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
• Attributes: Real-World Evidence, Pediatric

Real-World Evidence

Submission	Device	Sponsor	RWD Sources	RWE Use Summary	Key Tags
K213954 · Feb 3, 2023	BIOFIRE SPOTFIRE Respiratory (R) Panel	Biofire Diagnostics	Archived clinical nasopharyngeal swab specimens from various clinical laboratories	Archived clinical specimens were used to supplement the prospective clinical study for analytes that were not encountered in sufficient numbers during the prospective collection period.	Archived specimens; Clinical performance; Respiratory pathogens

Clinical Evidence

Study Design	Population	Comparator	Key Endpoints
Clinical Study with preselected, archived specimens; Retrospective analysis of archived clinical specimens	Subjects presenting with signs/symptoms of respiratory infection; Sample Size: 542 (included in analysis); Number of Sites: 4 U.S. sites	FDA-cleared molecular comparator methods	Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA)

Indications for 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 nucleic acid amplification test; uses disposable closed-system pouch; automated extraction, nested PCR, and fluorescent DNA melting analysis; LC Green Plus dye for detection; factory-calibrated instrument; requires BIOFIRE SPOTFIRE System; software-controlled interpretation; no user intervention during run.

Indications for Use

Indicated for individuals with signs and symptoms of respiratory tract infection, including COVID-19, to detect and identify 13 respiratory viral and bacterial pathogens in nasopharyngeal swab specimens. Rx only.

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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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION

# DECISION SUMMARY

# ASSAY AND INSTRUMENT

# I Background Information:

A 510(k) Number

K213954

B Applicant

BIOFIRE Diagnostics

C Proprietary and Established Names

BIOFIRE SPOTFIRE Respiratory Panel

D Regulatory Information

| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| QOF | Class II | 21 CFR 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 | MI - Microbiology |
| OEM | Class II | 21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay | MI - Microbiology |
| OOU | Class II | 21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay | MI - Microbiology |
| OTG | Class II | 21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay | MI - Microbiology |
| OZE | Class II | 21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay | MI - Microbiology |
| OZX | Class II | 21 CFR 866.3980 - Respiratory viral panel | MI - Microbiology |

Food and Drug Administration

10903 New Hampshire Avenue

Silver Spring, MD 20993-0002

www.fda.govK213954 - Page 1 of 59

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| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| | | multiplex nucleic acid assay | |
| OZY | Class II | 21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay | MI - Microbiology |
| OZZ | Class II | 21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay | MI - Microbiology |
| OCC | Class II | 21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay | MI - Microbiology |
| NSU | Class II | 21 CFR 862.2570 - Instrumentation for clinical multiplex test systems | CH – Clinical Chemistry |

## II Submission/Device Overview:

### A Purpose for Submission:

To obtain a substantial equivalence determination for the BIOFIRE SPOTFIRE Respiratory (R) Panel performed on the BIOFIRE SPOTFIRE System.

### B Measurand:

The BIOFIRE SPOTFIRE Respiratory (R) Panel detects and identifies nucleic acids from the following pathogens: Adenovirus, seasonal Coronavirus (229E, HKU1, OC43, NL63 not differentiated), Severe Acute Respiratory Syndrome (SARS)-Coronavirus-2, Human Metapneumovirus, Human Rhinovirus/Enterovirus (not differentiated), Influenza A virus with subtyping of H3 and H1-2009 (reported separately), Influenza B virus, Parainfluenza Virus (serotypes 1-4, not differentiated), Respiratory Syncytial Virus, Bordetella parapertussis, Bordetella pertussis, Chlamydia pneumoniae and Mycoplasma pneumoniae.

### C Type of Test:

Multiplex nucleic acid assay for use with the BIOFIRE SPOTFIRE System for the qualitative detection of viral and/or bacterial pathogens in patients with signs and symptoms of respiratory tract infection.

Food and Drug Administration

10903 New Hampshire Avenue

Silver Spring, MD 20993-0002

www.fda.govK213954 - Page 2 of 59

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### III Intended Use/Indications for Use:

#### A Intended Use(s):

See Indications for Use below.

#### B Indication(s) for Use:

The BIOFIRE SPOTFIRE Respiratory (R) Panel (SPOTFIRE R Panel) is a multiplexed polymerase chain reaction (PCR) test intended for use with the BIOFIRE SPOTFIRE 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:

*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*

Food and Drug Administration
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www.fda.govK213954 - Page 3 of 59

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

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.

# **C Special Conditions for Use Statement(s):**

Rx - For Prescription Use Only

# **D Special Instrument Requirements:**

BIOFIRE SPOTFIRE System

# **IV Device/System Characteristics:**

# **A Device Description:**

The BIOFIRE SPOTFIRE Respiratory (R) Panel (SPOTFIRE R Panel) is a multiplexed nucleic acid-amplification-based test that is intended to detect and identify 13 different respiratory viral and bacterial pathogens in nasopharyngeal swab (NPS) specimens from individuals with signs and symptoms of respiratory tract infection. The SPOTFIRE R Panel is performed on the BIOFIRE SPOTFIRE System, an automated polymerase chain reaction (PCR)-based *in vitro* diagnostic system for use with reagent pouches for specific indications.

The BIOFIRE SPOTFIRE System automates nucleic acid extraction and nested multiplex PCR in closed system. The resulting PCR products are evaluated using assay-specific DNA melting analysis. The BIOFIRE SPOTFIRE System Software executes the SPOTFIRE R Panel test and interprets and reports the test results in approximately 15 minutes without user intervention.

The SPOTFIRE System is comprised of between one and four modules that are connected to a single SPOTFIRE Control Station equipped with the SPOTFIRE System Software. The first module is placed on top of the Control Station and up to three additional modules may be stacked on top as required. Each module can be accessed at random to perform a test, independent of the other modules attached to the same Control Station.

The SPOTFIRE R Panel Reagent Kit includes the following components:

Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.govK213954 - Page 4 of 59

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- BIOFIRE SPOTFIRE R Panel Pouches
- Each packed under vacuum in a metal canister and outer bag
- BIOFIRE Sample Preparation Reagent Kit (SPRK)
- A fixed volume transfer pipette for addition of the test sample to the Sample Injection Vial
- BIOFIRE Sample Buffer ampoule for addition to the Sample Injection Vial
- BIOFIRE Sample Injection Vial for mixing of the test sample and Sample Buffer
- BIOFIRE Hydration Injection Vial containing Hydration Solution for pouch rehydration

**B Principle of Operation:**

The BIOFIRE SPOTFIRE Panel Pouch is a closed-system disposable that contains all the reagents needed for sample preparation, reverse transcription, PCR amplification and fluorescent detection, including melt curve analysis of amplified products. The rigid plastic “fitment” of the pouch contains reagents in freeze-dried form while the flexible plastic portion of the pouch is divided into discrete “blisters” in which different chemical processes are carried out. After sample collection, the user injects hydration solution and the sample combined with BIOFIRE Sample Buffer into the pouch, places the pouch into the SPOTFIRE System, and starts the run. All other operations are automated.

The SPOTFIRE System Software is designed to run on the SPOTFIRE Control Station which consists of an integrated computer and touchscreen. The SPOTFIRE Systems Software interfaces with the user via the touchscreen to operate the instrument, collect, save, analyze and retrieve data, report test results and view stored information. Meanwhile the BIOFIRE SPOTFIRE R Panel Software works with the System Software and specifies the assay-specific parameters to perform the test, interpret the data and display the reported results.

To perform a test with SPOTFIRE R Panel, the sample is first mixed with a denaturing Sample Buffer to inactivate nucleases. The user then loads the diluted sample into the SPOTFIRE R Panel Pouch via the injection port. The vacuum in the pouch draws the liquid into the sample well of the pouch fitment and in the process rehydrates the cell-based Process Control. The sample mixture is moved to the lysis blister where mechanical agitation with zirconia-silica beads is used to complete cell lysis. The lysate is then mixed with glass-coated magnetic beads which adsorb the nucleic acids liberated in the lysis step. The extracted nucleic acids are washed to remove potential inhibitors and then eluted into buffer that is used to rehydrate the contents of the first stage PCR blister, which includes all the components needed for reverse transcription (RT) and first stage PCR. RT-PCR is initiated upon achievement of the requisite temperature within the amplification chamber. Following completion of first-stage amplification, the products of the reaction are diluted and combined with fresh reagents that are distributed over a second stage PCR array. The second stage primers are internal (“nested”) with respect to the first stage primers and each analyte or control target is represented by three discrete amplification reactions. The second stage PCR array includes an assay control, the PCR2 Control, failure of which indicates a failure in the second stage PCR and invalidates the run.

Food and Drug Administration
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Included in the PCR master mix for the second stage PCR is a fluorescent dye (LC Green Plus) that is specific for double-stranded DNA. The dye intercalates with the double stranded-PCR products and fluoresces at a specific wavelength that is detected by the SPOTFIRE System. At the end of the second stage PCR, the temperature of the array is gradually raised at a controlled ramp rate and the fluorescence is monitored. Fluorescence decreases at the melting temperature of the amplicons, and the temperature at which this occurs is characteristic of each amplification product. The software automatically evaluates the calculated melting temperature (Tm) in comparison to the predefined range for each specific analyte. The results from each of the three amplification reactions for each analyte are combined by the software to determine the test result. For an assay to be called positive, at least two of the three wells must have a PCR product with a Tm that is within the specified range for that analyte. Assays that do not meet these criteria are reported as negative.

Positive and negative results for the assay controls (Process Control and PCR2 Control) are compared to their expected values and assigned a “pass” or “fail” result. Panel-specific rules define how control failures affect interpretation of test results. For the SPOTFIRE R Panel, failure of either the RNA Process Control or PCR2 Control invalidates the results for all analytes. The final sample result is determined by the interpretation rules defined in the panel software.

For most bacteria and viruses detected by the SPOTFIRE R Panel, a “positive” result is reported if two or more of the wells corresponding to a single assay contain detectable PCR product of the appropriate Tm. However, to ensure inclusivity, certain analytes are targeted by multiple assays, the results of which are interpreted in combination, as described below in **Tables 1 and 2**.

**Table 1.** Interpretation of results for analytes targeted by multiple assays

| Analyte | Number of Independent Assays | Positive Result Interpretation |
| --- | --- | --- |
| Adenovirus | 4 | ≥ 1 assay positive |
| Coronavirus (seasonal) | 4 | ≥ 1 assay positive |
| Influenza A | 4 | ≥ 2 assays positive ^{1} |
| Parainfluenza Virus | 4 | ≥ 1 assay positive |
| SARS-CoV-2 | 2 | ≥ 1 assay positive |

$^{1}$ At least one of the pan-influenza A assays and *either* the H1-2009- *or* the H3-specific assay must produce positive results to report a positive subtype result (**Table 2**)

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**Table 2.** Possible result permutations for influenza A from patient samples

| Assay | | | | Result Reported | Action Required |
| --- | --- | --- | --- | --- | --- |
| FluA-pan1 | FluA-pan2 | FluA-H1-2009 | FluA-H3 | | |
| Positive | Positive | Positive | Negative | POSITIVE Influenza A Virus Subtype H1-2009 | Report |
| Positive | Negative | Positive | Negative | | |
| Negative | Positive | Positive | Negative | | |
| Positive | Positive | Negative | Positive | POSITIVE Influenza A Virus Subtype H3 | Report |
| Positive | Negative | Negative | Positive | | |
| Negative | Positive | Negative | Positive | | |
| Positive | Positive | Positive | Positive | POSITIVE Influenza A Virus Multiple Subtypes | Report |
| Positive | Negative | Positive | Positive | | |
| Negative | Positive | Positive | Positive | | |
| Positive | Positive | Negative | Negative | POSITIVE Influenza A Virus No Subtype | Re-test ^{1} |
| Positive | Negative | Negative | Negative | UNCERTAIN Influenza A Virus | |
| Negative | Positive | Negative | Negative | | |
| Negative | Negative | Positive | Negative | | |
| Negative | Negative | Negative | Positive | | |
| Negative | Negative | Positive | Positive | | |
| Negative | Negative | Negative | Negative | NEGATIVE | Report |

$^{1}$ If the re-test result is the same, the user is instructed to contact public health authorities for confirmatory testing

### C Instrument Description Information:

#### 1. Instrument Name:

BIOFIRE SPOTFIRE System

#### 2. Specimen Identification:

Specimen identification can be entered manually or by scanning a barcode.

Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.govK213954 - Page 7 of 59

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# 3. Specimen Sampling and Handling:

Use of the SPOTFIRE R Panel requires a nasopharyngeal swab (NPS) to be collected according to standard procedures and placed in 1-3 mL of viral transport medium. The minimum sample volume required to perform a test is 300 μL. Specimens should be tested as soon as possible following collection but may be stored for up to 4 hours at room temperature (15-25 °C) or for up to 3 days at 2-8 °C or up to 30 days at ≤ -15 °C.

The SPOTFIRE System Software includes step-by-step on-screen instructions that guide the user through the process of starting a run on the instrument. After cleaning the work area and Pouch Loading Station, the user removes a SPOTFIRE R Panel pouch from its vacuum packaging and places it into the Pouch Loading Station. They then hydrate the pouch using the Hydration Injection Vial by injecting the contents through the Hydration Solution Injection Port, after which they transfer a fixed volume of sample to the Sample Injection Vial, together with the entire contents of the Sample Buffer ampoule. After mixing the Sample Injection Vial by insertion, the user injects the mixture into the pouch via the Sample Injection Port. The pouch is then inserted in the SPOTFIRE instrument, after which the run starts automatically and proceeds to completion without further user intervention, including interpretation of results.

# 4. Calibration:

The BIOFIRE SPOTFIRE System is factory calibrated. User calibration is not required.

# 5. Quality Control:

# Internal Controls

Two process controls are included in each SPOTFIRE R Panel pouch:

# 1) RNA Process Control

The RNA Process Control comprises freeze-dried Schizosaccharomyces pombe which is rehydrated when the sample is loaded. The control material is carried through all stages of the test process, including nucleic acid extraction and purification, reverse transcription, first and second stage PCR amplification and DNA melt analysis. A positive result indicates that all process steps carried out in the SPOTFIRE R Panel pouch were completed successfully.

# 2) PCR2 Control

The PCR2 control comprises a DNA target sequence that is dried into specific wells used for the second stage amplification and melting curve analysis. A positive result indicates that these processes were successful.

A positive result is required for both the RNA Process Control and the PCR2 Control for the results for an individual pouch to be considered valid. If either control fails, the sample should be retested using a new pouch.

Food and Drug Administration

10903 New Hampshire Avenue

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www.fda.govK213954 - Page 8 of 59

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### *External Controls*

BIOFIRE recommends testing External Positive and Negative Controls that are commercially available from Maine Molecular Quality Controls, Inc. when training a new operator, or when a new shipment of SPOTFIRE panels or a new control station is received:

- SPOTFIRE RSP Positive Control (M42638)
- SPOTFIRE RSP Negative Control (M42738)

These controls are designed to monitor the performance of each of the individual assays within the SPOTFIRE R Panel and should be tested in accordance with their FDA-cleared Instructions For Use (K221253).

The Instructions For Use of the SPOTFIRE R Panel indicate that an External Negative Control should be tested at least monthly to monitor for environmental contamination. Additional External Controls may be tested as needed.

External Positive and Negative Controls were tested during the Prospective Clinical Study described in **Section VII (C)**. In total, 252 External Controls were tested of which 8 were associated with instrument errors, 4 were tested using an incorrect version of the reagent pouch and 4 were tested using the workflow for patient samples rather than Quality Control. As a result, there were 236 External Controls with valid results (109 Positive and 131 Negative). Of these, 99.1% of the External Positive Controls (108/109) and 100% of the External Negative Controls (131/131) produced the expected results.

## **V Substantial Equivalence Information:**

### **A Predicate Device Name(s):**

BioFire Respiratory Panel 2.1 (RP2.1)

### **B Predicate 510(k) Number(s):**

DEN200031

### **C Comparison with Predicate(s):**

| **Device & Predicate Device(s):** | **K213954** | **DEN200031** |
| --- | --- | --- |
| Device Trade Name | BIOFIRE SPOTFIRE Respiratory (R) Panel | BIOFIRE Respiratory Panel 2.1 |
| **General Device Characteristic Similarities** | | |
| Intended Use/Indications For Use | The BIOFIRE SPOTFIRE Respiratory (R) Panel (SPOTFIRE R Panel) is a multiplexed polymerase chain reaction (PCR) test intended for use with the BIOFIRE SPOTFIRE System for the simultaneous, qualitative | The BIOFIRE Respiratory Panel 2.1 (RP2.1) is a PCR-based multiplexed nucleic acid test intended for use with the BIOFIRE FilmArray 2.0 or BIOFIRE FilmArray Torch systems for the simultaneous qualitative detection and |

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| Device & Predicate Device(s): | K213954 | DEN200031 |
| --- | --- | --- |
| | 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: *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. The detection and identification | identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals suspected of respiratory tract infections, including COVID-19. The following organism types and subtypes are identified using the BIOFIRE RP2.1: - Adenovirus, - Coronavirus 229E - Coronavirus HKU1 - Coronavirus NL63 - Coronavirus OC43 - Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) - Human Metapneumovirus - Human Rhinovirus/Enterovirus - Influenza A, including subtypes H1, H1-2009, and H3 - Influenza B - Parainfluenza Virus 1 - Parainfluenza Virus 2 - Parainfluenza Virus 3 - Parainfluenza Virus 4 - Respiratory Syncytial Virus - *Bordetella parapertussis* (IS1001) - *Bordetella pertussis* (ptxP) - *Chlamydia pneumoniae*, and - *Mycoplasma pneumoniae* Nucleic acids from the respiratory viral and |

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| Device & Predicate Device(s): | K213954 | DEN200031 |
| --- | --- | --- |
| | of specific viral and bacterial nucleic acids from individuals exhibiting signs and/or symptoms of respiratory infection are indicative of the presence of the identified microorganism and aids in diagnosis if used in conjunction with other clinical and epidemiological information, and laboratory findings. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. 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. | bacterial organisms identified by this test are generally detectable in NPS specimens during the acute phase of infection. The detection and identification of specific viral and bacterial nucleic acids from individuals exhibiting signs and/or symptoms of respiratory infection is indicative of the presence of the identified microorganism and aids in the diagnosis of respiratory infection if used in conjunction with other clinical and epidemiological information. 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 BIOFIRE RP2.1 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 |

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| Device & Predicate Device(s): | K213954 | DEN200031 |
| --- | --- | --- |
| | | a patient with possible respiratory tract infection. |
| Specimen Types | Same | Nasopharyngeal swabs |
| Patient Population | Individuals with signs and symptoms of respiratory tract infection | Individuals suspected of respiratory tract infections, including COVID-19 |
| Organisms Detected | Same except: Seasonal Coronaviruses are not differentiated Parainfluenza viruses are not differentiated No subtyping assay for Influenza Virus A/H1 (pre-pdm2009) | *Viruses:* Adenovirus Coronavirus 229E Coronavirus HKU1 Coronavirus NL63 Coronavirus OC43 Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Human Metapneumovirus Human Rhinovirus/Enterovirus Influenza A Subtypes: H1, H3, H1-2009 Influenza B Parainfluenza Virus 1 Parainfluenza Virus 2 Parainfluenza Virus 3 Parainfluenza Virus 4 Respiratory Syncytial Virus *Bacteria:* *Bordetella parapertussis* *Bordetella pertussis* *Chlamydia pneumoniae* *Mycoplasma pneumoniae* |
| Technology | Same | Highly multiplexed PCR with DNA melting analysis |
| **General Device Characteristic Differences** | | |
| Instrument | BIOFIRE SPOTFIRE System | BIOFIRE FilmArray 2.0 or BIOFIRE FilmArray Torch Systems |
| Disease Syndrome | Same | Respiratory tract infection |
| Time to Result | ~15 minutes | ~45 minutes |

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## VI Standards/Guidance Documents Referenced:

- Guidance for Clinical Laboratories, Commercial Manufactures, and FDA Staff – Policy for Coronavirus Disease-2019 Tests During the Public Health Emergency (2020)
- Guidance for Industry - Part 11, Electronic Records; Electronic Signatures - Scope and Application (August 2003)
- Guidance for Industry - Computerized Systems Used in Clinical Investigations (May 2007)
- Guidance for Industry - Oversight of Clinical Investigations - A Risk-Based Approach to Monitoring (August 2013)
- Guidance for Industry - Electronic Source Data in Clinical Investigations (September 2013)
- Guidance for Industry and FDA Staff - Acceptance of Clinical Data to Support Medical Device Applications and Submissions - Frequently Asked Questions (February 2018)
- Guidance for Industry and FDA Staff - Recommendations for Clinical Laboratory Improvement Amendments of 1988 (CLIA) Waiver Applications for Manufacturers of *In Vitro* Diagnostic Devices (February 2020)
- Guidance for Industry and FDA Staff - Recommendations for Dual 510(k) and CLIA Waiver by Application Studies (February 2020)
- Guidance for Industry and FDA Staff - Class II Special Controls Guidance Document: Respiratory Viral Panel Multiplex Nucleic Acid Assay (October 2009)
- Guidance for Industry and FDA Staff - Class II Special Controls Guidance Document: Testing for Detection and Differentiation of Influenza A Virus Subtypes Using Multiplex Nucleic Acid Assays (October 2009)
- Guidance for Industry and FDA Staff - Class II Special Controls Guidance Document: Testing for Human Metapneumovirus (hMPV) Using Nucleic Acid Assays (October 2009)
- Guidance for Industry and FDA Staff - Statistical Guidance on Reporting Results from Studies Evaluating Diagnostic Tests, (March 2007)
- Guidance for Industry and FDA Staff, Evaluation and Reporting of Age-, Race-, and Ethnicity-Specific Data in Medical Device Clinical Studies (September 2017)
- Guidance for Sponsors, Investigators, and IRBs - Impact of Certain Provisions of the Revised Common Rule on FDA-Regulated Clinical Investigations (October 2018)
- Guidance for Sponsors, Institutional Review Boards, C and FDA Staff - Guidance on Informed Consent for *In Vitro* Diagnostic Device Studies Using Leftover Human Specimens that are Not Individually Identifiable (April 2006)
- Guidance for Industry and Food and Drug Administration Staff - Highly Multiplexed Microbiological/Medical Countermeasure *In Vitro* Nucleic Acid Based Diagnostic Devices (August 2014)
- Guidance for Industry and FDA Staff - Statistical Guidance on Reporting Results from Studies Evaluating Diagnostic Tests (March 2007)
- Guidance for Industry and FDA Staff - Guidance for the Content of Premarket Submissions for Software Contained in Medical Devices (May 2005)
- Guidance for Industry and FDA Staff - Off-The-Shelf Software Use in Medical Devices (September 2019)
- Final Guidance for Industry and FDA Staff - General Principles of Software Validation (January 2002)
- Guidance for Industry and FDA Staff - Content of Premarket Submissions for

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Management of Cybersecurity in Medical Devices (October 2014)

- CLSI. User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline - Second Edition. CLSI document EP12-A2. Wayne, PA: Clinical and Laboratory Standards Institute; 2008.
- CLSI. Evaluation of Stability of *In Vitro* Diagnostic Reagents; Approved Guideline. CLSI document EP25-A. Wayne, PA: Clinical and Laboratory Standards Institute; 2009.
- CLSI. Interference Testing in Clinical Chemistry. 3rd ed. CLSI guideline EP07. Wayne, PA: Clinical and Laboratory Standards Institute; 2018.
- ICH Harmonised Guideline: Integrated Addendum to ICH E6(R1): Guideline for Good Clinical Practice E6(R2) (November 2016)
- GHTF: Clinical Evidence for IVD Medical Devices - Clinical Performance Studies for *In Vitro* Diagnostic Medical Devices (November 2012)
- WMA Declaration of Helsinki, Ethical Principles for Medical Research Involving Human Subjects
- ISO 14971:2019 Medical devices - Application of risk management to medical devices
- IEC 62366-1:2015 Medical devices - Part 1: Application of usability engineering to medical devices
- ISO 62304:2006 Medical device software - Software life-cycle processes
- ISO 15223-1:2016: Medical Devices - Symbols to be used with medical device labels, labeling and information to be supplied - Part 1: General requirements
- IEC 61010-1 Ed. 3.0 2010: Safety requirements for electrical equipment for measurement, control, and laboratory use - Part 1: General requirements
- EN 61010-2-101:2002/IEC 61010-2-101:2015: Safety requirements for electrical equipment for measurement, control and laboratory use - Part 2-101: Particular requirements for *in vitro* diagnostic (IVD) medical equipment.
- IEC 60601-1-2:2014 (Edition 4.0): Medical electrical equipment - Part 1-2: General requirements for basic safety and essential performance - Collateral Standard: Electromagnetic disturbances - Requirements and tests
- AIM 7351731 Rev 2.00 (2017-02-23): Medical Electrical Equipment and System Electromagnetic Immunity Test for Exposure to Radio Frequency Identification Readers
- UL 2900-1 Ed. 1 2017: Standard for Software Cybersecurity Network-Connectable Products, Part I: General Requirements
- ANSI UL 2900-2-1: Standard for Safety, Software Cybersecurity for Network-Connectable Products, Part 2-1: Particular Requirements for Network Connectable Components of Healthcare and Wellness Systems
- ISO 13485:2016/EN ISO 13485:2016; Medical devices - Quality Management System - Requirements for regulatory purposes
- ISO 20916:2019; *In vitro* diagnostic medical devices. Clinical performance studies using specimens from human subjects. Good study practice
- EN 13612:2002; Performance evaluation of *in vitro* diagnostic medical devices
- EN ISO 18113-1:2011; *In vitro* diagnostic medical devices - Information supplied by the manufacturer (labeling). Terms, definition and general requirements
- EN ISO 18113-2:2011; *In vitro* diagnostic medical devices - Information supplied by the manufacturer (labeling) – Part 2: *In vitro* diagnostic reagents for professional use
- EN ISO 18113-3:2011; *In vitro* diagnostic medical devices - Information supplied by the manufacturer (labeling) – Part 3: *In vitro* diagnostic instruments for professional use
- EN ISO 23640:2015; *In vitro* diagnostic medical devices - Evaluation of stability of *in vitro*

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diagnostic reagents

- EN 13641:2002; Elimination or reduction of risk of infection related to *in vitro* diagnostics reagents
- IEC 61326-1:2012; Electrical equipment for measurement control and laboratory use - EMC requirements - Part 1: General requirements
- EN 61326-2-6:2006/IEC 61326-2-6:2012; Electrical equipment for measurement control and laboratory use - EMC requirements - Part 2-6: Particular requirements - *In vitro* diagnostic (IVD) medical equipment

**VII Performance Characteristics (if/when applicable):**

**A Analytical Performance:**

1. Precision/Reproducibility:

The reproducibility and precision of the SPOTFIRE R Panel on different BIOFIRE SPOTFIRE Systems was evaluated with multiple operators who tested positive and negative samples over multiple days at three study sites, in addition to BIOFIRE Diagnostics. All analytes detected by the panel were evaluated using contrived positive specimens at concentrations equivalent to ≤ 3X their respective analytical limit of detection (LoD) (**Table 3**). Negative samples were prepared using artificial matrix only.

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**Table 3.** Contrived samples used to evaluate the precision/reproducibility of the SPOTFIRE R Panel

| Sample | Analyte/Strain | Source Identity | Concentration (per mL) |
| --- | --- | --- | --- |
| 1 | None | -- | -- |
| 2 | *Bordetella pertussis* A639 | Zeptometrix 0801459 | 990 CFU |
| | *Mycoplasma pneumoniae* M129 | Zeptometrix 0801579 | 10 CCU |
| | Adenovirus Species B Type 3 | Zeptometrix 0810062CF | 2.4 TCID_{50} |
| | Coronavirus NL63 | Zeptometrix 0810228CF | 0.0025 TCID_{50} |
| | Influenza B B/Florida/02/06 | Zeptometrix 0810037CF | 0.099 TCID_{50} |
| | Parainfluenza virus 4a | Zeptometrix 0810060CF | 200 TCID_{50} |
| 3 | *Bordetella parapertussis* E595 | Zeptometrix 0801462 | 120 CFU |
| | *Chlamydophila pneumoniae* AR-39 | ATCC 53592 | 20 IFU |
| | Human metapneumovirus 3 Type B1 | Zeptometrix 0810156CF | 0.75 TCID_{50} |
| | Parainfluenza virus 1 | Zeptometrix 0810014CF | 4.6 TCID_{50} |
| | Influenza A H1N1pdm Michigan/45/15 | Zeptometrix 0810538CF | 2.5 TCID_{50} |
| | Coronavirus SARS-CoV-2 2019-nCoV/USA-WA1/2020 | ATCC VR-1986HK | 250 copies |
| 4 | Human enterovirus D68 US/MO/14-18947 | ATCC VR-1823 | 11 TCID_{50} |
| | Parainfluenza virus 2 | Zeptometrix 0810015CF | 42 TCID_{50} |
| | Respiratory syncytial virus A 2006 | Zeptometrix 0810040ACF | 6.2 x 10^{-2} TCID_{50} |
| | Coronavirus 229E | ATCC VR-740 | 2.0 TCID_{50} |
| 5 | Parainfluenza virus 3 | Zeptometrix 0810016CF | 8.8 TCID_{50} |
| | Influenza A H3N2 Hong Kong/4801/14 | Zeptometrix 0810526CF | 2.6 TCID_{50} |
| | Coronavirus OC43 | Zeptometrix 0810024CF | 1.6 x 10^{-2} TCID_{50} |

CCU: Color Changing Units; CFU: Colony Forming Units; IFU: Inclusion Forming Units; TCID$_{50}$: Tissue Culture Infectious Dose-50%

At BIOFIRE Diagnostics, testing was performed on three SPOTFIRE Systems, using three lots of reagents over five consecutive days. On each day of testing, two operators each tested three replicates of each sample (one per reagent lot) on each SPOTFIRE System for a total of 90 replicates per sample overall (2 operators x 3 samples x 3 instruments x 5 days = 90 replicates).

At the external study sites, testing was performed with a single SPOTFIRE System at each site and occurred over five non-consecutive days using a single lot of reagents that was common across each of the sites. On each day, two operators each tested two replicates of each sample for a total of 60 replicates per sample overall (2 operators x 2 samples x 3 sites x 5 days = 60 replicates).

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For each analyte there were 150 positive and 600 negative samples. Samples that produced an invalid result on initial testing due to process control failure (n = 12) or instrument error (n = 2) were retested. Precision and reproducibility for each analyte were calculated by assessing the agreement between the reported test results and the expected results for each sample.

A summary of the results of the study, stratified by analyte and SPOTFIRE system/site is provided in Table 4. Positive agreement for all analytes was > 95% and there was negligible difference between instruments, sites, reagent lots or operators. Negative agreement was 100% in all cases.

Overall, the reproducibility and precision of the SPOTFIRE R Panel for detection of the target analytes was determined to be acceptable.

Table 4. Summary of results from the Precision and Reproducibility Study, stratified by SPOTFIRE instrument and study site

| Assay/Analyte | Expected Result | Agreement (%) Stratified by Site and SPOTFIRE System | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | BIOFIRE Diagnostics | | | External Site | | | Total |
| | | A | B | C | 1 | 2 | 3 | |
| **Adenovirus** Adenovirus Species B Type 3 | Positive | 30/30 (100) | 30/30 (100) | 30/30 (100) | 20/20 (100) | 20/20 (100) | 20/20 (100) | 150/150 (100) |
| | | 90/90 (100) | | | 60/60 (100) | | | |
| | Negative | 120/120 (100) | 120/120 (100) | 120/120 (100) | 80/80 (100) | 80/80 (100) | 80/80 (100) | 600/600 (100) |
| | | 360/360 (100) | | | 240/240 (100) | | | |
| **Bordetella parapertussis** *B. parapertussis* E595 | Positive | 30/30 (100) | 30/30 (100) | 30/30 (100) | 20/20 (100) | 20/20 (100) | 20/20 (100) | 150/150 (100) |
| | | 90/90 (100) | | | 60/60 (100) | | | |
| | Negative | 120/120 (100) | 120/120 (100) | 120/120 (100) | 80/80 (100) | 80/80 (100) | 80/80 (100) | 600/600 (100) |
| | | 360/360 (100) | | | 240/240 (100) | | | |
| **Bordetella pertussis** *B. pertussis* A639 | Positive | 29/30 (96.7) | 29/30 (96.7) | 30/30 (100) | 20/20 (100) | 20/20 (100) | 20/20 (100) | 148/150 (98.7) |
| | | 88/90 (97.8) | | | 60/60 (100) | | | |
| | Negative | 120/120 (100) | 120/120 (100) | 120/120 (100) | 80/80 (100) | 80/80 (100) | 80/80 (100) | 600/600 (100) |
| | | 360/360 (100) | | | 240/240 (100) | | | |
| **Chlamydia pneumoniae** *Chlamydophila pneumoniae* AR-39 | Positive | 30/30 (100) | 30/30 (100) | 30/30 (100) | 20/20 (100) | 20/20 (100) | 20/20 (100) | 150/150 (100) |
| | | 90/90 (100) | | | 60/60 (100) | | | |
| | Negative | 120/120 (100) | 120/120 (100) | 120/120 (100) | 80/80 (100) | 80/80 (100) | 80/80 (100) | 600/600 (100) |
| | | 360/360 (100) | | | 240/240 (100) | | | |
| **Seasonal Coronavirus** | Coronavirus 229E | Positive | 30/30 (100) | 30/30 (100) | 29/30 (96.7) | 20/20 (100) | 20/20 (100) | 20/20 (100) |
| | | | 89/90 (98.9) | | | 60/60 (100) | | |
| | Coronavirus OC43 | Positive | 30/30 (100) | 30/30 (100) | 30/30 (100) | 20/20 (100) | 20/20 (100) | 20/20 (100) |
| | | | 90/90 (100) | | | 60/60 (100) | | |
| | Coronavirus NL63 | Positive | 27/30 (90.0) | 30/30 (100) | 30/30 (100) | 18/20 (90.0) | 20/20 (100) | 18/20 (90.0) |
| | | | 87/90 (96.7) | | | 56/60 (93.3) | | |
| | None | Negative | 60/60 (100) | 60/60 (100) | 60/60 (100) | 40/40 (100) | 40/40 (100) | 40/40 (100) |
| | | | 180/180 (100) | | | 120/120 (100) | | |

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| Assay/Analyte | Expected Result | Agreement (%) Stratified by Site and SPOTFIRE System | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | BIOFIRE Diagnostics | | | External Site | | | Total | |
| | | A | B | C | 1 | 2 | 3 | | |
| **Coronavirus SARS-CoV-2** SARS-CoV-2 2019-nCoV/USA-WA1/2020 | Positive | 30/30 (100) | 30/30 (100) | 30/30 (100) | 20/20 (100) | 20/20 (100) | 20/20 (100) | 150/150 (100) | |
| | | 90/90 (100) | | | 60/60 (100) | | | | |
| | | Negative | 120/120 (100) | 120/120 (100) | 120/120 (100) | 80/80 (100) | 80/80 (100) | 80/80 (100) | 600/600 (100) |
| | | | 360/360 (100) | | | 240/240 (100) | | | |
| **Human Metapneumovirus** hMPV 3 Type B1 | Positive | 30/30 (100) | 30/30 (100) | 30/30 (100) | 19/20 (95.0) | 20/20 (100) | 19/20 (95.0) | 148/150 (98.7) | |
| | | 90/90 (100) | | | 58/60 (96.7) | | | | |
| | | Negative | 120/120 (100) | 120/120 (100) | 120/120 (100) | 80/80 (100) | 80/80 (100) | 80/80 (100) | 600/600 (100) |
| | | | 360/360 (100) | | | 240/240 (100) | | | |
| **Human Rhinovirus/Enterovirus** Enterovirus D68 US/MO/14-18947 | Positive | 30/30 (100) | 30/30 (100) | 30/30 (100) | 20/20 (100) | 20/20 (100) | 20/20 (100) | 150/150 (100) | |
| | | 90/90 (100) | | | 60/60 (100) | | | | |
| | | Negative | 120/120 (100) | 120/120 (100) | 120/120 (100) | 80/80 (100) | 80/80 (100) | 80/80 (100) | 600/600 (100) |
| | | | 360/360 (100) | | | 240/240 (100) | | | |
| **Influenza A Virus** | Influenza A H1N1pdm | Positive | 30/30 (100) | 30/30 (100) | 30/30 (100) | 20/20 (100) | 20/20 (100) | 20/20 (100) | 150/150 (100) |
| | | | 90/90 (100) | | | 60/60 (100) | | | |
| | Influenza A H3N2 | Positive | 30/30 (100) | 30/30 (100) | 30/30 (100) | 19/20^{3} (95.0) | 19/20 (95.0) | 20/20 (100) | 148/150 (98.7) |
| | | | 90/90 (100) | | | 58/60 (96.7) | | | |
| | None | Negative | 90/90 (100) | 90/90 (100) | 90/90 (100) | 60/60 (100) | 60/60 (100) | 60/60 (100) | 450/450 (100) |
| | | | 270/270 (100) | | | 180/180 (100) | | | |
| **Influenza A Virus Subtype H1-2009**^{1} Influenza A H1N1pdm Michigan/45/15 | Positive | 30/30 (100) | 30/30 (100) | 30/30 (100) | 20/20 (100) | 20/20 (100) | 20/20 (100) | 150/150 (100) | |
| | | 90/90 (100) | | | 60/60 (100) | | | | |
| | | Negative | 120/120 (100) | 120/120 (100) | 120/120 (100) | 80/80 (100) | 80/80 (100) | 80/80 (100) | 600/600 (100) |
| | | | 360/360 (100) | | | 240/240 (100) | | | |
| **Influenza A Virus Subtype H3**^{2} Influenza A H3N2 Hong Kong/4801/14 | Positive | 30/30 (100) | 30/30 (100) | 30/30 (100) | 19/20^{3} (95.0) | 19/20 (95.0) | 20/20 (100) | 148/150 (98.7) | |
| | | 90/90 (100) | | | 58/60 (96.7) | | | | |
| | | Negative | 120/120 (100) | 120/120 (100) | 120/120 (100) | 80/80 (100) | 80/80 (100) | 80/80 (100) | 600/600 (100) |
| | | | 360/360 (100) | | | 240/240 (100) | | | |
| **Influenza B Virus** Influenza B B/Florida/02/06 | Positive | 29/30 (96.7) | 30/30 (100) | 30/30 (100) | 20/20 (100) | 20/20 (100) | 20/20 (100) | 149/150 (99.3) | |
| | | 89/90 (98.9) | | | 60/60 (100) | | | | |
| | | Negative | 120/120 (100) | 120/120 (100) | 120/120 (100) | 80/80 (100) | 80/80 (100) | 80/80 (100) | 600/600 (100) |
| | | | 360/360 (100) | | | 240/240 (100) | | | |
| **Mycoplasma pneumoniae** *M. pneumoniae* M129 | Positive | 29/30 (96.7) | 30/30 (100) | 30/30 (100) | 20/20 (100) | 20/20 (100) | 20/20 (100) | 149/150 (99.3) | |
| | | 89/90 (98.9) | | | 60/60 (100) | | | | |
| | | Negative | 120/120 (100) | 120/120 (100) | 120/120 (100) | 80/80 (100) | 80/80 (100) | 80/80 (100) | 600/600 (100) |
| | | | 360/360 (100) | | | 240/240 (100) | | | |
| **Parainfluenza Virus** | Parainfluenza Virus 1 | Positive | 30/30 (100) | 30/30 (100) | 30/30 (100) | 20/20 (100) | 20/20 (100) | 20/20 (100) | 150/150 (100) |
| | | | 90/90 (100) | | | 60/60 (100) | | | |
| | Parainfluenza Virus 2 | Positive | 30/30 (100) | 29/30 (96.7) | 28/30 (93.3) | 20/20 (100) | 20/20 (100) | 20/20 (100) | 147/150 (98.0) |
| | | | 87/90 (96.7) | | | 60/60 (100) | | | |

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| Assay/Analyte | Expected Result | Agreement (%) Stratified by Site and SPOTFIRE System | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | BIOFIRE Diagnostics | | | External Site | | | Total | |
| | | A | B | C | 1 | 2 | 3 | | |
| | Parainfluenza Virus 3 | Positive | 28/30 (93.3) | 30/30 (100) | 30/30 (100) | 19/20 (95.5) | 18/20 (90.0) | 20/20 (100) | 145/150 (96.7) |
| | | | 88/90 (97.8) | | | 57/60 (95.0) | | | |
| | Parainfluenza Virus 4 | Positive | 29/30 (96.7) | 30/30 (100) | 30/30 (100) | 20/20 (100) | 20/20 (100) | 20/20 (100) | 149/150 (99.3) |
| | | | 89/90 (98.9) | | | 60/60 (100) | | | |
| | None | Negative | 30/30 (100) | 30/30 (100) | 30/30 (100) | 20/20 (100) | 20/20 (100) | 20/20 (100) | 150/150 (100) |
| | | | 90/90 (100) | | | 60/60 (100) | | | |
| Respiratory Syncytial Virus RSV A 2006 | Positive | 30/30 (100) | 30/30 (100) | 30/30 (100) | 20/20 (100) | 19/20 (95.0) | 20/20 (100) | 149/150 (99.3) | |
| | | 90/90 (100) | | | 59/60 (98.3) | | | | |
| | | Negative | 120/120 (100) | 120/120 (100) | 120/120 (100) | 80/80 (100) | 80/80 (100) | 80/80 (100) | 600/600 (100) |
| | | | 360/360 (100) | | | 240/240 (100) | | | |
| Positive Agreement By System/Site | 621/630 (98.6) | 628/630 (99.7) | 627/630 (99.5) | 414/420 (98.6) | 415/420 (98.8) | 417/420 (99.3) | 3122/ 3150 (99.1) | | |
| | | | 1876/1890 (99.3) | | | 1246/1260 (98.9) | | | |

¹ 1 positive sample was initially reported as Positive: Influenza A Virus (No Subtype Identified) and was therefore re-tested. The re-test result was as expected. The retest results from this sample for each analyte are included in the summary table.

² 2 positive samples were initially reported as Positive: Influenza A Virus (No Subtype Identified) and 1 positive sample was initially reported as Uncertain: Influenza A Virus. All 3 samples were retested and the retest results were as expected. The retest results from these samples for each analyte are included in the summary table.

³ 1 sample was reported as Uncertain Influenza A Virus but was not retested.

## 2. Linearity:

Not applicable.

## 3. Analytical Specificity/Interference:

The analytical specificity (exclusivity) of the SPOTFIRE R Panel was evaluated through a combination of in silico analysis and laboratory testing of on- and off-panel species, phylogenetically related viruses and bacteria and other microorganisms that may be found in the respiratory tract.

### In silico Analysis of Specificity (Exclusivity)

In silico analysis of assay specificity/exclusivity was performed by conducting searches with whole amplicon and paired inner primer sequences using the NCBI BLAST “blastn” tool. The location and orientation of regions of homology with the primer sequences were evaluated to determine the potential for amplification. A summary of the results from the analysis is provided in Table 5. The SPOTFIRE R Panel assays were shown to be specific for their respective analytes with the following exceptions, which are noted in the device labeling:

- Cross-reaction of the SARS-CoV-2 assays with closely related bat and pangolin coronavirus sequences
- Cross-reaction of the Human Rhinovirus/Enterovirus assay with B. pertussis, B. parapertussis and B. bronchiseptica, in addition to bovine and canine picornaviruses

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- Cross-reaction of the *B. parapertussis* assay with strains of *B. bronchiseptica* that carry IS1001
- Cross-reaction of the *C. pneumoniae* assay with *Chlamydia gallinacea*

**Table 5.** Summary of *in silico* BLAST analysis to evaluate exclusivity

| Assay | Predicted Cross-reaction |
| --- | --- |
| Adenovirus | None |
| | None |
| | None |
| | None |
| Seasonal Coronavirus | None |
| | None |
| | None |
| | None |
| SARS-CoV-2 | Bat coronavirus |
| | Pangolin coronavirus |
| | Bat SARS-like coronavirus |
| Human Metapneumovirus | None |
| Human Rhinovirus/Enterovirus | Bovine and canine picornavirus |
| | *B. pertussis*, *B. parapertussis* and *B. bronchiseptica* |
| Influenza A Virus A/H1-2009 ^{1} | Swine H1 sequences |
| Influenza B Virus | None |
| Parainfluenza Virus | None |
| Respiratory Syncytial Virus | None |
| *Bordetella parapertussis* | Strains of *B. bronchiseptica* carrying IS1001 |
| *Bordetella pertussis* | None |
| *Chlamydia pneumoniae* | *Chlamydia gallinacea* |
| *Mycoplasma pneumoniae* | None |

$^{1}$ Certain avian H1N2 sequences (AY233393 and AY038014) may also be detected and reported as H1-2009

**Note:** A sequence from *Protopolystoma xenopodis* (a parasitic worm found in frogs) exhibited significant homology to the pan-influenza A PCR primers and may be amplified but this organism is of no clinical relevance

#### *Laboratory Testing of Analytical Specificity*

The analytical specificity (exclusivity) of the SPOTFIRE R Panel was also evaluated by testing high concentrations of on- and off-panel viruses, bacteria and other microorganisms that are phylogenetically related or may be found in nasopharyngeal and throat swab specimens. Analysis of on-panel analytes was performed to evaluate the potential for intra-panel cross-reactivity (i.e., detection of an analyte by an assay other than the target-specific assay for that analyte). Analysis of off-panel analytes was performed to evaluate the potential for cross-reaction with other respiratory flora that are not detected by the panel.

Each on-panel species was tested in triplicate and the results are shown in **Tables 6** and **7**. The expected results were obtained with each on-panel virus and no cross-reaction was observed (**Table 6**). Based on *in silico* analysis, it was predicted that *B. parapertussis* and *B. pertussis* would produce positive results with the Human Rhinovirus/Enterovirus assay. *B. pertussis* produced false-positive results at 1.3 x 10$^{10}$ CFU/mL but was reported negative when tested at 1.3 x 10$^{9}$ CFU/mL (**Table 7**). False-positive results were not observed with *B.*

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parapertussis at the concentration tested (4.6 x 10⁹ CFU/mL). The potential for cross-reaction of the human Rhinovirus/Enterovirus assay with both B. parapertussis and B. pertussis is noted in the device labeling.

Table 6. Results from cross-reactivity testing with “on-panel” viruses

| Species | Source Identity | Concentration (per mL) | Observed Cross-reaction |
| --- | --- | --- | --- |
| Adenovirus A | Zeptometrix 0810073CF | 1.4 x 10⁵ TCID₅₀ | None |
| Adenovirus B | Zeptometrix 0810062CF | 1.2 x 10⁷ TCID₅₀ | None |
| Adenovirus C | Zeptometrix 0810110CF | 2.2 x 10⁶ TCID₅₀ | None |
| Adenovirus D | Zeptometrix 0810119CF | 1.7 x 10⁵ TCID₅₀ | None |
| Adenovirus E | Zeptometrix 081070CF | 1.5 x 10⁵ TCID₅₀ | None |
| Adenovirus F | Zeptometrix 0810085CF | 1.1 x 10⁶ TCID₅₀ | None |
| Coronavirus 229E | ATCC VR-740 | 8.9 x 10⁶ TCID₅₀ | None |
| Coronavirus HKU1 | Clinical specimen-1 ¹ | 4.5 x 10⁷ copies | None |
| | Clinical specimen-2 ² | | None ³ |
| Coronavirus NL63 | Zeptometrix 0810228CF | 5.0 x 10⁵ TCID₅₀ | None |
| Coronavirus OC43 | Zeptometrix 0810024CF | 3.6 x 10⁵ TCID₅₀ | None |
| Coronavirus SARS-CoV-2 | ATCC VR-1986HK | 7.6 x 10⁷ copies | None |
| Enterovirus D68 | ATCC VR-1823 | 1.6 x 10⁷ TCID₅₀ | None |
| Human Metapneumovirus A1 | Zeptometrix 0810161CF | 2.5 x 10⁵ TCID₅₀ | None |
| Human Metapneumovirus A2 | Zeptometrix 0810164CF | 3.6 x 10⁵ TCID₅₀ | None |
| Human Metapneumovirus B1 | Zeptometrix 0810156CF | 1.6 x 10⁴ TCID₅₀ | None |
| Human Metapneumovirus B2 | Zeptometrix 0810162CF | 1.3 x 10⁶ TCID₅₀ | None |
| Rhinovirus A1 | Zeptometrix 0810012CFN | 1.3 x 10⁶ TCID₅₀ | None |
| Influenza A H1N1pdm09 | Zeptometrix 08100538CF | 1.4 x 10⁵ TCID₅₀ | None |
| Influenza A H3N2 | Zeptometrix 010526CF | 7.2 x 10⁵ TCID₅₀ | None |
| Influenza B (Victoria) | BEI NR-44023 | 2.8 x 10⁸ CEID₅₀ | None |
| | Zeptometrix 0810037CF | 2.5 x 10⁵ TCID₅₀ | None |
| Influenza B (Yamagata) | Zeptometrix 0810256CF | 2.1 x 10⁴ TCID₅₀ | None |
| Parainfluenza Virus 1 | Zeptometrix 0810014CF | 4.2 x 10⁵ TCID₅₀ | None |
| Parainfluenza Virus 2 | Zeptometrix 0810015CF | 1.2 x 10⁷ TCID₅₀ | None |
| Parainfluenza Virus 3 | Zeptometrix 0810016CF | 3.4 x 10⁷ TCID₅₀ | None |
| Parainfluenza Virus 4a | Zeptometrix 0810060CF | 3.4 x 10⁷ TCID₅₀ | None |
| Respiratory Syncytial Virus A | Zeptometrix 0810040ACF | 4.2 x 10⁵ TCID₅₀ | None |
| Respiratory Syncytial Virus B | Zeptometrix 0810479CF | 4.2 x 10⁵ TCID₅₀ | None |

CEID₅₀: Chicken Egg Infectious Dose-50%; TCID₅₀: Tissue Culture Infectious Dose-50%

¹ Previously reported positive for Parainfluenza Virus 4 in addition to Coronavirus HKU1

² Previously reported positive for Adenovirus in addition to Coronavirus HKU1

³ 1/3 replicates positive for Adenovirus (not considered cross-reactivity based on previous test results for the source specimen)

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**Table 7.** Results from cross-reactivity testing with “on-panel” bacteria

| Species | Source Identity | Concentration (per mL) | Observed Cross-reaction |
| --- | --- | --- | --- |
| *Bordetella parapertussis* | Zeptometrix 0801462 | 4.6 x 10^{9} CFU | None ^{1} |
| *Bordetella pertussis* | Zeptometrix 0801459 | 1.3 x 10^{10} CFU | Human Rhinovirus/Enterovirus ^{2} |
| | | 1.3 x 10^{9} CFU | None |
| *Chlamydia pneumoniae* | ATCC 53592 | 2.9 x 10^{7} IFU | None |
| *Mycoplasma pneumoniae* | Zeptometrix 0801579 | 2.5 x 10^{7} CCU | None |

CCU: Color Changing Units; CFU: Colony Forming Units; IFU: Inclusion Forming Units

$^{1}$ *In silico* analysis predicted detection by the Human Rhinovirus/Enterovirus assay but none was observed

$^{2}$ Cross-reaction also predicted by *in silico* analysis

The potential for cross-reaction with off-panel analytes was evaluated by testing 87 stains of bacteria, 2 species of fungus and 7 viruses that are either phylogenetically related to the on-panel analytes or which are commonly found in the respiratory tract (**Table 8**). No cross-reaction was observed other than the detection of one strain of *B. bronchiseptica* by the assay for *B. parapertussis* that was predicted by *in silico* analysis.

**Table 8.** Results from cross-reactivity testing with “off-panel” bacteria, viruses and fungi

| Species | Source Identity | Concentration (per mL) | Observed Cross-reaction |
| --- | --- | --- | --- |
| **Bacteria** | | | |
| *Arcanobacterium bernardiae* | ATCC BAA-441 | 1.6 x 10^{9} cells | None |
| *Arcanobacterium haemolyticum* | ATCC 9345 | 1.5 x 10^{8} cells | None |
| *Arcanobacterium pyogenes* | ATCC 49698 | 6.7 x 10^{9} cells | None |
| *Bacillus cereus* | ATCC 7064 | 8.3 x 10^{9} cells | None |
| *Bordetella bronchiseptica* | ATCC 10580 | 8.3 x 10^{9} cells | None |
| | ATCC 4617 | 7.9 x 10^{9} cells | None |
| | ATCC 19395 | 7.9 x 10^{9} cells | None |
| | NRRL B-59914 | 7.1 x 10^{9} cells | None |
| | NRRL B-59909 | 28 cells | *B. parapertussis*^{1} |
| | | 2.8 cells | *B. parapertussis*^{1} |
| *Bordetella holmesii* | ATCC 700052 | 8.3 x 10^{9} cells | None |
| *Burholderia cepacia* | ATCC 51671 | 7.9 x 10^{9} cells | None |
| *Campylobacter rectus* | ATCC 33238 | 7.6 x 10^{7} cells | None |
| *Chlamydia trachomatis* | Zeptometrix 0801775 | 1.3 x 10^{8} IFU | None |
| *Corynebacterium diphtheriae* | ATCC 27010 | 8.0 x 10^{9} cells | None |
| *Corynebacterium pseudodiphtheriticum* | ATCC 10700 | 8.7 x 10^{9} cells | None |
| *Enterococcus casseliflavus* | ATCC 49605 | 8.0 x 10^{9} cells | None |
| *Enterococcus faecalis* | Zeptometrix 0801637 | 8.0 x 10^{9} cells | None |
| *Escherichia coli* | ATCC BAA-2196 | 7.2 x 10^{9} cells | None |
| *Fusobacterium nucleatum* | ATCC 25586 | 4.9 x 10^{8} cells | None |
| *Fusobacterium necrophorum* subsp. *funduliforme* | ATCC 51357 | 4.4 x 10^{8} cells | None |
| *Fusobacterium varium* | ATCC 27725 | 1.6 x 10^{8} cells | None |
| *Gemella haemolysans* | ATCC 10379 | 4.0 x 10^{9} cells | None |
| *Gemella morbillorum* | ATCC 27824 | 1.0 x 10^{8} cells | None |
| *Granulicatella adiacens* | ATCC 49175 | 1.3 x 10^{9} cells | None |
| *Haemophilus influenzae* | ATCC 10211 | 8.3 x 10^{9} cells | None |

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| Species | Source Identity | Concentration (per mL) | Observed Cross-reaction |
| --- | --- | --- | --- |
| Haemophilus parahaemolyticus | ATCC 49700 | 8.7 x 10⁹ cells | None |
| Klebsiella pneumoniae | CDC AR-BANK#0115 | 7.3 x 10⁹ cells | None |
| Lactobacillus rhamnosus | ATCC 7469 | 7.9 x 10⁹ cells | None |
| Lactobacillus lactis | ATCC 29146 | 6.2 x 10⁹ cells | None |
| Legionella pneumophila | ATCC 33215 | 7.0 x 10⁹ cells | None |
| Leptotrichia buccalis | ATCC 14201 | 4.4 x 10⁸ cells | None |
| Moraxella catarrhalis | ATCC 43627 | 7.2 x 10⁹ cells | None |
| Mycobacterium tuberculosis | Zeptometrix 0801660 | 6.1 x 10⁶ cells | None |
| Mycoplasma buccale | Mycoplasma Experience NC10136 | 1.4 x 10⁷ CFU | None |
| Mycoplasma faucium | Mycoplasma Experience NC10174 | 1.4 x 10⁶ CFU | None |
| Mycoplasma fermentans | Mycoplasma Experience NC10117 | 2.8 x 10⁷ CFU | None |
| Mycoplasma genitalium | Mycoplasma Experience NC10195 | 1.8 x 10⁶ CFU | None |
| Mycoplasma hominis | Mycoplasma Experience NC10111 | 1.2 x 10⁷ CFU | None |
| Mycoplasma lipophilum | Mycoplasma Experience NC10173 | 1.5 x 10⁶ CFU | None |
| Mycoplasma orale | Mycoplasma Experience NC10112 | 2.2 x 10⁷ CFU | None |
| Mycoplasma salivarium | Mycoplasma Experience NC10113 | 4.4 x 10⁶ CFU | None |
| Neisseria elongata | ATCC 25295 | 8.5 x 10⁹ cells | None |
| Neisseria gonorrhoeae | Zeptometrix 0801482 | 4.9 x 10⁷ CFU | None |
| Neisseria lactamica | ATCC 23971 | 2.7 x 10⁹ cells | None |
| Neisseria meningitidis | ATCC 13113 | 7.4 x 10⁹ cells | None |
| Neisseria sicca | ATCC 9913 | 7.2 x 10⁹ cells | None |
| Neisseria subflava | ATCC 49275 | 8.0 x 10⁹ cells | None |
| Parvimonas micra | ATCC 33270 | 6.0 x 10⁷ cells | None |
| Pneumocystis carinii | ATCC PRA-159 | 1.0 x 10⁷ nuclei | None |
| Porphyromonas endodontalis | ATCC 35406 | 1.6 x 10⁷ cells | None |
| Porphyromonas gingivalis | ATCC BAA-308 | 5.0 x 10⁸ cells | None |
| Prevotella histicola | BEI HM-471 | 9.0 x 10⁸ cells | None |
| Prevotella melaninogenica | ATCC 25845 | 6.9 x 10⁸ cells | None |
| Prevotella oralis | ATCC 33322 | 6.2 x 10⁸ cells | None |
| Pseudomonas aeruginosa | CDC AR-BANK#0092 | 8.3 x 10⁹ cells | None |
| Rhodococcus equi | ATCC 33706 | 7.3 x 10⁹ cells | None |
| Serratia marcescens | ATCC 27137 | 8.9 x 10⁹ cells | None |
| Staphylococcus aureus | ATCC BAA-1700 | 7.4 x 10⁹ cells | None |
| Staphylococcus epidermidis | ATCC 12228 | 8.0 x 10⁹ cells | None |
| Staphylococcus haemolyticus | ATCC 29968 | 8.0 x 10⁹ cells | None |
| Staphylococcus intermedius | ATCC 29663 | 8.2 x 10⁹ cells | None |
| Staphylococcus saprophyticus | ATCC 15305 | 8.1 x 10⁹ cells | None |
| Streptococcus agalactiae | ATCC 13813 | 6.0 x 10⁹ cells | None |
| Streptococcus anginosus | ATCC 700231 | 7.1 x 10⁹ cells | None |
| Streptococcus constellatus subsp. pharyngitis | NCTC 13122 | 5.6 x 10⁸ cells | None |
| Streptococcus dysgalactiae subsp. dysgalactiae | ATCC 43078 | 6.7 x 10⁹ cells | None |
| | NCTC 4669 | 7.4 x 10⁹ cells | None |
| | NCTC 4335 | 8.4 x 10⁹ cells | None |

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| Species | Source Identity | Concentration (per mL) | Observed Cross-reaction |
| --- | --- | --- | --- |
| | NCTC 4670 | 6.6 x 10^{9} cells | None |
| | CCUG 27665 | 7.4 x 10^{9} cells | None |
| | CCUG 28112 | 6.7 x 10^{9} cells | None |
| | CCUG 28114 | 7.5 x 10^{9} cells | None |
| Streptococcus dysgalactiae subsp. equisimilis (human) | Zeptometrix 0801516 | 7.8 x 10^{8} CFU | None |
| Streptococcus dysgalactiae subsp. equisimilis (pig) | CCUG 28117 | 7.1 x 10^{9} cells | None |
| Streptococcus dysgalactiae subsp. equisimilis (horse) | CCUG 27664 | 7.5 x 10^{9} cells | None |
| | ATCC 10009 | 6.9 x 10^{9} cells | None |
| Streptococcus gallolyticus | ATCC 43143 | 2.8 x 10^{9} cells | None |
| Streptococcus gordonii | ATCC 10558 | 4.5 x 10^{9} cells | None |
| Streptococcus intermedius | ATCC 27335 | 2.9 x 10^{9} cells | None |
| Streptococcus mitis | ATCC 15914 | 3.2 x 10^{9} cells | None |
| Streptococcus mutans | ATCC 25175 | 2.3 x 10^{9} cells | None |
| Streptococcus oralis | ATCC 10557 | 1.1 x 10^{9} cells | None |
| Streptococcus parasanguinis | ATCC 15912 | 7.8 x 10^{9} cells | None |
| Streptococcus pneumoniae | ATCC 49619 | 2.5 x 10^{8} cells | None |
| Streptococcus pyogenes | ATCC 12344 | 3.4 x 10^{8} cells | None |
| Streptococcus salivarius | ATCC 13419 | 6.6 x 10^{9} cells | None |
| Streptococcus sanguinis | ATCC 10556 | 1.1 x 10^{9} cells | None |
| Tannerella forsythia | ATCC BAA-2717 | 2.6 x 10^{8} cells | None |
| Treponema denticola | ATCC 33520 | 2.2 x 10^{8} cells | None |
| Ureaplasma urealyticum | ATCC 27618 | 5.7 x 10^{7} cells | None |
| Veillonella parvula | ATCC 10790 | 4.7 x 10^{8} cells | None |
| **Yeast** | | | |
| Candida albicans | ATCC MYA-2876 | 2.8 x 10^{8} cells | None |
| Saccharomyces cerevisiae | ATCC 18824 | 1.9 x 10^{8} cells | None |
| **Viruses** | | | |
| Cytomegalovirus | Zeptometrix 0810003CF | 1.9 x 10^{5} TCID_{50} | None |
| Epstein Barr Virus | Zeptometrix 0810008CF | 5.9 x 10^{6} copies | None |
| Human Herpes Simplex Virus 1 | ATCC VR-260 | 8.9 x 10^{6} TCID_{50} | None |
| Measles Virus | Zeptometrix 0810025CF | 2.5 x 10^{5} TCID_{50} | None |
| Middle East Respiratory Syndrome Coronavirus | Zeptometrix 0810575CFHI | 1.2 x 10^{5} TCID_{50} | None |
| Mumps virus | Zeptometrix 0810079CF | 2.0 x 10^{5} TCID_{50} | None |
| Severe Acute Respiratory Syndrome Coronavirus | BEI NR-52346 | 5.3 x 10^{5} genomes | None |

CFU: Colony Forming Units; IFU: Inclusion Forming Units; TCID$_{50}$: Tissue Culture Infectious Dose-50%

$^{1}$ B. bronchiseptica NRRL B-59909 carries IS1001 which is the target for the SPOTFIRE R Panel B. parapertussis assay. Positive results were obtained with all target levels ≥ 28 cells/mL and with 1/3 replicates at 2.8 cells/mL.

Based on a combination of the in silico and laboratory testing data, the potential for cross-reaction of the SPOTFIRE R Panel with certain on- and off-panel analytes is noted in the device labeling, as summarized in Table 9.

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**Table 9.** Summary of known or predicted cross-reactivity

| SPOTFIRE R Panel Assay | Cross-reactive Species | Description |
| --- | --- | --- |
| SARS-CoV-2 | Bat coronavirus Pangolin coronavirus Bat SARS-like coronavirus | Predicted cross-reaction with closely related sarbecoviruses isolated from bats and pangolin |
| *Bordetella parapertussis* | *Bordetella bronchiseptica* (isolates carrying IS1001) | Some strains of *B. bronchiseptica* carry IS1001, the target for the *B. parapertussis* assay |
| Human Rhinovirus/Enterovirus | *Bordetella bronchiseptica* *Bordetella parapertussis* *Bordetella pertussis* Bovine and canine picornaviruses | The Human Rhinovirus/Enterovirus assay may amplify homologous sequences in the listed organisms |
| Influenza A Virus A/H1-2009 | Influenza A H1N1 (swine) | The FluA-H1-2009 assay may detect influenza A H1 gene sequences of swine origin, that will be reported as Influenza A Virus (subtype H1-2009) or as Influenza A (No Subtype Found), depending on the strain and concentration present Certain strains of avian H1N2 may also be detected and reported as H1-2009 |
| *Chlamydia pneumoniae* | *Chlamydia gallinacea* | The *C. pneumoniae* assay may amplify homologous sequences in the listed species |

#### *Interference Testing*

A study was conducted to evaluate the potential for assay interference due to the presence of endogenous and exogenous substances that may be present in upper respiratory specimens. In addition, testing was also performed with representative on- and off-panel microorganisms at very high concentrations to evaluate the potential for competitive interference between analytes. Testing of each potentially interfering substance/microorganism was performed using contrived positive and negative specimens. The positive specimens contained representative on-panel analytes at concentrations equivalent to 3X their respective LoD (**Table 10**). The concentration of each potentially interfering substance tested was chosen to represent a high level that may be found in natural clinical specimens. The results of the study are summarized in **Tables 11-13**.

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**Table 10.** Representative on-panel analytes used to evaluate assay interference

| Assay | Analyte | Characteristics | Concentration (per mL)^{1} |
| --- | --- | --- | --- |
| Adenovirus | Adenovirus B Type 3 (ZeptoMetrix 0810062CF) | Non-enveloped DNA virus | 2.4 TCID_{50} |
| *Chlamydia pneumoniae* | *Chlamydia pneumoniae* AR-39 (ATCC 53592) | Gram negative bacterium | 60 IFU |
| Seasonal Coronavirus | Coronavirus NL63 (ZeptoMetrix 0810228CF) | Enveloped RNA virus | 0.0075 TCID_{50} |
| Human Rhinovirus/Enterovirus | Human Rhinovirus A1 ZeptoMetrix 0810012CFN | Non-enveloped RNA virus | 0.63 TCID_{50} |
| Influenza B Virus | Influenza B virus B/Florida/02/06 (Victoria) (ZeptoMetrix 0810255CF) | Enveloped RNA virus | 0.099 TCID_{50} |

IFU: Inclusion Forming Units; TCID$_{50}$: Tissue Culture Infectious Dose-50%

$^{1}$ 3X LoD

**Table 11.** Results from testing the effect of endogenous substances on the performance of the SPOTFIRE R Panel

| Potentially Interfering Substance | Concentration | Contrived Samples | Number of Positive Results by Assay | | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | Adenovirus | Seasonal Coronavirus | Coronavirus SARS-CoV-2 | Human Metapneumovirus | Human Rhinovirus / Enterovirus | Influenza A | Influenza A H1-2009 | Influenza A H3 | Influenza B Virus | *B. parapertussis* | *B. pertussis* | *C. pneumoniae* | *M. pneumoniae* |
| Whole human blood (sodium citrate) | 10% v/v | Negative | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| | | Positive | 3/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 |
| Human sputum/mucus | 1% v/v | Negative | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| | | Positive | 3/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 |
| Human genomic DNA | 20 ng/μL | Negative | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| | | Positive | 5/5 | 5/5 | 0/5 | 0/5 | 4/5^{1} | 0/5 | 0/5 | 0/5 | 5/5 | 0/5 | 0/5 | 5/5 | 0/5 |

Results from the representative assays for which Contrived Positive Samples were prepared are shown in red

Contrived Positive Samples contained the representative on-panel analytes at 3X LoD

Contrived Negative Samples contained only the potentially interfering substance

$^{1}$ Initially, 1/3 samples was reported as negative; therefore, 2 additional replicates were tested, both of which produced the expected results for all analytes

None of the endogenous substances tested caused reproducible interference with the SPOTFIRE R Panel.

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**Table 12.** Results from testing the effect of exogenous substances on the performance of the SPOTFIRE R Panel

| Potentially Interfering Substance | Concentration | Contrived Samples | Number of Positive Results by Assay | | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | Adenovirus | Seasonal Coronavirus | Coronavirus SARS-CoV-2 | Human Metapneumovirus | Human Rhinovirus / Enterovirus | Influenza A | Influenza A H1-2009 | Influenza A H3 | Influenza B Virus | B. parapsilosis | B. pertussis | C. pneumoniae | M. pneumoniae |
| Promethazine hydrochloride | 3.3 x 10^{-4} mg/mL | Negative | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| | | Positive | 3/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 |
| Acetaminophen (paracetamol) | 1.5 x 10^{-1} mg/mL | Negative | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| | | Positive | 3/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 |
| Acetylsalicylic acid (aspirin) | 3.0 x 10^{-2} mg/mL | Negative | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| | | Positive | 3/3 | 3/3 | 0/3 | 0/3 | 4/5^{1} | 0/3 | 0/3 | 0/3 | 5/5 | 0/3 | 0/3 | 5/5 | 0/3 |
| Ibuprofen | 2.2 x 10^{-1} mg/mL | Negative | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| | | Positive | 5/5 | 5/5 | 0/5 | 0/5 | 4/5^{1} | 0/5 | 0/5 | 0/5 | 5/5 | 0/5 | 0/5 | 5/5 | 0/5 |
| Albuterol sulfate | 5.4 x 10^{-5} mg/mL | Negative | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| | | Positive | 3/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 |
| Triple antibiotic ointment (neomycin/polymyxin B/bacitracin) | 2% w/v | Negative | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| | | Positive | 3/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 |
| Mucinex Severe Nasal Congestion Relief Clear & Cool Spray | 1% v/v | Negative^{2} | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| | | Positive | 3/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 |
| Saline nasal spray (0.65% disodium phosphate phenylcarbinol, monosodium phosphate, benzalkonium chloride) | 1% v/v | Negative | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| | | Positive | 3/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 |
| Vicks VapoRub Cough Suppressant Topical Analgesic (4.8% camphor, 1.2% eucalyptus oil, 2.6% menthol) | 1% w/v | Negative | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| | | Positive | 3/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 |
| Vaseline petroleum jelly (100% white petrolatum) | 1% w/v | Negative | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| | | Positive | 3/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 |
| Orajel (0.13% benzalkonium chloride, 20% benzocaine, 0.5% menthol, 0.15% zinc chloride) | 2% w/v | Negative | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| | | Positive | 3/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 |
| Chloraseptic Sore Throat Spray (1.4% phenol) | 1% v/v | Negative | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| | | Positive | 3/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 |
| Vicks Formula 44 DM | 1% v/v | Negative | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |

Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.gov K213954 - Page 27 of 59

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| Potentially Interfering Substance | Concentration | Contrived Samples | Number of Positive Results by Assay | | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | Adenovirus | Seasonal Coronavirus | Coronavirus SARS-CoV-2 | Human Metapneumovirus | Human Rhinovirus / Enterovirus | Influenza A | Influenza A H1-2009 | Influenza A H3 | Influenza B Virus | B. parapertussis | B. pertussis | C. pneumoniae | M. pneumoniae |
| (0.67 mg/mL dextromethorphan hydrobromide, 13 mg/mL) | | Positive^{2} | 3/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 |
| Phenylephrine hydrochloride | 1% w/v | Negative | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| | | Positive | 3/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 |
| Nasal spray (50 mg fluticasone propionate) | 1% v/v | Negative | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| | | Positive | 5/5 | 5/5 | 0/5 | 0/5 | 4/5^{1} | 0/5 | 0/5 | 0/5 | 5/5 | 0/5 | 0/5 | 5/5 | 0/5 |
| Sucrets Sore Throat (2.0 mg dyclonine hydrochloride/lozenge) | 1% w/v | Negative | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| | | Positive | 3/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 |
| Benadryl Allergy Liqui-gels (25 mg/capsule diphenhydramine hydrochloride) | 1% v/v | Negative | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| | | Positive^{2} | 3/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 |
| Zican Cold Remedy (galphimia glauca, luffa operculata, sabadilla) | 1% v/v | Negative | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| | | Positive | 3/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 |
| Cold-eeze (2.3% zinc gluconate) | 1% w/v | Negative | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| | | Positive | 3/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 |
| HALLS lozenge (5 mg menthol/lozenge) | 1% w/v | Negative | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| | | Positive | 3/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 |
| Listerine Cool Mint (0.042% menthol, 0.064% thymol, 0.06% methyl salicylate, 0.092% eucalyptol) | 6.5% v/v | Negative | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| | | Positive | 3/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 |
| Copenhagen Snuff (tobacco) | 1% w/v | Negative | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| | | Positive | 5/5 | 5/5 | 0/5 | 0/5 | 4/5^{1} | 0/5 | 0/5 | 0/5 | 5/5 | 0/5 | 0/5 | 5/5 | 0/5 |
| Juice Head (30% polypropylene glycol, 70% vegetable glycerin) | 1% v/v | Negative | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| | | Positive | 3/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 |

Results from the representative assays for which Contrived Positive Samples were prepared are shown in red

Contrived Positive Samples contained the representative on-panel analytes at 3X LoD

Contrived Negative Samples contained only the potentially interfering substance

$^{1}$ Initially, 1/3 samples was reported as negative; therefore, 2 additional replicates were tested, both of which produced the expected results for all analytes
$^{2}$ Initially, 1/3 samples was reported as invalid; the sample was retested and the results for all analytes were as expected

None of the endogenous substances tested caused reproducible interference with the SPOTFIRE R Panel.

Food and Drug Administration

10903 New Hampshire Avenue

Silver Spring, MD 20993-0002

www.fda.gov K213954 - Page 28 of 59

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**Table 13.** Results from testing the effect of substances associated with specimen collection on the performance of the SPOTFIRE R Panel

| Potentially Interfering Substance | Concentration | Contrived Samples | Number of Positive Results by Assay | | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | Adenovirus | Seasonal Coronavirus | Coronavirus SARS-CoV-2 | Human Metapneumovirus | Human Rhinovirus / Enterovirus | Influenza A | Influenza A H1-2009 | Influenza A H3 | Influenza B Virus | B. parapsilosis | B. pertussis | C. pneumoniae | M. pneumoniae |
| Rayon swab (Copan Diagnostics, Inc) | 1 swab, 15 min^{1} | Negative | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| | | Positive | 3/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 |
| Nylon flocked swab (Copan Diagnostics, Inc.) | 1 swab, 15 min^{1} | Negative | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| | | Positive | 3/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 |
| Polyester swab (Copan Diagnostics, Inc.) | 1 swab, 15 min^{1} | Negative | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| | | Positive | 3/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 |
| Calcium alginate swab (Puritan) | 1 swab, 15 min^{1} | Negative | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| | | Positive | 3/3 | 3/3 | 0/3 | 0/3 | 3/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 3/3 | 3/3 |
| Cary-Blair Medium | 90% v/v | Negative | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| | | Positive | 3/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 |
| Dulbecco's Modified Eagles Medium (DMEM) | 90% v/v | Negative | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| | | Positive | 3/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 0/3 | 3/3 | 0/3 | 0/3 | 3/3 | 0/3 |
| 0.9% Normal Saline | 90% v/v | Negative | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 |
| | | Positive | 3/3 | 3/3 |…