OC43 | Same, except:<br>No assay for Influenza A subtype H1<br>Coronavirus 229E/HKU1/NL63/OC43<br>combined call (coronavirus seasonal) |

A table comparing the SPOTFIRE R Panel to the BioFire RP2.1 is provided in Table 2.

| Table 2. Similarities and differences between the BioFire RP2.1 and the SPOTFIRE R Panel | | |
|------------------------------------------------------------------------------------------|--|--|
| | | |

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| | Severe Acute Respiratory Syndrome<br>Coronavirus 2 (SARS-CoV-2)<br>Human Metapneumovirus<br>Human Rhinovirus/Enterovirus<br>Influenza A<br>Subtypes: H1, H3 and H1-2009<br>Influenza B<br>Parainfluenza Virus 1<br>Parainfluenza Virus 2<br>Parainfluenza Virus 3<br>Parainfluenza Virus 4<br>Respiratory Syncytial Virus<br>Bacteria<br>Bordetella parapertussis<br>Bordetella pertussis<br>Chlamydia pneumoniae<br>Mycoplasma pneumoniae | Combined Parainfluenza call for serotypes<br>1-4 |
|--------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------|
| Analytes | DNA/RNA | Same |
| Technological Principles | Highly multiplexed nested nucleic acid<br>amplification test with melt analysis | Same |
| Instrumentation | BioFire FilmArray 2.0 or BioFire FilmArray<br>Torch Systems | SPOTFIRE System (this submission) |
| Time to result | About 1 hour | About 15 minutes |
| Reagent Storage | Room Temperature | Same |
| Test Interpretation | Automated test interpretation and reporting.<br>User cannot access raw data. | Same |
| Controls | Two controls are included in each reagent<br>pouch to control for sample processing and<br>both stages of PCR and melt analysis. | Same |
| User complexity | Moderate | Low (CLIA-waived) |
| | Defines panel-specific parameters,<br>instrument protocols and report<br>requirements. | Same |
| Panel Software Functions | Analyzes processed image data<br>(fluorescence and temperature data) and<br>provides test results. | Same |

{8}------------------------------------------------

## Summary of Performance Data

## Clinical Performance

### Prospective Clincal Evaluation

The clinical performance (encompassing both accuracy and ease of use) of the SPOTFIRE R Panel was established during a prospective multi-center study that was further supplemented with archived specimens. Five geographically distinct urgent care or emergency department study sites representative of the intended use setting (four in the US and one in the UK) participated in these studies from December 2020 to June 2021. All SPOTFIRE R Panel testing was performed according to the manufacturer's instructions by minimally trained operators. No hands-on training was provided to the SPOTFIRE R Panel test operators; rather, training was limited to written materials (i.e. quick reference guides) that were intended to be included with the BioFire SPOTFIRE System.

A total of 1215 NPS specimens were enrolled from consented volunteers or obtained as residual leftover specimens from subjects of all ages for the prospective clinical study; 95 of these NPS specimens were excluded. The most common reason for specimen exclusion was that a valid SPOTFIRE R Panel test was not obtained due to an invalid SPOTFIRE R Panel test. The final data set consisted of 1120 NPS specimens. Across the five study sites, 259 NPS specimens were initially collected and immediately frozen for later testing at the source study site. The remaining 861 NPS specimens were collected and tested fresh (without freezing). No difference in performance was observed when fresh and frozen specimen results were compared. Therefore, the data collected from 259 valid frozen specimens are combined with data from the valid 861 fresh specimens for all analyses.

Table 3 provides a summary of demographic information for the specimens included in the study.

| | Category | Prospective NPS Specimens |
|-------|-------------|---------------------------|
| Sex | Male | 587 (52.4%) |
| | Female | 533 (47.6%) |
| Age | ≤5 years | 457 (40.8%) |
| | 6-18 years | 258 (23.0%) |
| | 19-40 years | 160 (14.3%) |
| | 41-60 years | 147 (13.1%) |
| | 61+ years | 98 (8.8%) |
| Total | | 1120 |

Table 3. Demographic Summary for Prospective SPOTFIRE R Panel Clinical Evaluation

The performance of the SPOTFIRE R Panel was evaluated by comparing the test results with those from FDAcleared multiplexed respiratory pathogen panels. The prospective study is summarized in Table 4. Positive percent agreement (PPA) for each analyte was calculated as 100% x (TP / (TP + FN)). True positive (TP) indicates that both the SPOTFIRE R Panel and the comparator method had a positive result for the specific analyte, and false negative (FN) indicates that the SPOTFIRE R Panel was negative while the comparator result was positive. Negative percent agreement (NPA) was calculated as 100% x (TN / TN + FP)). True negative (TN) indicates that both the SPOTFIRE R Panel and the comparator method had negative results, and false positive (FP) indicates that the SPOTFIRE R Panel was positive while the comparator method was negative. The exact binomial two-sided 95% confidence interval (95%Cl) was calculated. Investigations of discrepant results are summarized in the footnotes.

{9}------------------------------------------------

| Table 4. SPOTFIRE R Panel Clinical Performance Summary for NPS Specimens | | | | | | |
|--------------------------------------------------------------------------|----------------------------|------|------------|----------------------------|------|------------|
| SPOTFIRE R Panel Analyte | Positive Percent Agreement | | | Negative Percent Agreement | | |
| | TP/(TP + FN) | % | 95%CI | TN/(TN + FP) | % | 95%CI |
| Viruses | | | | | | |
| Adenovirusa | 32/33 | 97.0 | 84.7-99.5% | 1058/1082 | 97.8 | 96.7-98.5% |
| Coronavirus SARS-CoV-2b | 71/73 | 97.3 | 90.5-99.2% | 1031/1037 | 99.4 | 98.7-99.7% |
| Coronavirus (seasonal)c | 101/102 | 99.0 | 94.7-99.8% | 1000/1013 | 98.7 | 97.8-99.2% |
| Human metapneumovirus | 1/1 | 100 | - | 1114/1114 | 100 | 99.7-100% |
| Human rhinovirus/enterovirusd | 345/348 | 99.1 | 97.5-99.7% | 695/767 | 90.6 | 88.3-92.5% |
| Influenza A virus | 0/0 | - | - | 1115/1115 | 100 | 99.7-100% |
| Influenza A virus A/H1-2009 | 0/0 | - | - | 1115/1115 | 100 | 99.7-100% |
| Influenza A virus A/H3 | 0/0 | - | - | 1115/1115 | 100 | 99.7-100% |
| Influenza B virus | 0/0 | - | - | 1110/1110 | 100 | 99.7-100% |
| Parainfluenza viruse | 96/98 | 98.0 | 92.9-99.4% | 1006/1017 | 98.9 | 98.1-99.4% |
| Respiratory syncytial virusf | 26/27 | 96.3 | 81.7-99.3% | 1086/1088 | 99.8 | 99.3-99.9% |
| Bacteria | | | | | | |
| Bordetella parapertussis | 0/0 | - | - | 1110/1110 | 100 | 99.7-100% |
| Bordetella pertussis | 0/0 | - | - | 1115/1115 | 100 | 99.7-100% |
| Chlamydia pneumoniae | 0/0 | - | - | 1115/1115 | 100 | 99.7-100% |
| Mycoplasma pneumoniae | 0/0 | - | - | 1115/1115 | 100 | 99.7-100% |

Adenovirus was not detected in the single FN specimen upon SPOTFIRE R Panel retest. Adenovirus was detected in 21/24 FP specimens using an additional molecular method.

SARS-Col-2 was detected in 1/2 FN specimens upon SPOTFIRE R Panel retest. SARS-CoV-2 was detected in 2/6 FP specimens using an additional molecular method.

Seasonal coronavirus (229E/HKU1/NL63/OC43) was detected in the single FN specimen upon SPOTFIRE R Panel retest. Seasonal coronavirus (229E/HKU1/NL63/OC43) was detected in 8/13 FP specimens using an additional mothod.

4 Human rhinovirus/enterovirus was detected in 1/3 FN specimens upon SPOTFIRE R Panel retest. Human rhinovirus was detected in 48/72 FP specimens using an additional molecular method.

Parainfluenza virus was detected in both FN specimens during discrepancy investigation upon SPOTFIRE R Parainfluenza virus was detected in 9/11 FP specimens using an additional molecular method.

f Respiratory syncytial virus was detected in the single FN specimen upon SPOTFIRE R Panel retest. Respiratory syncytial virus was detected in 1/2 FP specimens using an additional molecular method.

The overall success rate for initial specimen tests was 96.7% (1158/1198). Eight tests (8/1198; 0.7%) did not complete on the initial test attempt, resulting in an instrument success rate of 99.3% (1190/1198) for initial specimen tests. Retests were not possible due to insufficient specimen volume. Of the 1190 tests that successfully produced a completed run on the initial test, 1158 had valid internal process controls. This represents a 97.3% (1158/1190) success rate for internal process controls in completed runs in the initial specimen tests.

### Testing of Preselected Archived Specimens

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

A total of 562 frozen archived NPS specimens were obtained from 15 external laboratories world-wide and retrospectively tested at the four US clinical sites. Of these, 542 NPS specimens had valid results that were included in performance analysis. The analyte composition of the archived specimens was confirmed using the same comparator methods as the prospective study (described above) for the analyte result to be included in the performance analysis.

The specimens were randomized such that the users performing both the confirmation and the SPOTFIRE R Panel testing were blinded to the expected test result. A summary of the available demographic information of the tested specimens is provided in Table 5, and the results of the SPOTFIRE R Panel performance for these archived specimens is shown in Table 6.

{10}------------------------------------------------

| Table 5. Demographic Summary for Valid Archived NPS Specimens | | |
|---------------------------------------------------------------|--|--|
| | | |

| Category | Archived NPS Specimens |
|----------|------------------------|
| Sex | Male 254 (46.9%) |
| | Female 185 (34.1%) |
| | Unknown 103 (19.0%) |
| Age | ≤5 years 234 (43.2%) |
| | 6-18 years 98 (18.1%) |
| | 19-40 years 36 (6.6%) |
| | 41-60 years 35 (6.5%) |
| | 61+ years 39 (7.2%) |
| | Unknown 100 (18.5%) |
| Total | 542 |

#### Table 6. SPOTFIRE R Panel Archived Performance Summary for NPS Specimens

| | Positive Percent Agreement | | | Negative Percent Agreement | | |
|-------------------------------|----------------------------|------|------------|----------------------------|------|------------|
| SPOTFIRE R Panel Analyte | TP/(TP + FN) | % | 95% CI | TN/(TN + FP) | % | 95% CI |
| Viruses | | | | | | |
| Adenovirusa | 31/31 | 100 | 89.0-100% | 439/453 | 96.9 | 94.9-98.2% |
| Coronavirus SARS-CoV-2 | 0/0 | - | | 0/0 | - | |
| Coronavirus (seasonal)b | 95/96 | 99.0 | 94.3-99.8% | 381/388 | 98.2 | 96.3-99.1% |
| Human metapneumovirusc | 32/33 | 97.0 | 84.7-99.5% | 451/451 | 100 | 99.2-100% |
| Human rhinovirus/enterovirusd | 29/30 | 96.7 | 83.3-99.4% | 439/454 | 96.7 | 94.6-98.0% |
| Influenza A viruse | 58/59 | 98.3 | 91.0-99.7% | 423/423 | 100 | 99.1-100% |
| Influenza A virus A/H1-2009e | 31/32 | 96.9 | 84.3-99.4% | 450/450 | 100 | 99.2-100% |
| Influenza A virus A/H3 | 27/27 | 100 | 87.5-100% | 455/455 | 100 | 99.2-100% |
| Influenza B virus | 30/30 | 100 | 88.6-100% | 28/28 | 100 | 87.9-100% |
| Parainfluenza virusf | 116/118 | 98.3 | 94.0-99.5% | 359/366 | 98.1 | 96.1-99.1% |
| Respiratory syncytial virusg | 37/37 | 100 | 90.6-100% | 440/447 | 98.4 | 96.8-99.2% |
| Bacteria | | | | | | |
| Bordetella parapertussish | 24/25 | 96.0 | 80.5-99.3% | 33/33 | 100 | 89.6-100% |
| Bordetella pertussisi | 27/28 | 96.4 | 82.3-99.4% | 452/456 | 99.1 | 97.8-99.7% |
| Chlamydia pneumoniaej | 30/30 | 100 | 88.6-100% | 452/454 | 99.6 | 98.4-99.9% |
| Mycoplasma pneumoniaek | 33/33 | 100 | 89.6-100% | 446/451 | 98.9 | 97.4-99.5% |

ª Adenovirus was detected in 6/12 FP specimens during discrepancy investigation: one was detected by standard of care and five were detected using an additional mothod; two additional FP specimens were unable to be investigated.

b The single FN specimen was unable to be investigated. Seasonal coronavirus (229E/HKU1/NL63/OC43) was detected in 3/6 FP specimens during discrepancy investigation using an additional molecular method; one additional FP specimen was unable to be investigated.

° Human metapneumovirus was detected in the single FN specimen by standard of care.

d The single FN specimen was unable to be investigated. Human rhinovirus/enterovirus was detected in 4/14 FP specimens during discrepancy investigation using an additional mothod; one additional FP specimen was unable to be investigated.

· Influenza A virus A/H1-2009 was detected in the single FN specimen by standard of care.

' Parainfluenza virus was detected in the single FN specimen by standard of care; one additional FN specimen was unable to be investigated. Parainfluenza virus was detected in all seven FP specimens during discrepancy

investigation: six were detected by standard of care and one was detected using an additional mothod. 9 Respiratory syncytial virus was detected in 4/6 FP specimens during discrepancy investigation using an additional

molecular method; one additional FP specimen was unable to be investigated.

b Bordetella parapertussis was detected in the single FN specimen by standard of care.

i Bordetella pertussis was detected in the single FN specimen by standard of care. Bordetella pertussis was detected in 3/4 FP specimens by standard of care.

i Chlamydia pneumoniae was detected in both FP specimens by standard of care.

* Mycoplasma pneumoniae was detected in all five FP specimens during discrepancy investigation: four were detected by standard of care and one was detected using an additional molecular method.

{11}------------------------------------------------

## Analytical Performance Characteristics

### Limit of Detection

The limit of detection (LoD) for SPOTFIRE R Panel analytes was estimated by testing dilutions of contrived samples containing known concentrations of organism in artificial nasopharyngeal swab matrix in VTM. The LoD concentrations were confirmed by testing at least 20 replicates at the estimated LoD. Confirmation of LoD required detection in at least 95% of replicates tested. The confirmed LoD concentrations for the SPOTFIRE R Panel are listed in Table 7.

| Analyte | Isolate<br>Source ID | Viruses | LoD Concentrationa |
|--------------------------------------|-------------------------------------------------------------------|------------------------------------------|------------------------------------------|
| Adenovirus | Species A Serotype 31<br>ZeptoMetrix 0810073CF | | 4.1E-03 TCID50/mL<br>(1.0E+02 copies/mL) |
| | Species B Serotype 3<br>ZeptoMetrix 0810062CF | | 8.0E-01 TCID50/mL<br>(8.4E+02 copies/mL) |
| | Species C Serotype 2<br>WHO I.S. NIBSC 16-324 | | 8.2E+02 IU/mLb<br>(8.2E+02 copies/mLb) |
| | Species D Serotype 37<br>ZeptoMetrix 0810119CF | | 1.1E-02 TCID50/mL<br>(4.5E+02 copies/mL) |
| | Species E Serotype 4<br>ZeptoMetrix 0810070CF | | 1.8E-02 TCID50/mL<br>(1.0E+04 copies/mL) |
| | Species F Serotype 41<br>ZeptoMetrix 0810085CF | | 1.4E-02 TCID50/mL<br>(1.0E+02 copies/mL) |
| | 229E<br>ATCC VR-740 | | 6.5E-01 TCID50/mL<br>(1.1E+01 copies/mL) |
| | HKU1<br>Clinical Specimenc | | 1.8E+04 copies/mL |
| Coronavirus (seasonal) | OC43<br>ZeptoMetrix 0810024CF | | 1.6E-02 TCID50/mL<br>(6.3E+01 copies/mL) |
| | NL63<br>ZeptoMetrix 0810228CF | | 2.5E-03 TCID50/mL<br>(4.7E+00 copies/mL) |
| | USA-WA1/2020<br>(heat inactivated)<br>ATCC VR-1986HK | | 1.1E-01 TCID50/mL<br>(2.5E+02 copies/mL) |
| | | | |
| Coronavirus SARS-CoV-2 | A1-16 Iowa 10/2003<br>ZeptoMetrix 0810161CF | | 3.2E+00 TCID50/mL<br>(2.4E+02 copies/mL) |
| Human metapneumovirus | B1-3 Peru2-2002<br>ZeptoMetrix 0810156CF | | 2.5E-01 TCID50/mL<br>(5.4E+02 copies/mL) |
| | A2-27 Iowa A/2004<br>ZeptoMetrix 0810164CF | | 5.8E-01 TCID50/mL<br>(1.8E+03 copies/mL) |
| | B2-18 IA18-2003<br>ZeptoMetrix 0810162CF | | 2.0E+00 TCID50/mL<br>(7.7E+02 copies/mL) |
| | Human Rhinovirus 1A<br>ZeptoMetrix 0810012CFN | | 2.1E-01 TCID50/mL<br>(1.1E+00 copies/mL) |
| Human rhinovirus/enterovirus | Enterovirus D68 US/MO/14-18947<br>ATCC VR-1823 | | 1.1E+01 TCID50/mL<br>(5.4E+01 copies/mL) |
| Influenza A virus<br>Subtype H1-2009 | Influenza A H1N1 pdm<br>A/Michigan/45/15<br>ZeptoMetrix 0810538CF | | 8.2E-01 TCID50/mL<br>(3.4E+02 copies/mL |
| | | | |
| Influenza A virus<br>Subtype H3 | Influenza A H3N2<br>A/Hong Kong/4801/14<br>ZeptoMetrix 0810526CF | | 8.6E-01 TCID50/mL<br>(3.4E+02 copies/mL) |
| | | | |
| Influenza B virus | B/Florida/02/06 (Victoria Lineage)<br>ZeptoMetrix 0810037CF | | 3.3E-02 TCID50/mL<br>(1.6E+02 copies/mL) |
| | B/Nevada/03/2011 (Victoria Lineage)<br>BEI NR-44023 | | 1.6E+00 CEID50/mL<br>(4.3E+00 copies/mL) |
| | B/Florida/04/06 (Yamagata Lineage)<br>ZeptoMetrix 0810255CF | | 4.0E-01 TCID50/mL<br>(3.2E+01 copies/mL) |
| | | | |
| Parainfluenza virus | Serotype 1<br>ZeptoMetrix 0810014CF | | 4.6E+00 TCID50/mL<br>(1.4E+03 copies/mL) |
| | Serotype 2<br>ZeptoMetrix 0810015CF | | 1.4E+01 TCID50/mL<br>(1.6E+02 copies/mL) |
| | Serotype 3<br>ZeptoMetrix 0810016CF | | 2.6E+01 TCID50/mL<br>(6.1E+01 copies/mL) |
| Analyte | Isolate<br>Source ID | LoD Concentrationa | |
| Respiratory syncytial virus | Serotype 4<br>ZeptoMetrix 0810060CF | 2.0E+02 TCID50/mL<br>(1.1E+03 copies/mL) | |
| Respiratory syncytial virus | Type A 2006<br>ZeptoMetrix 0810040ACF | 6.2E-02 TCID50/mL<br>(2.2E+01 copies/mL) | |
| Respiratory syncytial virus | Type B 3/2015 Isolate #1<br>ZeptoMetrix 0810479CF | 2.8E-02 TCID50/mL<br>(2.4E+01 copies/mL) | |
| Bacteria | | | |
| Chlamydia pneumoniae | AR-39<br>ATCC 53592 | 2.0E+01 IFU/mL<br>(1.4E+02 copies/mL) | |
| Mycoplasma pneumoniae | M129<br>ZeptoMetrix 0801579 | 1.0E+01 CCU/mL<br>(2.1E+03 copies/mL) | |
| Bordetella parapertussis | E595<br>ZeptoMetrix 0801462 | 4.0E+01 CFU/mL | |
| Bordetella pertussis | A639<br>ZeptoMetrix 0801459 | 3.3E+02 CFU/mL<br>(3.8E+02 copies/mL) | |

#### Table 7. Limit of Detection (LoD) for the SPOTFIRE R Panel Analytes

{12}------------------------------------------------

' LoD concentration may vary from what is listed based on the accuracy and precision of the quantification method.

* U = International Units: BioFire Diagnostics quantitative real-time PCR to demonstrate that 8,2E+02 U/ml=8,2E+02 conies/ml

· Testing for Coronavirus HKU utilized a clinicality of a cultured isolate. Viral concentration was determined in RNA copies/mL by quantitative real-time RT-PCR.

NOTE: LoD concentrations in copies/mL in Error! Reference source not found. above are based on extraction of nucleic acids from isolate cultures followed by quantitative real-time PCR (qPCR) or digital PCR (dPCR). The accuracy of concentrations may be affected by extraction efficiency, standard curve accuracy (qPCR only), assay conditions, inhibitors, and/or sequence variance. The quantification has not been compared to a reference material or other quantification methods.

NOTE: LoD concentrations of cultured viruses provided in units of TCIDso (50% Tissue Culture Infectious Dose) or CEIDso (50% Chicken Embryo Infectious Dose) are not a direct count of viral particles or nucleic acid, but an indirect measure of viral concentration based on infectivity and cytotoxicity. TCIDs/mL and CEIDso/mL will therefore vary depending on technique and methodology (including cell type, culture media and conditions, cytotoxicity of the virus, etc.). It is not appropriate to make determinations on relative sensitivity of detection for different cultures and/or different molecular assays based on LoD values measured in TCID50/mL or CEID50/mL.

### Analytical Reactivity (Inclusivity)

The analytical reactivity (inclusivity) of the SPOTFIRE R Panel assessed by testing viral and bacterial isolates that represented clinical and genetic diversity and included the available phylogenetic, geographic, and temporal diversity of each SPOTFIRE R Panel analyte. Isolates were tested in triplicate at concentrations near the LoD.

A summary of analytical reactivity is included in Table 8 to Table 19.

NOTE: Influenza A assays will react variably with non-human influenza A viruses and rarely encountered human influenza A viruses that are not H1-2009 or H3; generally producing Uncertain: Influenza A virus or Influenza A virus (No Subtype Identified) results.

NOTE: The SPOTFIRE R Panel assays may react with vaccines that contain specific segments of the pathogen genome or full genome or vaccines containing attenuated/inactivated pathogen, including vaccines for SARS-CoV-2, influenza A (various subtypes), influenza B, poliovirus (human rhinovirus/enterovirus), and Bordetella pertussis. Care should be taken to minimize contamination of samples with vaccines, and clinical history of vaccine administration should be considered in the interpretation of results, particularly for vaccines administered by nasal spray.

{13}------------------------------------------------

| Table 8. Summary of Reactivity to Adenovirus Isolates | | | | |
|-------------------------------------------------------|-------|-----------------------|----------------------------|---------------------|
| Species | Type | Source/Isolate ID | [Strain/Location/Year] | Result |
| A | 12 | ATCC VR-863 | [Huie/Massachusetts] | |
| A | 18 | ATCC VR-19 | [Washington DC/1954] | |
| A | 31 | ZeptoMetrix 0810073CF | - | |
| A | 3 | ZeptoMetrix 0810062CF | - | |
| A | 7 | ATCC<br>VR-7 | [Gomen/California/1954] | |
| A | 7a | ZeptoMetrix 0810021CF | - | |
| A | 7d/d2 | UIRF | [Iowa/2001] | |
| B | 11 | ATCC VR-12 | [Slobitski/Massachusetts] | |
| B | 14 | ATCC VR-15 | [De Wit/Netherlands/1955] | |
| B | 16 | ATCC VR-17 | [CH.79/Saudi Arabia/1955] | |
| B | 21 | ATCC VR-1833 | [128/Saudi Arabia/1956] | |
| B | 34 | ATCC VR-716 | [Compton/1972] | |
| B | 35 | ATCC VR-718 | [Holden] | |
| B | 50 | ATCC VR-1602 | [Wan, RIVM no. 88-1773] | Adenovirus Positive |
| C | 1 | ZeptoMetrix 0810050CF | - | |
| C | 2 | W.H.O NIBSC 16/324 | - | |
| C | 5 | ZeptoMetrix 0810020CF | - | |
| C | 6 | ATCC VR-6 | [Tonsil 99] | |
| D | 8 | ZeptoMetrix 0810069CF | - | |
| D | 20 | ZeptoMetrix 0810115CF | [KB] | |
| D | 37 | ZeptoMetrix 0810119CF | - | |
| E | 4 | ZeptoMetrix 0810070CF | - | |
| E | 4 | ATCC VR-1572 | [RI-67/Missouri/1952-1953] | |
| E | 4a | UIRF | [S.Carolina/2004] | |
| F | 40 | NCPV 0101141v | - | |
| F | 40 | ZeptoMetrix 0810084CF | - | |
| F | 41 | ATCC VR-930 | [Tak (73-3544)] | |
| F | 41 | ZeptoMetrix 0810085CF | [Tak] | |

#### Table 9. Summary of Reactivity to Bordetella parapertussis Isolates

| Source/Isolate ID | Strain/Location/Year | Result |
|-----------------------------------|----------------------|--------|
| ZeptoMetrix 0801462 | [E595] | |
| ATCC 9305 | [517] | |
| ATCC 53892 | [PT28G] | |
| ATCC 53893 | [PT 26/28G] | |
| ATCC 15237 | [NCTC 10853] | |
| ATCC 15311 | [NCTC 5952] | |
| ATCC BAA-587 | [12822/Germany/1993] | |
| ZeptoMetrix 0801461 | [A747] | |
| ZeptoMetrix 0801643 | [C510] | |
| ZeptoMetrix 0801644 | [E838] | |
| Bordetella parapertussis Positive | | |

#### Table 10. Summary of Reactivity to Bordetella pertussis Isolates

| Source/Isolate ID | Strain/Location/Year | Result |
|---------------------|------------------------|-------------------------------|
| ZeptoMetrix 0801459 | [A639] | |
| ATCC 10380 | [10-536] | |
| ATCC 51445 | [CNCTC Hp 12/63 [623]] | |
| ATCC 8467 | [F] | |
| ATCC 9340 | [5 [17921] | Bordetella pertussis Positive |
| ATCC 9797 | [18323 [NCTC 10739] | |
| ATCC BAA-1335 | [MN2531] | |
| ATCC BAA-589 | [Tohama] | |
| ZeptoMetrix 0801460 | [E431] | |

#### Table 11. Summary of Reactivity to Chlamydia pneumoniae Isolates

| Source/Isolate ID | Strain/Location/Year | Result |
|-------------------|-----------------------------------|----------------------------------|
| ATCC 53592 | [AR-39] | |
| ATCC VR-1310 | [CWL-029] | |
| ATCC VR-1356 | [TWAR strain 2023] | |
| ATCC VR-1360 | [CM-1] | Chlamydia pneumoniae<br>Positive |
| ATCC VR-1435 | [J-21] | |
| ATCC VR-1452 | [A03] | |
| ATCC VR-2282 | [TWAR strain, TW-183/Taiwan/1965] | |

#### Table 12. Summary of Reactivity to Coronavirus Isolates

| Type | Source/Isolate ID | Strain/Location/Year | Result |
|------|-----------------------|----------------------|------------------------|
| NL63 | ZeptoMetrix 0810228CF | - | Coronavirus (seasonal) |

{14}------------------------------------------------

| Type | Source/Isolate ID | Strain/Location/Year | Result |
|--------------|---------------------------------------|-----------------------------|------------------------------------|
| 229E | BEI NR-470 | [Amsterdam/2003] | Positive |
| | ATCC VR-740 | - | |
| | ZeptoMetrix 0810229CF | - | |
| HKU1 | Clinical Specimen | [Columbus OH, 2016] | |
| | Clinical Specimen | [South Carolina/2010] | |
| | Clinical Specimen | [France/2016] | |
| | Clinical Specimen | [France/2016] | |
| OC43 | ZeptoMetrix 0810024CF | - | |
| | ATCC VR-759 | - | |
| | ATCC VR-1986HK | [USA-WA1/2020] | |
| SARS-CoV-2 a | ATCC VR-1991D | [Hong Kong/VM20001061/2020] | |
| | ATCC VR-1992D | [2019-nCoV/Italy-INMI1] | |
| | ATCC VR-1994D | [Germany/BavPat1/2020] | |
| | ATCC VR-3326D | [USA/CA_CDC_5574/2020] | |
| | BEI NR-52499 b | [England/02/2020] | |
| | BEI NR-52501 c | [Singapore/2/2020] | Coronavirus SARS-CoV-2<br>Positive |
| | BEI NR-52503 d | [USA-IL1/2020] | |
| | BEI NR-52505 e | [USA-AZ1/2020] | |
| | BEI NR-52507 f | [USA-CA3/2020] | |
| | BEI NR-52510 g | [Chile/Santiago_op4d1/2020] | |
| | BEI NR-53518 h | [New York-PV08410/2020] | |
| | LGC SeraCare AccuPlex™<br>0505-0298 i | [Omicron B.1.1.529 Variant] | |

a See Error! Reference source not found. for additional SARS-CoV-2 reactivity predictions based on in silico analysis.

9 The following reagent was deposited by Professor Maria Zambon and obtained through BEI Resources, NAID, NH: Genomic RNA from SARS-Related Coronavirus 2, Isolate England/02/2020, NR-52499.

· The following reagent was contributed by Duke-National University of Singapore, Programme in Emerging Infectious Diseases for distribution through BEI Resources, NIAID, NIH: Genomic RNA from SARS-Related Coronavirus 2, Isolate Singapore/2/2020, NR-52501.

9 The following reagent was deposited by the Centrol and Prevention and obtained through BEI Resources, NAID, NH: Genomic RNA from SARS-Related Coronavirus 2, Isolate USA-IL1/2020, NR-52503.

® The following reagent was deposited by the Centrol and Prevention and obtained through BEI Resources, NIAID, NH: Genomic RNA from SARS-Related Coronavirus 2, Isolate USA-AZ1/2020, NR-52505.

' The following reagent was deposited by the Centrol and Prevention and Prevention and obtained through BEI Resources, NAID, NH: Genomic RNA from SARS-Related Coronavirus 2, Isolate USA-CA3/2020, NR-52507.

9 The following reagent was obtained through BEI Resources, NAD, NH: Genomic RNA from SARS-Related Coronavirus 2, Isolate Chile/Santiago_qp4d1/2020, NR-52510.

h The following reagent was obtained through BEI Resornic RNA from SARS-Related Coronavirus 2, Isolate New York-PV08410/2020, NR-53518

' Recombinant alphavirus that contains the full SARS-Col-2 genome with mutations identified in the SARS-CoV-2 Omicron variant B.1.1.529.

| Species | Serotype | Source/Isolate ID | [Strain/Location/Year] | Result |
|------------------|--------------------|---------------------------|------------------------|------------------------------------------|
| Human Rhinovirus | | | | |
| | 1 | ZeptoMetrix<br>0810012CFN | [1A] | Human rhinovirus/enterovirus<br>Positive |
| | 77 | ATCC VR-1187 | [130-63] | |
| | 85 | ATCC VR-1195 | [50-525-CV54] | |
| A | 34 | ATCC VR-1365 | [137-3] | |
| | 57 | ATCC VR-1600 | [Ch47] | |
| | 7 | ATCC VR-1601 | [68-CV11] | |
| | 16 | ATCC VR-283 | [11757] | |
| | 2 | ATCC VR-482 | [HGP] | |
| | 17 | ATCC VR-1663 | [33342] | |
| | 14 | ATCC VR-284 | [1059] | |
| | 42 | ATCC VR-1950 | [56822] | |
| B | 3 | ATCC VR-483 | [FEB] | |
| | 27 | ATCC VR-1137 | [5870] | |
| | 83 | ATCC VR-1193 | [Baylor 7] | |
| Enterovirus | | | | |
| A | Enterovirus 71 | ATCC VR-1432 | [71 H] | Human rhinovirus/enterovirus<br>Positive |
| | Coxsackievirus 10 | ATCC VR-168 | [NY/1950] | |
| | Coxsackievirus 9 | ZeptoMetrix 0810017CF | - | |
| | Echovirus 11 | ZeptoMetrix 0810023CF | - | |
| B | Coxsackievirus B3 | ZeptoMetrix 0810074CF | - | |
| | Coxsackievirus B4 | ZeptoMetrix 0810075CF | - | |
| | Echovirus 6 | ZeptoMetrix 0810076CF | - | |
| | Echovirus 9 | ZeptoMetrix 0810077CF | - | |
| C | Coxsackievirus A24 | ATCC VR-583 | [DN-19/TX/1963] | |
| | Coxsackievirus A21 | ATCC VR-850 | [Kuykendall/CA/1952] | |
| D | Enterovirus D68 | ATCC VR-1823 | [US/MO/14-18947] | |

#### Table 13. Summary of Reactivity to Human Rhinovirus and Enterovirus Isolates

{15}------------------------------------------------

| Table 14. Summary of Reactivity to Human Metapneumovirus Isolates | | | | |
|-------------------------------------------------------------------|----------|-----------------------|-----------------|-----------------------------------|
| Genotype | Serotype | Source/Isolate ID | [Location/Year] | Result |
| A1 | 9 | ZeptoMetrix 0810160CF | [lowa 3/2002] | Human metapneumovirus<br>Positive |
| A1 | 16 | ZeptoMetrix 0810161CF | [lowa 10/2003] | |
| A2 | 20 | ZeptoMetrix 0810163CF | [lowa 14/2003] | |
| A2 | 27 | ZeptoMetrix 0810164CF | [lowa 27/2004] | |
| B1 | 3 | ZeptoMetrix 0810156CF | [Peru2-2002] | |
| B1 | 5 | ZeptoMetrix 0810158CF | [Peru 3/2003] | |
| B2 | 4 | ZeptoMetrix 0810157CF | [Peru 1/2002] | |
| B2 | 8 | ZeptoMetrix 0810159CF | [Peru 6/2003] | |
| B2 | 18 | ZeptoMetrix 0810162CF | [IA18-2003] | |
| B2 | Unknown | BEI NR-22232 | [TN/91-316] | |

#### Table 15. Summary of Reactivity to Influenza A Isolates

| Type | Host | Source/Isolate ID | Strain/Location/Year | Result |
|-----------|-------------|------------------------|-----------------------------------------------------------------------------|----------------------------------------------------------|
| H1N1pdm09 | Human | ZeptoMetrix 0810538CF | [Michigan/45/15] | Influenza A virus<br>(Subtype H1-2009) Positive |
| | | BEI NR-19823 | [Netherlands/2629/2009] | |
| | | BEI NR-42938 | [Georgia/F32551/2012] | |
| | | BEI NR-44345 | [Hong Kong/H090-761-V1(0)/2009] | |
| H3N2 | Human | ZeptoMetrix 0810109CFJ | [Canada/6294/2009] | Influenza A virus<br>(Subtype H3) Positive |
| | | ZeptoMetrix 0810165CF | [California/07/2009] | |
| | | ZeptoMetrix 0810166CF | [Mexico/4108/2009] | |
| | | ZeptoMetrix 0810249CF | [SwineNY/03/2009] | |
| | | ATCC VR-544 | [Hong Kong/8/1968] | |
| H3N2 | Human | ATCC VR-547 | [Aichi/2/1968] | Influenza A virus<br>Positive (No Subtype<br>Identified) |
| | | ATCC VR-776 | [Alice] | |
| | | ATCC VR-810 | [Port Chalmers/1/1973] | |
| H3N2 | Human | ATCC VR-822 | [Victoria/3/1975] | |
| H3N2 | Human | ZeptoMetrix 0810138CF | [Brisbane/10/2007] | |
| H3N2 | Human | ZeptoMetrix 0810238CF | [Texas/50/2012] | |
| H3N2 | Human | ZeptoMetrix 0810252CF | [Wisconsin/67/2005] | |
| H3N2 | Human | ZeptoMetrix 0810526CF | [Hong Kong/4801/14] | |
| H1N1 | Human | ZeptoMetrix 0810036CF | [New Caledonia/20/1999] | |
| H1N1 | Human | ZeptoMetrix 0810036CFN | [Solomon Islands/3/2006] | |
| H1N1 | Human | ZeptoMetrix 0810244CF | [Brisbane/59/2007] | |
| H1N1 | Swine | ATCC VR-333 | [Swine/Iowa/15/1930] | |
| H1N1 | Swine | ATCC VR-897 | [Swine/A/New Jersey/8/76] | |
| H1N1 | Swine | ATCC VR-99 | [Swine/1976/1931] | |
| H2N2 | Human | BEI NR-2775 a | [A/Japan/305/1957] | |
| H5N3 | Avian | BEI NR-9682 b | [A/Duck/Singapore/645/97] | Uncertain: Influenza A virus |
| H1N2 | Recombinant | BEI NR-3478 c | [Kilbourne F63 A/NWS/1934<br>(HA) x A/Rockefeller<br>Institute/5/1957 (NA)] | |
| H10N7 | | Avian | BEI NR-2765 d | |

® The following reagent was obtained through BEI Resources, NIAID, NH: Genomic RNA from Influenza A Virus, A Japan/305/1957 (H2N2), NR-2775. ® The following reagent was obtained through BEI Resources, NJAD, NH: Genomic RNA from Kilbourne F181: Adusk Singapore/645/1997 (H5N3), Wild Type, NR-9682.

® The following reagent was obtained through BEI Resources, NAID, NH: Kilbourne F63: ANWS/1934 (HA) x Althute/5/1957 (NA) (HN2),

Reassortant NWS-F, NR-3478. d The following reagent was obtained through BEI Resources, NAID, NH: Genomic RNA from Influenza A Virus, NR-2765.

Table 16. Summary of Reactivity to Influenza B Isolates

| Lineage | Source/Isolate ID | [Strain/Location/Year] | Result |
|----------|-----------------------|------------------------|-------------------------------|
| Yamagata | ZeptoMetrix 0810255CF | [Florida/04/06] | Influenza B virus<br>Positive |
| | ZeptoMetrix 0810239CF | [2/Massachusetts/2012] | |
| | ZeptoMetrix 0810241CF | [1/Wisconsin/2010] | |
| | ZeptoMetrix 0810256CF | [07/Florida/2004] | |
| | ZeptoMetrix 0810037CF | [B/Flo…