Predicate Devices

Not Found

Reference Devices

AI/ML (Artificial Intelligence / Machine Learning)

No
The description focuses on PCR-based nucleic acid detection and automated instrument processes, with no mention of AI or ML for data analysis or interpretation beyond standard automated result reporting.

Therapeutic

No

This device is designed for the detection and identification of microbial nucleic acids to aid in the diagnosis of respiratory infections, not for treatment or therapy.

Diagnostic

Yes

The "Intended Use / Indications for Use" states that the device aids in the diagnosis of respiratory infection and is for in vitro diagnostic use. The purpose is to detect and identify multiple respiratory viral and bacterial nucleic acids in samples from individuals suspected of respiratory tract infections.

SaMD (Software as a Medical Device)

No

The device description clearly states that the BioFire RP2.1 is a PCR-based multiplexed nucleic acid test that requires the use of the BioFire FilmArray 2.0 or BioFire FilmArray Torch systems, which are described as instruments that interact mechanically, thermally, and optically with a physical pouch containing reagents. While software is used to control the process and interpret results, the core functionality relies on hardware components and physical reagents.

IVD (In Vitro Diagnostic)

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

Here's why:

Intended Use: The document explicitly states the device is "intended for use... for the simultaneous qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals suspected of respiratory tract infections, including COVID-19." This describes a test performed in vitro (outside the body) on a biological sample (NPS) to provide information for diagnosis.
Device Description: The description details a "PCR-based in vitro diagnostic BioFire FilmArray 2.0 and BioFire FilmArray Torch systems for infectious disease testing." It also describes the use of reagents and a closed system disposable pouch to perform sample preparation, PCR, and detection of nucleic acids from patient samples. These are all characteristics of an in vitro diagnostic device.
Intended User / Care Setting: The statement "For in vitro diagnostic use only" directly confirms its classification as an IVD.

The entire description aligns with the definition of an In Vitro Diagnostic device, which is a medical device intended for use in vitro for the examination of specimens derived from the human body solely or principally for the purpose of providing information concerning a physiological or pathological state, or a congenital abnormality, or to determine the safety and compatibility with potential recipients, or to monitor therapeutic measures.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

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 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 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 a patient with possible respiratory tract infection.

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

QOF

Device Description

The BioFire Respiratory Panel 2.1 is designed to simultaneously identify 22 different potential pathogens of the respiratory tract infection, including the novel coronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), from a single NPS specimen in transport medium or saline. BioFire RP2.1 is compatible with BioFire's PCR-based in vitro diagnostic BioFire FilmArray 2.0 and BioFire FilmArray Torch systems for infectious disease testing. A specific software module (i.e., BioFire RP2.1 Pouch Module Software) is used to perform BioFire RP2.1 testing on these systems.

The RP2.1 reagent kit contains all the materials required to complete tests and includes the RP2.1 pouch, hydration solution, sample buffer, and sample handling components such as transfer pipettes. The RP2.1 pouches are used to test patient samples and is a closed-system disposable that stores all the necessary reagents for sample preparation reverse transcription. polymerase chain reaction (PCR), and detection in order to isolate, amplify, and detect nucleic acid from multiple pathogens within a single NPS specimen. The rigid plastic component ("fitment") of the pouch contains reagents in freeze-dried form. The flexible plastic portion of the pouch is divided into discrete segments ("blisters") where the required chemical processes are carried out. After sample collection, the user injections hydration solution and sample combined with BioFire Sample Buffer into the pouch, places the pouch into a FilmArray instrument, and starts the run. All other operations are automated.

The FilmArray instruments (FilmArray 2.0 and FilmArray Torch systems) interact with the pouch mechanically, thermally, and optically to drive the multi-step chemical process required for purification and detection of specific nucleic acid targets from the patient sample. FilmArray instruments follow a protocol defined in the BioFire RP2.1 Pouch Module Software that is downloaded from the host computer prior to runtime. The instrument protocol defines the specific sequence of the testing process, including the times and temperatures, as the instrument performs bead-based extraction/isolation/purification of nucleic acids, performs reverse transcription and a 2-stage nested PCR reaction, executes DNA melt and fluorescent signal detection, and monitors system performance in real time, and communicates results and errors to the user via software. The primary difference between the FilmArray 2.0 and FilmArray Torch systems is the external configuration of multiple modules in a system. Up to eight FilmArray 2.0 modules can be connected to one computer and pouch loading station, while up to 12 FilmArray Torch modules can be connected to one system base in a vertical stack to a computer and pouch loading station. In addition, the pouches are front-loaded via an automated mechanism for the Torch system whereas the pouches are manually inserted, removed, and there is pouch and lid sensing in the FilmArray 2.0.

Once a test run is completed, the software automatically interprets the results and displays a test report. The report can be printed and/or saved as a file. The test report is a single page containing three sections: Run Summary, Result Summary, and Run Details. An additional section, Change Summary, is present in specific situations. The overall layout of the report was previously described in the BioFire RP2 510(k) [K170604] and remains unchanged for the BioFire RP2.1.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

nasopharyngeal swabs (NPS)

Indicated Patient Age Range

Not Found

Intended User / Care Setting

For prescription use only.
For in vitro diagnostic use only.

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

Not Found

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

Description of the test set:
Clinical studies were performed using prospectively collected nasopharyngeal swab (NPS) specimens and natural retrospective leftover (archived) clinical specimens. Additionally, contrived clinical specimens were used.

Sample Size:

Prospective Clinical Study: 524 valid specimens
Retrospective Clinical Study: 98 evaluable specimens (50 positive for SARS-CoV-2, 50 negative for SARS-CoV-2, with 2 exclusions for instrument error)
Contrived Clinical Specimen Study: 50 contrived specimens (spiked with SARS-CoV-2) and 10 non-spiked specimens.

Data Source:

Prospective Clinical Study: specimens collected from three collection sites.
Retrospective Clinical Study: 50 natural retrospective leftover (archived) clinical specimens from three geographically distinct laboratories in the United States. SARS-CoV-2 positive specimens collected during March and April 2020. SARS-CoV-2 negative specimens collected before December 2019.
Contrived Clinical Specimen Study: unique NPS specimens collected before December 2019.

Annotation Protocol:

Prospective Clinical Study: The BioFire RP2.1 was evaluated by comparing the test results for SARS-CoV-2 with a composite comparator of three U.S. FDA EUA tests. Concordance for two out of three of the EUA tests were considered the final result for the comparator.
Retrospective Clinical Study: SARS-CoV-2 positive specimens had been previously characterized as positive for SARS-CoV-2 using the respective Emergency Use Authorization (EUA) assay employed at the collection site. NPA determined by comparing observed test result for SARS-CoV-2 negative specimens to the expected result of Not Detected.
Contrived Clinical Specimen Study: PPA determined by comparing the observed test results for samples contrived in unique clinical specimens to the expected Detected result.

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

1. Analytical Performance:
a. Precision/Reproducibility:
- Study Type: Multi-variable study evaluating reproducibility of BioFire RP2.1 analyte detection on FilmArray 2.0 and FilmArray Torch systems using contrived samples.
- Sample Size: Each sample type (negative, 1x LoD, 3x LoD) was tested repeatedly in three different testing sites over five days (20 replicates per sample at each site on both FilmArray systems), for a total of 120 valid runs per sample and 360 valid runs in total for the entire study.
- Key Results:
- Valid results were obtained in 361 of 363 runs (99.4%).
- Reproducibility of detection (percent agreement with expected results) ranged from 90.0% to 100% for various analytes at 1x LoD and 3x LoD concentrations, and 100% for negative samples. For SARS-CoV-2, detection was 96.7% at 1x LoD and 100% at 3x LoD (60/60) on FilmArray 2.0, and 100% for both on FilmArray Torch.
- No pattern in "Not Detected" results was observed in study variables.
b. Detection limit (LoD):
- Study Type: LoD estimation and confirmation testing for all analytes, including detailed analysis for SARS-CoV-2 in both inactivated and infectious forms, and for other analytes in VTM and saline matrices.
- Sample Size: Typically 20 replicates at 1x LoD and 20 replicates at 0.1x LoD for confirmation testing.
- Key Results:
- SARS-CoV-2 (inactivated): Confirmed LoD of 5.0E+02 copies/mL (100% detection at 1x LoD, 25% at 0.1x LoD).
- SARS-CoV-2 (infectious): Confirmed LoD of 1.6E+02 copies/mL (100% detection at 1x LoD, 55% at 0.1x LoD).
- Other analytes in VTM: LoD concentrations confirmed, generally with >=95% detection at 1x LoD and

Regulation Number and Section

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

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

Summary

0

EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR BIOFIRE RESPIRATORY PANEL 2.1 DECISION SUMMARY

A. De Novo Number:

DEN200031

B. Purpose for Submission:

De Novo request for evaluation of automatic class III designation for the BioFire Respiratory Panel 2.1 (RP2.1).

C. Measurands:

The assay detects and identifies nucleic acids of the following respiratory pathogens: Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2), Adenovirus, Coronavirus 229E, Coronavirus HKU1, Coronavirus NL63, Coronavirus OC43, 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.

D. Type of Test:

A multiplexed nucleic acid test intended for use with the BioFire FilmArray Torch systems for the simultaneous qualitative detection of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals suspected of respiratory tract infections, including COVID-19.

E. Applicant:

BioFire Diagnostics, LLC

F. Proprietary and Established Names:

BioFire Respiratory Panel 2.1 (RP2.1)

G. Regulatory Information:

1. Regulation section:
  21 CFR 866.3981
1. Classification:
  Class II (special controls)

1

3. Product code(s):

QOF

1. Panel:
  Microbiology (83)

H. Indications for Use:

1. Indication(s) for use:
  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 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 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

2

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 a patient with possible respiratory tract infection.

2. Special conditions for use statement(s):

For prescription use only.

For in vitro diagnostic use only.

1. Special instrument requirements:
  FilmArray Respiratory Panel 2.1 (RP2.1) is performed on the FilmArray 2.0 or the FilmArray Torch systems.

I. Device Description:

The BioFire Respiratory Panel 2.1 is designed to simultaneously identify 22 different potential pathogens of the respiratory tract infection, including the novel coronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), from a single NPS specimen in transport medium or saline. BioFire RP2.1 is compatible with BioFire's PCR-based in vitro diagnostic BioFire FilmArray 2.0 and BioFire FilmArray Torch systems for infectious disease testing. A specific software module (i.e., BioFire RP2.1 Pouch Module Software) is used to perform BioFire RP2.1 testing on these systems.

The RP2.1 reagent kit contains all the materials required to complete tests and includes the RP2.1 pouch, hydration solution, sample buffer, and sample handling components such as transfer pipettes. The RP2.1 pouches are used to test patient samples and is a closed-system disposable that stores all the necessary reagents for sample preparation reverse transcription. polymerase chain reaction (PCR), and detection in order to isolate, amplify, and detect nucleic acid from multiple pathogens within a single NPS specimen. The rigid plastic component ("fitment") of the pouch contains reagents in freeze-dried form. The flexible plastic portion of the pouch is divided into discrete segments ("blisters") where the required chemical processes are carried out. After sample collection, the user injections hydration solution and sample combined with BioFire Sample Buffer into the pouch, places the pouch into a FilmArray instrument, and starts the run. All other operations are automated.

The FilmArray instruments (FilmArray 2.0 and FilmArray Torch systems) interact with the pouch mechanically, thermally, and optically to drive the multi-step chemical process

3

required for purification and detection of specific nucleic acid targets from the patient sample. FilmArray instruments follow a protocol defined in the BioFire RP2.1 Pouch Module Software that is downloaded from the host computer prior to runtime. The instrument protocol defines the specific sequence of the testing process, including the times and temperatures, as the instrument performs bead-based extraction/isolation/purification of nucleic acids, performs reverse transcription and a 2-stage nested PCR reaction, executes DNA melt and fluorescent signal detection, and monitors system performance in real time, and communicates results and errors to the user via software. The primary difference between the FilmArray 2.0 and FilmArray Torch systems is the external configuration of multiple modules in a system. Up to eight FilmArray 2.0 modules can be connected to one computer and pouch loading station, while up to 12 FilmArray Torch modules can be connected to one system base in a vertical stack to a computer and pouch loading station. In addition, the pouches are front-loaded via an automated mechanism for the Torch system whereas the pouches are manually inserted, removed, and there is pouch and lid sensing in the FilmArray 2.0.

Once a test run is completed, the software automatically interprets the results and displays a test report. The report can be printed and/or saved as a file. The test report is a single page containing three sections: Run Summary, Result Summary, and Run Details. An additional section, Change Summary, is present in specific situations. The overall layout of the report was previously described in the BioFire RP2 510(k) [K170604] and remains unchanged for the BioFire RP2.1-

BioFire® Respiratory Panel 2.1
www.BioFireDx.com
Run Summary
Sample ID:	RP2.1example	Run Date:	04 April 2020
5:21 PM
Detected:	Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)	Controls:	Passed
Equivocal:	➔Influenza A
	Result Summary
	Viruses
Not Detected	Adenovirus
Not Detected	Coronavirus 229E
Not Detected	Coronavirus HKU1
Not Detected	Coronavirus NL63
Not Detected	Coronavirus OC43
✓ Detected	Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)
Not Detected	Human Metapneumovirus
Not Detected	Human Rhinovirus/Enterovirus
➔ Equivocal	Influenza A
Not Detected	Influenza B
Not Detected	Parainfluenza Virus 1
Not Detected	Parainfluenza Virus 2
Not Detected	Parainfluenza Virus 3
Not Detected	Parainfluenza Virus 4
Not Detected	Respiratory Syncytial Virus
	Bacteria
Not Detected	Bordetella parapertussis (IS1001)
Not Detected	Bordetella pertussis (ptxP)
Not Detected	Chlamydia pneumoniae
Not Detected	Mycoplasma pneumoniae
	Run Details
Pouch:	RP2.1 v1.0	Protocol:	NPS2 v3.2
Run Status:	Completed	Operator:	JDoe
Serial No.:	01234567	Instrument:	TM8CCF3

Test results for the organisms included in the BioFire RP2.1 are provided in two locations on the report. The Result Summary section provides a complete list of the test results. Possible results include "Detected," "Not Detected," "Equivocal," and "Invalid." Positive (Detected) and Equivocal results are also displayed in the Run Summary section. The following table

4

provides an explanation for each interpretation and any follow-up necessary to obtain a final result.

Result	Explanation	Action
Detecteda	The run was successfully
completed AND
The pouch controls were successful (Passed)
AND
The assay(s) for the organism were
POSITIVE
(i.e., met the requirements for a positive
result)	Report results.
Not Detected	The run was successfully
completed AND
The pouch controls were successful (Passed)
AND
The assay(s) for the organism were NEGATIVE
(i.e., did not meet the requirements for a positive
result)	Report results.
Equivocal	The run was successfully
completed AND
The pouch controls were successful (Passed)
AND
The combination of positive and negative assay results
for Influenza A were inconclusive	Retest the original sample ONCE and report
the result of the retest.
Invalid	The pouch controls were not successful
(Failed)
OR
The run was not successful (Run Status displayed as:
Aborted, Incomplete, Instrument Error, or Software Error)	See Interpretation of control fields on the
BioFire RP2.1 test report for instruction.

Table 1. Explanation of Reported Results and Required Actions

a If four or more organisms are detected in a specimen, retesting is recommended to confirm the polymicrobial result.

For most organisms detected by the BioFire RP2.1. the organism is reported as Detected if a single corresponding assay is positive. For example, Human Metapneumovirus will have a test report result of "Human Metapneumovirus Detected" if at least two of the three replicates of the one Human Metapneumovirus assay (hMPV) have similar positive melt peaks with Tm values that are within the assay-specific Tm range.

In contrast, the test results for SARS-CoV-2, Adenovirus, and Influenza A depend on the interpretation of results from more than one assay. Interpretation results for all organisms detected by the BioFire RP2.1, except for SARS-CoV-2, are previously described in the BioFire RP2 510(k) submission [K170604] and remain unchanged for the BioFire RP2.1.

The BioFire RP2.1 pouch contains two different assays for the detection of the SARS-CoV-2 microorganism. The assays each target a spike protein (S) gene and membrane protein (M) gene respectively. The BioFire FilmArray software interprets each of these assays independently and the results are combined as a final test result for the virus. An assay is called positive if at least two of the three replicates within the pouch have similar positive melt peaks with Tm values that are within the assay-specific Tm range. If either one or both of the assays is called

5

positive, the test report will show Severe Acute Respiratory Coronavirus 2 (SARS-CoV-2) as Detected. If all assays are called negative. the test report will be Severe Acute Respiratory Coronavirus 2 (SARS-CoV-2) Not Detected.

J. Standard/Guidance Document Referenced (if applicable):

General

Guidance for Clinical Laboratories, Commercial Manufactures, and FDA Staff Policy for ● Coronavirus Disease-
2019 Tests During the Public Health Emergency (2020) ●
. 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
. 2017/746 Regulation EU 2017/746 of the European Parliament and of the Council of 5 April 2017 on in vitro diagnostic medical devices and repealing Directive 98/79/EC and Commission Decision 2010/227/EU
2016/679 GDPR, Regulation EU 2016/679 of the European Parliament and of the Council of 27 April 2016 on the protection of natural persons with regard to the processing of personal data and on the free movement of such data, and repealing Directive 95/46/EC (General Data Protection Regulation)
. Guidance for Industry - Part 11. Electronic Records: Electronic Signatures - Scone 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 IRBs, Clinical Investigators, and Sponsors - Informed Consent Information Sheet (July 2014)
FDA Draft Guidance Use of Electronic Records and Electronic Signatures in Clinical . Investigations Under 21 CFR Part 11 - Questions and Answers (June 2017)
. Guidance for Industry and FDA Staff – Acceptance of Clinical Data to Support Medical Device Applications and Submissions - Frequently Asked Questions (February 2018)
Guidance for Sponsors, Investigators, and IRBs – Impact of Certain Provisions of the Revised Common Rule on FDA-Regulated Clinical Investigations (October 2018)
ICH E6(R1) Guideline for Good Clinical Practice E6(R1) June 1996 ●
ICH E6(R2) Integrated Addendum to ICH E6(R1): Guideline for Good Clinical Practice E6(R2) ● – November 2016
. Guidance for Industry and Food and Drug Administration Staff – Highly Multiplexed Microbiological/Medical Countermeasure In Vitro Nucleic Acid Based Diagnostic Devices, (August 27, 2014)
. Statistical Guidance on Reporting Results from Studies Evaluating Diagnostic Tests, FDA Guidance Document (March 13, 2007)
User Protocol for Evaluation of Qualitative Test Performance, Clinical and Laboratory Standards ● Institute (CLSI) Approved Guideline - Second Edition, EP12-A2 (January 2008)
Molecular Diagnostic Methods for Infectious Diseases, Clinical and Laboratory Standards ● Institute (CLSI) Proposed Guideline, MM3-P2 (February 2006)

6

. Interference Testing in Clinical Chemistry, 3rd Edition, Clinical and Laboratory Standards Institute (CLSI) Approved Guideline, EP07 (April 2018).
. CLSI EP25-A, 'Evaluation of stability of in vitro diagnostic reagents; Approved Guidelines'.
Guidance for Sponsors. Institutional Review Boards. Clinical Investigators and FDA Staff . Guidance on Informed Consent for In Vitro Diagnostic Device Studies Using Leftover Human Specimens that are Not Individually Identifiable, (April 2006)

Software

Guidance for Industry and FDA Staff, Guidance for the Content of Premarket Submissions for Software Contained in Medical Devices (May 11, 2005)
. Off-The-Shelf Software Use in Medical Devices, Guidance for Industry and Food and Drug Administration Staff (September 27, 2019)
. General Principle of Software Validation; Final Guidance for Industry and FDA Staff (January 11, 2002)
. Content of Premarket Submissions for Management of Cybersecurity in Medical Devices. Guidance for Industry and Food and Drug Administration Staff (October 2, 2014)

Labeling

Use of Symbols on Labels and in Labeling of In Vitro Diagnostic Devices Intended for Professional Use, FDA Guidance Document (November 30, 2004)
. Guidance for Industry and FDA on Alternative to Certain Prescription Device Labeling Requirements (January 1, 2000)

FDA-recognized Standards

ISO 14971:2007 'Medical devices – Application of risk management to medical devices'
EN 62366:2008/IEC 62366-1:2015, 'Medical device Application of usability engineering to . medical devices'
. ISO 62304:2006, 'Medical device software – Software life-cycle processes' – IEC 62304:2006, November 27, 2008
. ISO 15223-1:2012, 'Medical Devices – Symbols to be used with medical device labels, labeling and information to be supplied - Part 1: General requirements'

Non-recognized Standards

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 (European Commission)
EN ISO 18113-1:2011, 'In vitro diagnostic medical devices - Information supplied by the manufacturer (labeling) - Part 1: 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 23640:2015, 'In vitro diagnostic medical devices – Evaluation of stability of in vitro diagnostic reagents'

7

K. Test Principle:

The BioFire RP2.1 test takes approximately 2 minutes of hands-on-time from the point of collection to the initiation of the automated test. Once the test is initiated, a test result is produced in approximately 45 minutes.

During a test. the FilmArray instrument. software, and pouch work together to generate assay results. The test works through automated sample processing and nested multiplex nucleic acid amplification (including reverse transcription as appropriate) followed by highresolution melt analysis to confirm the identity of the amplified product. The basic sequence of actions and their associated instrument functions are outlined in Figure 1-

Image /page/7/Figure/3 description: The image shows a diagram of a multi-step process. The process starts with "Sample Lysis", which involves bead beating, reagent addition, and liquid movement. The next step is "Nucleic Acid Isolation", which involves reagent addition, liquid handling, and magnetic beads. The process continues with "Reverse Transcription and 1st Stage PCR", which involves temperature control, reagent addition, and liquid handling, and ends with "2nd Stage PCR and Detection", which involves temperature control, LED control, and camera control.

Figure 1. Basic steps performed during BioFire RP2.1 testing

The pouch contains all the necessary PCR reagents and is where samples are automatically processed to generate test results. The instrument communicates with the host computer and the FilmArray software. The software provides instructions to the instrument to control the various test steps. The instrument drives the testing process by applying mechanical force on the pouch exterior to actuate liquid movement to various compartments and to seal or block off flow in particular channels. The instrument also thermally interacts with the pouch to perform the subsequent 2-stage nested PCR reactions.

Optical systems on-board the instrument that include a LED and digital camera allow illumination and recording of fluorescence generated in the second stage PCR. The fluorescence signal generated during DNA melting is automatically analyzed by the FilmArray software from replicate wells of each assay for the detection of amplicons with a specific Tm. The detection denotes the presence of specific bacterial or viral targets.

The BioFire RP2.1 pouch contains the same sample preparation and PCR reaction chemistry as the previously cleared BioFire FilmArray Respiratory Panel 2 (RP2) (K170604; cleared for use on both FilmArray 2.0 and FilmArray Torch Systems). The PCR1 primer multiplex is also the same, with the addition of SARS-CoV-2 primers. The PCR2 array is similar except with the additions and minor reconfiguration of wells to accommodate the two SARS-CoV-2 assays. In addition, the instrument protocol and the analysis parameters in the panel-specific pouch module are the same as for FilmArray RP2, with the additional analysis of the SARS-CoV-2 assays.

8

The BioFire RP2.1 procedure occurs in six steps below. This simple procedure minimizes specimen manipulation and reduces operator error.

. Step 1 - Place pouch into the FilmArray Pouch Loading Station.
Step 2 Hydrate pouch using a blue Hydration Injection Vial. ●
Step 3 Prepare sample in the red Sample Injection Vial: ●
- o Dispense the Sample Buffer tube into the Sample Injection Vial.
- With a transfer pipette, draw the NPS in transport media or saline sample to O the third line, then add it to the Sample Injection Vial.
- o Mix by inversion.
Step 4 Load sample mix in pouch. .
Step 5 Insert pouch into the instrument. ●
Step 6 Enter sample information and start the run. The BioFire RP2.1 protocol will ● be automatically selected upon scanning the pouch barcode.

The FilmArray software uses the following steps to interpret the melt curve data generated from each FilmArray RP2.1 assay-

Analysis of Melt Curves ●
- o The FilmArray RP2.1 Melt Detector first performs basic calculations on the melt data to determine if a PCR reaction occurred in each well. If the melt profile indicates that a PCR product is present, then the analysis software calculates one or two Tm values, depending on the number of melt curves present in the data, and the Tm values are compared against an expected melt range for the associate assay. If the software determines that the melt is positive and the melt curve falls inside the assay's specific melt range, thent he curve is called positive. If the software determines that the melt is negative or that it is not in the appropriate range, then the curve is called negative.
Analysis of Replicates ●
- o The analysis software evaluates the replicates for each assay (target and control) to determine if the assay is positive or negative. For a positive, at least two of the three wells associated with an assay must have a positive melt curve and the Tm for the positive curves must be similar (i.e., within 1°C). Assays that do not meet these criteria are called negative.
Analysis of Controls ●
- Results for control assays are compared to their expected values and are o reported as "Passed", "Failed" or "Invalid". Passed control result is for successful run completion AND both pouch controls were successful. Failed result is when the run was successfully completed BUT at least one of the pouch controls (RNA Process Control and/or PCR2 Control) failed. If the instrument detects an out-of-specification condition or a significant error, it will automatically abort the run. If this happens or if user aborts the run, the control result will display "Invalid" and all results in the Result Summary of the report will also be displayed as "Invalid." A Run Status indicating "Incomplete", "Aborted", "Software Error", or "Instrument Error" will be reported to the user and the operator is asked to consult with the manual for

9

specific instructions on resolving the error. The test should be repeated once error is corrected.

Interpretation of Assay Results ●
- Once the results for the individual assays are determined, the software applies o interpretation rules to determine the final test results. For most organisms detected by the BioFire RP2.1, the organism is reported as Detected if a single corresponding assay is positive. The BioFire RP2.1 also includes test results for organisms (i.e., SARS-CoV-2, Adenovirus, and Influenza A) that depend on interpretation of results from more than one assay. See the Interpretation of Results section for more information on interpreting these test results.

L. Performance Characteristics (if/when applicable):

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

A multi-variable study was performed to evaluate the reproducibility of BioFire RP2.1 analyte detection on FilmArray 2.0 and FilmArray Torch systems. This study was additive to the reproducibility evaluation performed for the BioFire RP2 device. with overlapping data for certain analytes to bridge results from the two panels and collect data for select analytes including the newly added SARS-CoV-2.

Contrived samples were used in this study to evaluate variability in between run, system, site, day, or lot. Three samples were prepared in a matrix of viral transport medium (Table 2) and data were collected representing a negative (no analyte) and those containing analytes at low positive (1x LoD) or moderate positive (3x LoD) concentrations. The positive samples included inactivated SARS-CoV-2, Coronavirus NL63, Influenza A H1-2009, and three analytes that had been previously evaluated for the BioFire RP2 reproducibility study (i.e., Adenovirus, Bordetella parapertussis (IS1001) and Respiratory Syncytial Virus).

Each sample was tested repeatedly in three (3) different testing sites over five days by different operators (at least two per site), on different systems (60 per system) and modules, using three different reagent kit lots. Twenty replicates per sample were tested at each site on both FilmArray systems for a total of 120 valid runs per sample and 360 valid runs in total for the entire study. Reproducibility of analyte detection was assessed as percent agreement with the expected Detected and Not Detected results for the positive and negative samples.

The performance of the FilmArray systems and BioFire RP2.1 Controls are summarized as follow. Valid results were obtained in 361 of the 363 runs that were initiated (361/363, 99.4%). There were 181 and 182 runs initiated on the FilmArray 2.0 and FilmArray Torch systems, respectively. There was one instrument error (FilmArray 2.0) and one aborted run (FilmArray Torch). This showed that performance of the controls was reproducible (no control failures) and valid results were obtained for all completed runs.

10

Reproducibility data for each BioFire RP2.1 analyte are summarized in Table 2. Results are organized by system type (i.e., FilmArray 2.0 or Torch), test site (Site A, B. C), and all sites/systems with the corresponding 95% confidence interval. The summary data are presented as a combination of results collected for reproducibility studies with the BioFire RP2.1 (gray highlight) and the previous RP2 devices.

Table 2. Reproducibility of Detection Results for BioFire RP2.1 Analytes

Highlighted data were collected with the BioFire RP2.1. Non-highted data was collected with the BioFire FilmArray RP2. The same number of replicates (120) were tested per sample on both panels, but testing was distributed differently between sites and systems.

| Analyte
(Isolate Source
ID) | Concentration
Tested | FilmArray 2.0 | | | | FilmArray Torch | | | |
|----------------------------------------------------------------------|--------------------------------------------------------------------------------------|-----------------|------------------|------------------|-------------------|------------------|-----------------|------------------|-------------------|
| | | Site A | Site B | Site C | System
Total | Site A | Site B | Site C | System
Total |
| Adenovirusa
(NIBSC
16/324)
WHO
International
Standard | Negative
(no analyte) | 20/20
(100%) | 20/20
(100%) | 20/20
(100%) | 60/60
(100%) | 20/20
(100%) | 20/20
(100%) | 20/20
(100%) | 60/60
(100%) |
| | Moderate
Positive
(3x LoD)
$9.0E+03$
IU/mL | 20/20
(100%) | 20/20
(100%) | 20/20
(100%) | 60/60
(100%) | 20/20
(100%) | 20/20
(100%) | 20/20
(100%) | 60/60
(100%) |
| | Low Positive
(1x LoD)
$3.0E+03$
IU/mL | 20/20
(100%) | 20/20
(100%) | 20/20
(100%) | 60/60
(100%) | 20/20
(100%) | 20/20
(100%) | 20/20
(100%) | 60/60
(100%) |
| Coronavirus
229E | Negative
(no analyte) | 60/60
(100%) | 60/60
(100%) | 60/60
(100%) | 180/180
(100%) | 60/60
(100%) | 60/60
(100%) | 60/60
(100%) | 180/180
(100%) |
| Coronavirus
HKU1 | Negative
(no analyte) | 60/60
(100%) | 60/60
(100%) | 60/60
(100%) | 180/180
(100%) | 60/60
(100%) | 60/60
(100%) | 60/60
(100%) | 180/180
(100%) |
| Coronavirus
NL63
(BEI NR-470) | Negative
(no analyte) | 20/20
(100%) | 20/20
(100%) | 20/20
(100%) | 60/60
(100%) | 20/20
(100%) | 20/20
(100%) | 20/20
(100%) | 60/60
(100%) |
| | Moderate
Positive
(3x LoD)
$7.5E-01$
TCID50/mL
(1.6E+02
copies/mL) | 20/20
(100%) | 20/20
(100%) | 20/20
(100%) | 60/60
(100%) | 20/20
(100%) | 20/20
(100%) | 20/20
(100%) | 60/60
(100%) |
| | Low Positive
(1x LoD)
$2.5E-01$
TCID50/mL
(5.4E+01
copies/mL) | 20/20
(100%) | 20/20
(100%) | 20/20
(100%) | 60/60
(100%) | 20/20
(100%) | 20/20
(100%) | 20/20
(100%) | 60/60
(100%) |
| Coronavirus
OC43 | Negative
(no analyte) | 60/60
(100%) | 60/60
(100%) | 60/60
(100%) | 180/180
(100%) | 60/60
(100%) | 60/60
(100%) | 60/60
(100%) | 180/180
(100%) |
| | Moderate
Positive
(3x LoD)
$9.0E+01$
TCID50/mL | - | 29/30
(96.7%) | 30/30
(100%) | 59/60
(98.3%) | 29/30
(96.7%) | - | 29/30
(96.7%) | 58/60
(96.7%) |
| Coronavirus OC43
(ATCC
VR-759) | Low Positive | - | 30/30
(100%) | 27/30
(90.0%) | 57/60
(95.0%) | 30/30
(100%) | - | 30/30
(100%) | 60/60
(100%) |

11

			Agreement with Expected Result
	Analyte
(Isolate Source	Concentration			Film Array 2.0		FilmArray Torch
ID)		Tested	Site A	Site B	Site C	System
Total	Site A	Site B	Site C	System
Total
		(1× LoD)
3.0E+01
TCID50/mL
man Metapneumovi	Human
Metapne
umovirus	Negative
(no analyte)	60/60
(100%)	60/60
(100%)	60/60
(100%)	180/180
(100%)	60/60
(100%)	60/60
(100%)	60/60
(100%)	180/180
(100%)
	Human
Metapne
umovirus
(Zeptome	Moderate
Positive
(3× LoD)
3.0E+01
TCID50/mL	-	30/30
(100%)	30/30
(100%)	60/60
(100%)	30/30
(100%)	-	30/30
(100%)	60/60
(100%)
	trix
0810161C
F)	Low Positive
(1× LoD)
1.0E+01
TCID50/mL	-	28/30
(93.3%)	30/30
(100%)	58/60
(96.7%)	30/30
(100%)	-	30/30
(100%)	60/60
(100%)
nan Rhinovirus/Enterovi	Human
Rhinovir
us/
Enterovir
us	Negative
(no analyte)	60/60
(100%)	60/60
(100%)	60/60
(100%)	180/180
(100%)	60/60
(100%)	60/60
(100%)	60/60
(100%)	180/180
(100%)
	Human
Rhinovir
us	Moderate
Positive
(3× LoD)
3.0E-01
TCID50/mL
(1.1E+02
copies/mL)	-	28/30
(93.3%)	30/30
(100%)	58/60
(96.7%)	30/30
(100%)	-	30/30
(100%)	60/60
(100%)
	(Zeptome
trix
0810012C
FN)	Low Positive
(1× LoD)
1.0E-01
TCID50/mL
(3.8E+01
copies/mL)	-	30/30
(100%)	30/30
(100%)	60/60
(100%)	30/30
(100%)	-	30/30
(100%)	60/60
(100%)
	Influenza A H1	Negative
(no analyte)	60/60
(100%)	60/60
(100%)	60/60
(100%)	180/180
(100%)	60/60
(100%)	60/60
(100%)	60/60
(100%)	180/180
(100%)
		Negative
(no analyte)	20/20
(100%)	20/20
(100%)	20/20
(100%)	60/60
(100%)	20/20
(100%)	20/20
(100%)	20/20
(100%)	60/60
(100%)
Influenza A
H1-2009
(Zeptometrix
0810109CFN)		Moderate
Positive
(3× LoD)
1.5E+00
TCID50/mL
(9.9E+02
copies/mL)	20/20
(100%)	20/20
(100%)	20/20
(100%)	60/60
(100%)	20/20
(100%)	20/20
(100%)	20/20
(100%)	60/60
(100%)
		Low Positive
(1× LoD)
5.0E-01
TCID50/mL
(3.3E+02
copies/mL)	20/20
(100%)	20/20
(100%)	20/20
(100%)	60/60
(100%)	20/20
(100%)	20/20
(100%)	20/20
(100%)	60/60
(100%)
Infl	Influenza
A H3	Negative
(no analyte)	60/60
(100%)	60/60
(100%)	60/60
(100%)	180/180
(100%)	60/60
(100%)	60/60
(100%)	60/60
(100%)	180/180
(100%)
	Analyte
(Isolate Source ID)	Concentration
Tested	FilmArray 2.0				FilmArray Torch
			Site A	Site B	Site C	System
Total	Site A	Site B	Site C	System
Total
	Influenza
A H3
(ATCC
VR-810)	Moderate
Positive
(3× LoD)
$3.0E-01$
TCID50/mL		29/30
(96.7%)	30/30
(100%)	59/60
(98.3%)	30/30
(100%)	-	30/30
(100%)	60/60
(100%)
		Low Positive
(1× LoD)
$1.0E-01$
TCID50/mL		30/30
(100%)	30/30
(100%)	60/60
(100%)	30/30
(100%)	-	30/30
(100%)	60/60
(100%)
Influenza B	Influenza
B	Negative
(no analyte)	60/60
(100%)	60/60
(100%)	60/60
(100%)	180/180
(100%)	60/60
(100%)	60/60
(100%)	60/60
(100%)	180/180
(100%)
	Influenza
B
(Zeptome
trix
0810037C
F)	Moderate
Positive
(3× LoD)
$1.5E+01$
TCID50/mL	-	30/30
(100%)	30/30
(100%)	60/60
(100%)	30/30
(100%)	-	30/30
(100%)	60/60
(100%)
		Low Positive
(1× LoD)
$5.0E+00$
TCID50/mL	-	30/30
(100%)	30/30
(100%)	60/60
(100%)	30/30
(100%)	-	30/30
(100%)	60/60
(100%)
	Parainfluenza
Virus 1	Negative
(no analyte)	60/60
(100%)	60/60
(100%)	60/60
(100%)	180/180
(100%)	60/60
(100%)	60/60
(100%)	60/60
(100%)	180/180
(100%)
Parainfluenza Virus 2	Parainflu
enza
Virus 2	Negative
(no analyte)	60/60
(100%)	60/60
(100%)	60/60
(100%)	180/180
(100%)	60/60
(100%)	60/60
(100%)	60/60
(100%)	180/180
(100%)
	Parainflu
enza
Virus 2
(Zeptome
trix
0810015C
F)	Moderate
Positive
(3× LoD)
$1.5E+00$
TCID50/mL	-	29/30
(96.7%)	30/30
(100%)	59/60
(98.3%)	30/30
(100%)	-	29/30
(96.7%)	59/60
(98.3%)
		Low Positive
(1× LoD)
$5.0E-01$
TCID50/mL	-	30/30
(100%)	27/30
(90.0%)	57/60
(95.0%)	30/30
(100%)	-	29/30
(96.7%)	59/60
(98.3%)
	Parainfluenza
Virus 3	Negative
(no analyte)	60/60
(100%)	60/60
(100%)	60/60
(100%)	180/180
(100%)	60/60
(100%)	60/60
(100%)	60/60
(100%)	180/180
(100%)
	Parainflu
enza
Virus 4	Negative
(no analyte)	60/60
(100%)	60/60
(100%)	60/60
(100%)	180/180
(100%)	60/60
(100%)	60/60
(100%)	60/60
(100%)	180/180
(100%)
Parainfluenza Virus 4	Parainflu
enza
Virus 4
(
Zeptomet
rix
0810060C
F)	Moderate
Positive
(3× LoD)
$1.5E+02$
TCID50/mL	-	30/30
(100%)	30/30
(100%)	60/60
(100%)	30/30
(100%)	-	30/30
(100%)	60/60
(100%)
		Low Positive
(1× LoD)
$5.0E+01$
TCID50/mL		29/30
(96.7%)	30/30
(100%)	59/60
(98.3%)	30/30
(100%)		29/30
(96.7%)	59/60
(98.3%)
		Negative
(no analyte)	20/20
(100%)	20/20
(100%)	20/20
(100%)	60/60
(100%)	20/20
(100%)	20/20
(100%)	20/20
(100%)	60/60
(100%)
Analyte
(Isolate Source
ID)	Concentration
Tested	FilmArray 2.0			Agreement with Expected Result	FilmArray Torch
		Site A	Site B	Site C	System
Total	Site A	Site B	Site C	System
Total
Respiratory
Syncytial
Virusb
(Zeptometrix
0810040ACF)	Moderate
Positive
(3× LoD)
6.0E-02
TCID50/mL
(2.7E+01
copies/mL)	20/20
(100%)	20/20
(100%)	20/20
(100%)	60/60
(100%)	20/20
(100%)	20/20
(100%)	20/20
(100%)	60/60
(100%)
	Low Positive
(1× LoD)
2.0E-02
TCID50/mL
(9.0E+00
copies/mL)	19/20
(95%)	20/20
(100%)	18/20
(90%)	57/60
(95%)	20/20
(100%)	20/20
(100%)	19/20
(95%)	59/60
(98.3%)
	Negative
(no analyte)	20/20
(100%)	20/20
(100%)	20/20
(100%)	60/60
(100%)	20/20
(100%)	20/20
(100%)	20/20
(100%)	60/60
(100%)
Severe Acute
Respiratory
Syndrome
Coronavirus 2
(SARS-CoV-2)
(ATCC VR-
1986HK)	Moderate
Positive
(3× LoD)
1.5E+03
copies/mL	20/20
(100%)	20/20
(100%)	20/20
(100%)	60/60
(100%)	20/20
(100%)	20/20
(100%)	20/20
(100%)	60/60
(100%)
	Low Positive
(1× LoD)
5.0E+02
copies/mL	20/20
(100%)	19/20
(95%)	19/20
(95%)	58/60
(96.7%)	20/20
(100%)	20/20
(100%)	20/20
(100%)	60/60
(100%)
	Negative
(no analyte)	20/20
(100%)	20/20
(100%)	20/20
(100%)	60/60
(100%)	20/20
(100%)	20/20
(100%)	20/20
(100%)	60/60
(100%)
Bordetella
parapertussisc
(IS1001)
(Zeptometrix

```
                        | Moderate
```

Positive
(3× LoD)
1.8E+02
IS1001
copies/mL | 19/20
(95%) | 20/20
(100%) | 20/20
(100%) | 59/60
(98.3%) | 19/20
(95%) | 19/20
(95%) | 20/20
(100%) | 58/60
(96.7%) | | |
| | Low Positive
(1× LoD)
6.0E+01
IS1001
copies/mL | 20/20
(100%) | 20/20
(100%) | 20/20
(100%) | 60/60
(100%) | 20/20
(100%) | 19/20
(95%) | 20/20
(100%) | 59/60
(98.3%) | | |
| Bordetella pertussis (ptxP) | Bordet
ella
pertuss
is
(ptxP) | Negative
(no analyte) | 60/60
(100%) | 60/60
(100%) | 60/60
(100%) | 180/180
(100%) | 60/60
(100%) | 60/60
(100%) | 60/60
(100%) | 180/180
(100%) | |
| | Bordetell
a
pertussis
(ptxP)
(Zeptome
trix
0801459) | Moderate
Positive
(3× LoD)
3.0E+03
CFU/mL | | 30/30
(100%) | 30/30
(100%) | 60/60
(100%) | 30/30
(100%) | | 30/30
(100%) | 60/60
(100%) | |
| | Low Positive
(1× LoD)
1.0E+03
CFU/mL | | 30/30
(100%) | 30/30
(100%) | 60/60
(100%) | 28/30
(93.3%) | | 30/30
(100%) | 58/60
(96.7%) | | |
| Analyte
(Isolate Source
ID) | Concentration
Tested | Agreement with Expected Result | | | | | | | | | |
| | | | | Film Array 2.0 | | FilmArray Torch | | | | | |
| | | Site A | Site B | Site C | System
Total | Site A | Site B | Site C | System
Total | | |
| Chlamydia
pneumoniae | Negative
(no analyte) | 60/60
(100%) | 60/60
(100%) | 60/60
(100%) | 180/180
(100%) | 60/60
(100%) | 60/60
(100%) | 60/60
(100%) | 180/180
(100%) | | |
| Mycoplasma
pneumoniae | Negative
(no analyte) | 60/60
(100%) | 60/60
(100%) | 60/60
(100%) | 180/180
(100%) | 60/60
(100%) | 60/60
(100%) | 60/60
(100%) | 180/180
(100%) | | |

12

13

14

a Reproducibility of Adenovirus detection with the BioFire RP2 was 98.3% and 99.2% for the low and moderate positive samples, respectively and 100% for the negative sample(s).

b Respiratory Syncytial Virus results in the BioFire RP2 reproducibility study agreed with the expected result in 98.3 - 100% of the positive sample replicates in 100% of the negative sample replicates.

· Bordetella parapertussis (IS 1001) was detected in 93.3% of the low positive sample replicates tested and in 99.2% of the moderate positive sample replicates tested in the BioFire RP2 reproducibility study. Agreement with the expected result was 100% for the negative sample(s).

For the three analytes that had been evaluated in both studies, the reproducibility of detection observed for the BioFire RP2.1 was overall similar to what was observed for BioFire RP2. Overall, there were ten Not Detected results when the analyte was known to be present in the test sample in the reproducibility evaluations for the BioFire RP2.1. The observed Not Detected frequency is consistent with the test levels (BACTERIA | | | | | | | | | | | | |
| Bordetella
parapertussis | Zeptometrix
0801461 | 3.0E+02
copies/mL | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 |
| Bordetella
pertussis | Zeptometrix
0801459 | 1.0E+04
CFU/mLe | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 |
| Chlamydia
pneumoniae | ATCC
53592 | 6.7E+02
copies/mL | 9/10 | 30/30 | 8/10 | 30/30 | 9/10 | 30/30 | 10/10 | 30/30 | 8/10 | 30/30 |
| Mycoplasma
pneumoniae | Zeptometrix
0801579 | 4.7E+03
copies/mLe | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 |

" Unless otherwise noted, the concentration tested is 5× LoD was determined in the LoD study for saline sample type with the BioFre Respiratory Panel 2.1 (RP2.1).

"The storage claim is that samples can be frozen for up to 30 days. Although the T0 time point was collected on different analytes, the frozen ime point for all analytes was collected on this resulted in analytes being stored for variable durations and tested at different intervals after To. All analytes were tested at least 30 days containing Adenovins, Bordetella parapertussis, Coronavious NL63, Influenza A HINI-2009, Parainfluenza Virus 3, and Severe Acute Respiratory Syndrome Coronavirus 2 were tested 45 days after T0. Samples containing Chlamycia preumoniae, Coronavins OC43, Human Entervirus, Paranfluenza Virus 2 were tested 31 days after T0. Samples contaning Bordetella pertussis, Coronavins 229E, Coronavirus HKU1, Human Rhinoviros, Influenza A H1N1, Influenza A H1N1, Influenza B, Mycoplasma preumoniae, Parainfluenza Virus 1, Parainfinenza Virus 4, Middle East Respiratory Syndrome Coronavirus, and Respiratory Syncytal Virus were tested 30 days after TO

WHO IS = World Health Organization International Standard

4 TU = Intenational Unis. BioFire Diagnostics quantitive real-time PCR and denonstrated that 1.5E+04 IUinLis equivalent to 1.5E+04 copies/mL.

e Indicated concentration is 10× LoD.

4 Catalog or Source ID from Zeptometrix was previously 0810109CFN, as indicated on the stock was received.

6 Human Enterovins and Human Rhinovinus cannot be distingussed by the BloFire RP2.1 Panel. A detection of ether virus is reported as Hunan

Rhinovins Enterovins. Two organism mither virus, resulting in 20 expected Not Detected results at each time point.

4 Influenza A H3 was noorrectly spiked into one sample ma lower than expected organism concentration. It was then neluded in an additional mix at the appropriate concentration. Influenza A H3 results for the incorrectly formulated sample mix were excluded resulting in 10 expected Detected results and only 20 expected Not Detected results at each time point.

Additional saline specimen stability study (Mycoplasma pneumoniae, Parainfluenza Virus 2, and Respiratory Syncytial Virus (RSV))

In the original saline specimen stability study, three analytes were evaluated in concentrations that were not in accordance with the original study design and therefore vielded results that did not clarify possible stability trends. Therefore, an additional study was performed at the indicated 5x LoD as originally intended for those three analytes. A summary of the additional data collected for the three analytes are summarized in the below table (Table 6). Note that the conditions that were evaluated corresponded to sample storage claims, and included no storage control (TO), ambient (4 hours at 25℃), refrigerated (2-3 days at 8℃), and frozen (30 days at LoD testing for Adenovirus was performed with a World Health Organization (WHO) International Standard developed by NIBSC (National Institute for Biological

29

Standards and Control) that was released in late 2018 (NIBSC 16/324, 1st WHO International Standard for human adenovirus DNA for nucleic acid amplification techniques, version 1.0, dated 12/13/2018). This is a whole virus preparation of HAdV type 2 with a standardized concentration expressed in International Units (IU)}. All adenovirus cultures previously quantified by different species-specific qRT-PCR kits and assays were re-evaluated using a single quantitative assay (Adenovirus R-GENE) that was verified against the international standard.

LoD confirmation for the Adenovirus international standard was first performed in 2019 with the FilmArray RP2 to establish an Adenovirus LoD concentration of 3.0E+03 IU/mL (data provided in Table 13 and Table 14; equivalent to 3.0E+03 copies/mL when quantified with the Adenovirus R-GENE (bioMerieux) quantitative real-time PCR kit). The same samples used in establishing the Adenovirus LoD on the FilmArray RP2 with the international standard was tested with the BioFire RP2.1. Similar results were observed (Table 15), confirming identical LoD for Adenovirus (3.0E+03 IU/mL or 3.0E+03 copies/mL) on each panel.

Table 13. LoD Estimate Test Results for WHO Adenovirus International Standard (NIBSC 16/324) on BioFire FilmArray

The boxed data indicates the estimated LoD concentration
(b) (4)

Table 14. LoD Confirmation Results for WHO Adenovirus International Standard (NIBSC 16/324) on BioFire FilmArrav (b) (4)

1 Fryer JF, Hockley JG, Govind S, Morris CL and the Collaborative Study Group. Collaborative Study to Evaluate the Proposed 1st WHO International Standard for Human Adenovirus (HAdV) DNA for Nucleic Acid Amplification Techniques (NAT). WHO ECBS Report 2018; WHO/BS/2018.2346

30

(b) (4)

Table 15. Adenvirus LoD Verification and Confirmation Results (Species C Serviype 2; WHO International Standard, M
NIBSC 16/32)

(b) (4)

31

Additional Adenovirus LoD Data

The BioFire RP2.1 contains five assays for Adenovirus detection and the WHO International Standard (type C2) is amplified by the Adeno2 and Adeno6 assays. Therefore, for completeness two additional Adenovirus isolates (serotypes B7A and F41) were also tested at the RP2 1x LoD concentrations (8.7E+02 copies/mL and 1.1E+03 copies/mL. respectively by R-GENE quantification) and at 0.1x LoD. in order to collect data for each of the five adenovirus assays. In these Adenovirus isolates testing, twenty replicates of each sample were evaluated with both panels and demonstrated 100% detection of replicates (20/20) at the 1x LoD concentration for the FilmArray RP2 and BioFire RP2.1 devices. The inclusion of two additional isolates in the testing allowed for data to be collected from each assay (Adeno2 and Adeno7.1 for B7 and Adeno 3 and Adeno8 for F41). Both of the additional isolates were tested and detected as expected (Table 16) at a copies/mL concentration lower than that of the WHO International Standard. The LoD for the Adenovirus WHO International Standard is the Adenovirus LoD claim for the panel.

| Analyte | Isolate | LoD
Concentration a,b | FilmArray RP2
Detection Results | | BioFire RP2.1
Detection Results | |
|------------|------------------------------------------------|-----------------------------------------------------------------|------------------------------------|----------------|------------------------------------|----------------|
| | | | 1×LoD | 0.1×LoD | 1×LoD | 0.1×LoD |
| Adenovirus | Species B Serotype 7A
Zeptometrix 0810021CF | 5.0E-02 TCID50/mL
3.9E+01 copies/mLa
[8.7E+02 copies/mL]b | 20/20
100% | 12/20
60.0% | 20/20
100% | 14/20
70.0% |
| | Species F Serotype 41
ATCC VR-930 | 1.0E+00 TCID50/mL
1.2E+02 copies/mLa
[1.1E+03 copies/mL]b | 20/20
100% | 14/20
70.0% | 20/20
100% | 13/20
65.0% |

Table 16. Additional Adenovirus LoD Testing Data on FilmArray RP2 and BioFire RP2.1

4 Concentration based on quantification with the Genesig Adenovirus B kit (PrimerDesign, Ltd.) as described in K170604 b Concentration based on quantification with Adenovirus R-GENE (BioMerieux, ref. 69-010B)

The sample containing Human Metapneumovirus required a substitute culture that is only quantified in TCID50/mL units (same isolate, different culture event/lot#). Attempts to accurately quantify the substitute culture in copies/mL (similar to the original stock used in the RP2 LoD study) were not successful and therefore the sample could only be prepared based on the RP2 TCID50/mL LoD (1.0E+01 TCID50/mL). Testing at this concentration resulted in detection of the analyte in 19/20 and 20/20 replicates on FilmArray RP2 and BioFire RP2.1, respectively. However, the analyte was also detected in all replicates (20/20) on both panels at the 0.1× LoD concentration.

(b) (4)
(b) (4)	more robust detection at the 0.1x
level (Table 19) suggests that the substitute culture has a slightly higher nucleic acid
concentration compared to the original culture at the same TCID50/mL. Based on the

32

data provided, it appears that the BioFire RP2.1 hMPV amplification and detection are equivalent to FilmArray RP2 and the existing claimed LoD concentration (1.0E+01 TCID50mL and 1.2E+03 copies/mL) appears to be applicable to FilmArray RP2 and BioFire RP2.1.

Table 17. Comparison of Results at 1.0E+01 TCIDss/mL for Different Cultures of Human Metapneumovirus (Zeptometrix 0810161CF) (b) (4)

Table 18. Human Metapneumovirus LoD Verification and Confirmation Results (Zeptometrix 0810161CF) (BioFire RP2 and BioFire RP2 1)

33

(b) (4)

For Influenza A detection, the BioFire RP2.1 contains five assays, and including three assays used to differentiate the Influenza A hemagglutinin type. Testing for Influenza A H3 with BioFire FilmArray RP2 at the BioFire FilmArray RP2 LoD concentration generated 18/20 Influenza H3 Detected results, with Influenza A H3 Equivocal results for the other two replicates. Retesting is typically recommended for an Equivocal result, however, the retesting was not performed for these two sample replicates (Table 19). The BioFire RP2.1 data (20/20 at the 1× concentration and 4/20 at the 0.1× concentration) confirm that the concentration tested is an appropriate LoD for Influenza A H3 detection by BioFire RP2.1. The 2.1E+01 copies/mL confirmed LoD concentration is the same for each panel.

Table 19. Influenza A H3 LoD Verification and Confirmation Results (A/Port Chalmers/1/73, ATCC VR-810)

NSD = Influenza A (no subtype detected), InfA E = Influenza A Equivocal, H3 E = Influenza A H3 Equivocal (b) (4)

34

Image /page/34/Figure/0 description: The image shows the label '(b) (4)' in the upper left corner. The rest of the image is a gray rectangle. The OCR text indicates that the image is related to Chlamydia pneumoniae.

Chlannydia pneumoniae is the only analyte tested where results indicate a potential difference in LoD between the panels. The first sample prepared was detected in 20/20 replicates (100%) with the BioFire FilmArray RP2 test and in only 16/20 replicates in the BioFire RP2.1 testing. (b) (4)

, a second sample was prepared at the same concentration and tested on both panels again, with the same detection results (20/20 BioFire FilmArray RP2 and 16/20 BioFire RP2.1).

Table 20. Chlamydia pneumoniae LoD Testing Data on BioFire FilmArray RP2 and BioFire RP2.1 Pouches for Two Samples Prepared at the Same Concentration (1x LoD: 6.6E+01 copies/mL, 1.0E-01 TCID50/mL)

35

Testing was then performed with the BioFire RP2.1 pouches with a sample containing C. pneumoniae at a 2-fold higher concentration (1.3E+02 copies/mL) and the analyte was detected in all replicates (20/20, 100%) at the 1× concentration and in Limit of Detection for other (non-SARS-CoV-2) BioFire RP2.1 Analytes (Saline specimens)

36

When possible, testing was completed with the same stock/lot used to verify and confirm the LoD of the BioFire RP2.1 in VTM (results for 18/25 analytes of the RP2.1 panel are shown in Table 22). Among these, five analytes did not initially meet the acceptance criteria. These are indicated in the table by the addition of results from comparing to samples prepared in VTM. Additional evaluations were performed for these analytes including Adenovirus C, Coronavirus HKU1, and Respiratory Syncytial Virus. The SARS-CoV-2 detection rate in aNPSs is detailed above.

| Analyte | Isolate | LoD
Concentration | Detection Results | | | |
|-----------------------------------------------------------------------|-------------------------------------------------------------------------|--------------------------------------------|-------------------|---------------------|---------------|--------------|
| | | | Salinea | VTMb | | |
| | | | 1× LoD | 0.1×
LoD | 1× LoD | 0.1×
LoD |
| Viruses | | | | | | |
| Adenovirus | Species C Serotype 2
WHO Int'l Standard
NIBSC 16/324 | $3.0E+03 IU/mLc$
$(3.0E+03 copies/mL)e$ | 20/20d
100% | 20/20d
100% | 20/20
100% | 18/20
90% |
| | Species F Serotype 41
ATCC VR-930 | 1.0E+00 TCID50/mL
1.1E+03 copies/mL | 20/20
100% | 7/20
35% | | |
| Coronavirus
229E | ATCC VR-740 | 4.0E-01 TCID50/mL
6.5E+01 copies/mL | 20/20
100% | 8/20
40% | | |
| Coronavirus
HKU1 | Clinical NPS specimen
(53727) | 2.0E+03 copies/mL | 18/20
90% | 6/20
30% | 17/20
85% | 5/20
25% |
| Coronavirus
NL63 | BEI NR-470 | 2.5 E-01 TCID50/mL
5.4E+01 copies/mL | 20/20
100% | 10/20
50%
10% | | |
| Severe Acute
Respiratory Syndrome
Coronavirus 2
(SARS-CoV-2) | USA-WA1/2020
ATCC VR-1986HK
(heat inactivated) | $5.0E+02 copies/mLf$
2.1E-01 TCID50/mL | 17/20g
85% | 10/20g
50% | 20/20
100% | 5/20
25% |
| Human Metapneumovirus | 16, Type A1 IA10-2003
Zeptometrix 0810161CF | $1.0E+01 TCID50/mLh$ | 20/20
100% | 20/20
100% | | |
| Human Rhinovirus/
Enterovirus | Enterovirus D68
ATCC VR-1823 | 3.0E+02 TCID50/mL
2.6E+01 copies/mL | 19/20
95% | 6/20
30% | | |
| Influenza A H1 | Influenza A H1N1
A/New Caledonia/20/99
Zeptometrix 0810036CF | 1.0E+03 TCID50/mL
1.4E+02 copies/mL | 20/20
100% | 14/20
70% | | |
| Influenza A H1-2009i | Influenza A
H1N1pdm09
A/Swine/NY/03/2009
Zeptometrix 0810249CF | 5.0E-01 TCID50/mL
3.3E+02 copies/mL | 19/20
95% | 0/20
0% | | |
| Influenza B | B/FL/04/06
Zeptometrix 0810255CF | 5.0E+00 TCID50/mL
3.4E+01 copies/mL | 19/20
95% | 10/20
50% | | |
| Parainfluenza Virus 2 | Type 2
Zeptometrix 0810015CF | 5.0E-01 TCID50/mL
3.0E+01 copies/mL | 20/20
100% | 6/20
30% | | |
| Parainfluenza Virus 4 | Type 4a
Zeptometrix 0810060CF | 5.0E+01 TCID50/mL
1.6E+03 copies/mL | 20/20
100% | 4/20
20% | | |
| Respiratory Syncytial Virus | Type A
Zeptometrix
0810040ACF | 2.0E-02 TCID50/mL
9.0E+00 copies/mL | 16/20i
80% | 2/20J
10% | 16/20
80% | 7/20
35% |
| Bacteria | | | 20/20 | 11/20 | | |

Table 22. Limit of Detection (LoD) for BioFire Respiratory Panel 2.1 (RP2.1) Analytes in Saline.

37

| Bordetella parapertussis
(IS1001) | A747
Zeptometrix 0801461 | 6.0E+01 IS1001
copies/mL
4.1E+01 CFU/mL | 100% | 55% |
|--------------------------------------|-----------------------------|-----------------------------------------------|---------------|--------------|
| Bordetella pertussis
(ptxP) | A639
Zeptometrix 0801459 | 1.0E+03 CFU/mL | 20/20
100% | 12/20
60% |
| Mycoplasma pneumoniae | M129
Zeptometrix 0801579 | 4.6E+02 copies/mL | 19/20
95% | 9/20
45% |

a Testing was performed in aNPSs.

b Comparison to samples prepared in VTM are shown when initial testing results did not meet the acceptance criteria

C IU = International Units.

d Results shown are from a second sample containing Adenovirus C tested alongside sample prepared in VTM. In the first 0.1× sample

tested on the BioFire RP2.1/RP2.1plus. Adenovirus was Detected in 19/20 (95%) replicates (20/20.100% for the 1× sample). BioFire Diagnostics quantified the WHO International Standard by quantitative real-time PCR to demonstrate that 3.0E+03 IU/mL=3.0E+03 copies/mL.

4 Concentration of viral RNA in the culture was determined by digital droplet PCR, as indicated on the Certificate of Analysis from ATCC.

8 Results shown a second sample containing SARS-CoV-2 tested alongside sample prepared in VTM. In the first 1 × sample tested on the BioFire RP2.1. SARS-CoV-2 was Detected in 18/20 (90%) replicates (3/20, 15% for the 0.1× sample).

A copies/mL concentration for the culture of Human Metapneumovinus that was tested has not been determined.

1 Detection for Influenza A subtypes is calculated based only on the correct Influenza A (subtype) Detected result. An Equivocal or Influenza A (no subtype detected) result is tallied as a Not Detected result.

J Results shown are from a second sample containing Respiratory Syncytial Virus tested alongside sample prepared in VTM. In the first 1× sample tested on the BioFire RP2.1, Respiratory Syncytial Virus was Detected in 15/20 (75%) replicates (4/20, 20% for the 0.1× sample).

For seven analytes (Adenovirus, Coronavirus OC43, Human Rhinovirus/Enterovirus, Influenza A H3, Parainfluenza Virus 1, Parainfluenza Virus 3, and Chlamydia pneumoniae), it was necessary to perform testing using different organism stock cultures or lots than were previously evaluated on the RP2.1 device. For these analytes, LoD verification was completed by testing organism at a range of concentrations intended to bracket the LoD in samples prepared in both VTM and aNPSs. This procedure was used to preclude potential quantification differences in stocks used in the original study and subsequent saline media evaluations, that might confound a determination of the potential impact of media type on device performance. Detection results for these analytes indicated overall concordance at similar organism concentrations in both saline and VTM (Table 23).

Table 23. Limit of Detection (LoD) for BioFire Respiratory Panel 2.1 (RP2.1) Analytes that were Evaluated with New
Isolates in Saline and VTM.

		1× LoD Concentration	Detection Results
Analyte	Isolate		Salinea		VTM
			1× LoD	0.1× LoD	1× LoD	0.1× LoD
Adenovirus	Species B Serotype 3
Zeptometrix
0810062CF	1.2E+07 TCID50/mL
3.9E+02 copies/mLb	20/20
100%	9/20
45%	19/20
95%	12/20
60%
Coronavirus OC43	Zeptometrix
0810024CF	3.6E+05 TCID50/mL
5.6E+01 copies/mLc	20/20
100%	11/20
55%	19/20
95%	12/20
60%
Human Rhinovirus/
Enterovirus	Human Rhinovirus
Type 1 A
Zeptometrix
0810012CFN	1.3E+06 TCID50/mL
3.8E+01 copies/mLd	20/20
100%	20/20
100%	20/20
100%	15/20
75%
Influenza A H3	Hong Kong/4801/14
ZeptoMetrix
0810526CF	7.2E+05 TCID50/mL
2.1E-01 copies/mLe	19/20
95%	1/20
5%	20/20
100%	3/20
15%	Parainfluenza Virus 1	Type 1
Zeptometrix	4.2E+05 TCID50/mL
1.0E+02 copies/mLc	20/20
100%	3/20
15%	19/20
95%	8/20
40%
Influenza A H3	Hong Kong/4801/14
ZeptoMetrix
0810526CF	7.2E+05 TCID50/mL
2.1E-01 copies/mLe	19/20
95%	1/20
5%	20/20
100%	3/20
15%
Parainfluenza Virus 1	Type 1
Zeptometrix	4.2E+05 TCID50/mL
1.0E+02 copies/mLc	20/20
100%	3/20
15%	19/20
95%	8/20
40%

38

Analyte	Isolate	Detection Results
		Salinea	VTM
		1× LoD	0.1× LoD	1× LoD	0.1× LoD
Parainfluenza Virus 3	0810014CF
Type 3
Zeptometrix
0810016CF	3.4E+07 TCID50/mL
3.8E+01 copies/mLd	20/20
100%	9/20
45%	20/20
100%	12/20
60%
Chlamydia
pneumoniae	AR-39
ATCC 53592	2.9E+07 IFU/mL
1.3E+02 copies/mLd	19/20
95%	7/20
35%	18/20
90%	0/20
0%

a Testing was performed in aNPSs.

b 1× LoD value is approximately 2-fold lower than the value established when testing original stock in VTM.

e 1× LoD value is 10-fold lower than value established when testing original stocks in VTM.

d 1× LoD value is equivalent to value established when testing original stocks in VTM.

e 1× LoD value is 100-fold lower than value established when testing an original stock in VTM.

e. Analytical Reactivity (Inclusivity):

The BioFire RP2.1 contains assays for the detection of SARS-CoV-2 and multiple other viral and bacterial respiratory pathogens. Assays are designed to detect sequences of clinically relevant strains, serotypes, and/or genotypes of species that cause respiratory illness.

The reactivity of each assay in the BioFire RP2.1 device has been previously evaluated by in silico analysis and laboratory testing in studies for the performance evaluation of the BioFire RP2, except for SARS-CoV-2. The reactivity assessment and limitations defined in the previous studies also apply to the same assays in the BioFire RP2.1 because the sample type, assay primers, pouch chemistry and reaction conditions are unchanged. In brief, similar to the previous BioFire RP2 analytical reactivity study, when testing the BioFire RP2.1, each isolate that was evaluated with RP2.1 was prepared as a contrived sample in transport medium at a concentration near (3x) LoD and then tested in triplicate. Reactivity was established when the isolate was detected at a near-LoD concentration in 3/3 or 4/5 replicates, and any isolate or sequence with an observed or predicted issue with detection (i.e., requiring more than 10-fold LoD concentration) is defined as an assay limitation.

The Detection Limit section describe the limits of detection for the analytes in the RP2.1 panel. The focus of this reactivity study for the RP2.1 device was on evaluating the reactivity of the SARS-CoV-2 assays and revising or expanding the reactivity assessment for a small number of analytes with updated limits of detection (i.e., Adenovirus and C. pneumoniae).

In Silico Reactivity Analysis of SARS-CoV-2 Sequences

Evaluation of inclusivity (analytical reactivity) for the BioFire RP2.1 SARS-CoV-2 assays (SARSCoV2-1 and SARSCoV2-2) was based on in silico analysis of sequences from the NCBI and GISAID databases as of February 21, 2021.

In total, 467,066 sequences from around the globe were aligned to the assay primers. The sequences evaluated included the following lineages and variants of

39

concern (VOC) or variants under investigation (VUI) that may have important epidemiological, immunological, or pathogenic properties from a public health perspective:

· A.23 lineage (Uganda)
- o VUI-202102/01 (A.23.1 with E484K in Spike)
B.1.1, B1.1.7, B.1.258 lineages (United Kingdom; Δ69-70 and N501Y in Spike)
- o VOC-202012/01 (B.1.1.7)
- o VOC-202102/02 (B.1.1.7 with E484K in Spike)
B.1.1.28 lineage (Brazil) ●
- o VOC-202101/02 P1 variant (Brazil/Japan)
- o VUI-202101/01 P2 variant (Brazil)
B.1.1.318 (United Kingdom) ●
- o VUI-202102/04
B.1.351 lineage (South Africa) ●
- o VOC-202012/02 (501Y.V2 in Spike)
B.1.429 lineage (United States) ●
- o CAL.20C variant
B.1.525 lineage (United Kingdom) ● o VUI-202102/03 or UK1188
- B.1.526 (United States)

●

All lineages and variants of public health interest identified as of February 2021 are predicted to be detected. Approximately 1.2% of the sequences (5.405/467,066) have a mismatched base within the 3' half of a primer that may affect one assay, but will be detected by the second assay. Both SARS-CoV-2 assays of the BioFire RP2.1 device are predicted to be impaired for nine sequences (9/467,066) evaluated. In summary, this analysis determined that 99,998% (467,057/467,066) of the database sequences evaluated will be amplified by at least one of the SARS-CoV-2 assays in the BioFire RP2.1 device.

The analysis is summarized in the following table-

| Predicted Assay Result | SARSCoV2-1 | | # (%) sequences predicted to be
detected with no limitations
(one or both assays positive) | |
|------------------------|------------|---------|--------------------------------------------------------------------------------------------------|-----------------|
| | + | - | | |
| SARSCoV2-2 | + | 461,652 | 4581b | 467,057/467,066 |
| | - | 824 | 9c | (99.998%)** |

Table 24. In silico Prediction of SARS-CoV-2 Detection by the BioFire RP2.1 Assays®

a +/+ indicates detected by both assays with no impairment, +/- indicates detection by one assay with no impairment and potential for impaired detection by the other assay, -- indicates potential for impaired detection by both assays. b Includes sequences of lineage B.1.525 (VUI-202102/03), which has a mutation in the Spike gene that is predicted to impair detection by the SARS-CoV2-1 assay, but detection by the SARSCoV2-2 (Membrane gene) assay is predicted to be unaffected.

· Nine sequences have mismatches in the 3' half of primer(s) for both the SARSCoV2-1 and SARSCoV2-2 assays. The mismatches are predicted to impair detection for these sequences.

Empirical Evaluations for the Reactivity Analysis of SARS-CoV-2 Sequences

40

The in silico reactivity predictions were supplemented with empirical experiments to estimate the actual detection impact of the mismatches observed in two sequences (as indicated in the above In silico Prediction of SARS-CoV-2 Detection by the BioFire RP2.1 Assays table summary). Briefly, synthetic double-stranded gene fragments, (i.e., gBlocks) were synthesized for the regions of the Spike (S) gene amplified by the SARSCoV2-1 assay and the Membrane (M) gene amplified by the SARSCoV2-2 assay. One construct for each gene carried the consensus sequence with a perfect match to the assay primers (control) while the other was generated with the mismatches to primers, as indicated by the Oct 24, 2020 database alignment. Dilutions of the control and mismatch constructs were tested with the BioFire RP2.1 device.

Testing verified that the 3' terminal mismatch to one of the inner primers of the SARSCoV2-1 assay had a significant (10,000-fold) impact on amplification and detection relative to the control sequence. Further, a mismatch that is 7 bases in from the 3' end of a SARS-CoV2-2 inner primer had a much less impact (10-fold) on amplification and detection relative to the control sequence.

The analysis indicated that as of Oct 24, 2020, the BioFire RP2.1 device can detect the analyzed sequences though an impairment or limit of detection is predicted at low concentrations (≤10x LoD) for 2/130,788 sequences.

Reactivity for Other BioFire RP2.1 Analytes

The BioFire RP2.1 differs from the BioFire FilmArray RP2 by the addition of primers for the detection of SARS-CoV-2. The assay primers and reaction conditions for the viral and bacterial analytes shared between panels are unchanged, and testing has demonstrated that, with the exception of the updated Adenovirus evaluations based on the WHO International Standard and C. pneumoniae, the analytical LoD is unchanged for the assays shared between the two panels. The analytical inclusivity testing performed for the assessment of FilmArray RP2 assays (see K170604) were applied to the corresponding BioFire RP2.1 analytes with the following exceptions-

Testing of adenovirus isolates (same as in the RP2 study) but based on the . BioFire RP2.1 Adenovirus LoD newly established with the WHO International Standard (3.0E+03 IU/mL or copies/mL)
Testing of C. penumoniae isolates (same isolates as in RP2) but based on the ● LoD established for the BioFire RP2.1

Analytical reactivity testing for adenovirus with the BioFire RP2.1 included evaluating over 25 different isolates representing all but two for the known serotypes within the species associated with respiratory infection (i.e., B, C, and E) and representative serotypes for the other species. Adenovirus B serotype 55 and Adenovirus C serotype 57 were not tested but predicted to be detected efficiently with sequence analysis. At least one representative of each serotype within the Species A and F was tested, while only three different serotypes were tested as representative of species D supplemented with sequence analysis that predicted all other D serotypes

41

would be detected by the Adenovirus assays. An isolate of species G was not available for testing but in silico evaluation predicts that the Adenovirus assays react with species G, serotype 52. Overall, testing in triplicate demonstrated that all isolates were detected as expected on the BioFire RP2.1 at a concentration at or below 3x LoD (Table 26). Note that isolates tested in the Detection Limit study were not retested in this inclusivity testing but are listed for reference.

Table 25. Results for Adenovirus Inclusivity Testing on BioFire RP2.1 Isolates highlighted grey were tested in the BioFire RP2.1 LoD study and results are compiled from 20 replicates.

Isolates highlighted grey were tested in the BioFire RP2.1 LoD study and results are compiled from 20 replicates.
Species	Serotype	Isolate ID	Strain	xLoD	Test Concentration
(copies/mL)a	#Detected
/Total	Result	(b) (4)
A	Serotype 12	ATCC VR-863	Huie	3x	9.0E+03 copies/mL	3/3	D
	Serotype 18	ATCC VR-19	Washington D.C./1954	3x	9.0E+03 copies/mL	3/3	D
	Serotype 31	Zeptometrix 0810073CF	-	3x	9.0E+03 copies/mL	3/3	D
	Serotype 3	Zeptometrix 0810062CF	-	3x	9.0E+03 copies/mL	3/3	D
	Serotype 7A	Zeptometrix 0810021CF	-	0.3x	8.7E+02 copies/mL	20/20	D
	Serotype 7d/d2	UIRF	Iowa/2001	3x	9.0E+03 copies/mL	3/3	D
	Serotype 7h	UIRF	Iowa/1999	3x	9.0E+03 copies/mL	3/3	D
	B	Serotype 11	ATCC VR-12	-	3x	9.0E+03 copies/mL	3/3
Serotype 14		ATCC VR-15	-	3x	9.0E+03 copies/mL	3/3	D
Serotype 16		ATCC VR-17	CH.79/Saudi Arabia/1955	3x	9.0E+03 copies/mL	3/3	D
Serotype 21		ATCC VR-1833	-	3x	9.0E+03 copies/mL	3/3	D
Serotype 34		ATCC VR-716	Compton/19 72	3x	9.0E+03 copies/mL	3/3	D
Serotype 35		ATCC VR-718	Holden	3x	9.0E+03 copies/mL	3/3	D

42

	Serotype
	50	ATCC VR-
1602	Wan/Amster
dam/1988	3x	9.0E+03 copies/mL	3/3	D
D
D
C	1	Zeptometrix
0810050CF	-	3x	9.0E+03 copies/mL	3/3	D
D
D
	2	ATCC VR-
846	Adenoid 6	3x	9.0E+03 copies/mL	3/3	D
D
D
		NIBSC
16/324	WHO
International
Standard	1x	3.0E+03 copies/mL	20/20	D
D
	5	Zeptometrix
0810020CF	-	3x	9.0E+03 copies/mL	3/3	D
D
D
	6	ATCC VR-6	Tonsil
99/Wash DC	3x	9.0E+03 copies/mL	3/3	D
D
D
D	8	Zeptometrix
0810069CF	-	3x	9.0E+03 copies/mL	3/3	D
D
D
	20	Zeptometrix
0810115CF	-	3x	9.0E+03 copies/mL	3/3	D
D
D
	37	Zeptometrix
0810119CF	-	3x	9.0E+03 copies/mL	3/3	D
D
D
E	4a	UIRF 4a	S.
Carolina/200
4	3x	9.0E+03 copies/mL	3/3	D
D
D
	4	Zeptometrix
0810070CF	-	3x	9.0E+03 copies/mL	3/3	D
D
D
F	40	Zeptometrix
0810084CF	-	3x	9.0E+03 copies/mL	4/5	D
ND
D
	40	NCPV
0101141v	-	3x	9.0E+03 copies/mL	3/3	D
D
	41	ATCC VR-
930	Tak/73-
3544/Netherl
ands/1973	0.4x	1.1E+03	20/20	D
D
		Zeptometrix
0810085CF	-	3x	9.0E+03 copies/mL	3/3	D
D

a All isolates were quantified by a quantitative real-time PCR assay (R-GENE, BioMerieux).

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Analytical reactivity testing of C. pneumoniae isolates was performed according to the testing procedure followed for the FilmArray RP2 inclusivity study. Each isolate was prepared in VTM matrix at 3× the 1.3E+02 copies/mL BioFire RP2.1 LoD for this analyte and each was detected in the requisite 3/3 or 4/5 replicates tested (Table 27). The LoD isolate data (in grey) are shown for reference.

| Source/
Isolate ID | Strain/Location/
Year Isolated | Concentration
Tested | xLoD | # Detected
/Total | Result | (b) (4) |
|-----------------------|-----------------------------------|-------------------------|------|----------------------|--------|---------|
| ATCC a
VR-2282 | TW-183 | 1.3E+02
copies/mL | 1x | 20/20 | D | |
| ATCC
VR-1310 | CWL-029 | 3.9E+02
copies/mL | 3x | 4/5 | ND | |
| | | | | | D | |
| | | | | | D | |
| | | | | | D | |
| ATCC
VR-1360 | CM-1/Georgia | 3.9E+02
copies/mL | 3x | 3/3 | D | |
| | | | | | D | |
| | | | | | D | |
| ATCC
53592 | AR-39/Seattle/1983 | 3.9E+02
copies/mL | 3x | 3/3 | D | |
| | | | | | D | |
| | | | | | D | |

Table 26. BioFire RP2.1 Inclusivity Testing Results for C. pneumoniae Based on the Revised LoD

Summary of BioFire RP2.1 Analytical Reactivity

Cumulatively, through the in silico and empirical reactivity assessments, including those from shared assays in the BioFire RP2, 179 different viral and bacterial isolates were detected within 10x LoD.

It is notable that the Influenza A assays will react variably with non-human influenza A viruses and some rarely encountered human influenza A viruses that are not H1, H1-2009, or H3; generally producing Influenza A Equivocal or Influenza A (no subtype detected) results. Also, not evaluated in this study but in previous BioFire RP2 evaluations, the BioFire RP2.1 Bordetella pertussis. Influenza A (subtype). Influenza B and Human Rhinovirus/Enterovirus assays are predicted to react with the viral nucleic acids in B. pertussis, influenza, and poliovirus vaccines respectively. Thus, results should be interpreted with caution for specimens obtained from patients with such vaccinations.

The BioFire RP2.1 analytical reactivity test data is summarized as follows-

RP2.1 Analyte/Result	# of Isolates Detected	xLoD Detected	Description of Isolates Tested and Detected	Limitations
Viruses
Adenovirus	Species A	3	3x	A12, A18, A31	Noneb
	Species B	11	3x	B3, B7, B11, B14, B16, B21, B34, B35, B50
	Species C	5	3x	C1, C2, C5, C6
	Species D	3	3x	D8, D20, D37
RP2.1 Analyte/Result	# of Isolates Detected	xLoD Detected	Description of Isolates Tested and Detected	Limitations
Species E	2	3x	E4 and E4a
Species F	4	3x	F40 and F41
Coronavirus 229E	2	3x	Group 1 Coronavirus CoV-229E
Coronavirus HKU1	5	3x	Group 2 Coronavirus HKU1
Clinical specimens from 2010 and 2015	None
Coronavirus NL63	2	3x	Group 1 Coronavirus CoV-NL63
Coronavirus OC43	2	3x	Group 2 Coronavirus CoV-OC43
Severe Acute Respiratory Syndrome
Coronavirus 2 (SARS-CoV-2)	1c	1x	USA-WA1/2020
Human Metapneumovirus	11	3x	Genotypes: A1, A2, B1, B2
Subtypes: 3, 4, 5, 8, 9, 13, 16, 18, 20,
22, 27	None
Human Rhinovirus/
Enterovirus	Human
Rhinovirus	14	3x	Species A:
A1, A2, A7, A16, A34, A57, A77, A85
Species B:
B3, B14, B17, B27, B42, B83
	Enterovirus	11	3x	Species: A, B, C, D
Coxsackievirus A9, A10, A21, A24
Coxsackievirus B3, B4
Echovirus 6, 9, 11
Enterovirus 71, D68 (2014)	Noned
Influenza A H1	13	Up to
10xe	Human and swine Influenza A H1N1
isolates from the 1930s to 2007 and
H1N2 recombinant virus	None for Human
isolates.
Variable detection
of swine and avian
isolates, depending
on strain and
concentration
Influenza A H1-2009	9	Up to
10xf	Influenza A H1N1pdm09 isolates from
2009 and 2012	None
Influenza A H3	10	3x	Influenza A H3N2 isolates from the
1960s to 2012, attenuated
vaccine/recombinant H3N2 viruses, and
swine variant H3N2 (H3N2v)	None
Influenza A
(no subtype detected)
or
Influenza A (Equivocal)	10	3xg	Human H2N2 recombinant
Avian H2N3, H5N1, H5N2, H5N3,
H5N8, H7N7, H7N9, H10N7 (including
isolates from human infection)	Variable results
reported,
depending on
strain and
concentration
Influenza B	13	3x	Influenza B isolates from the 1940s to
2012 and attenuated
vaccine/recombinant viruses	None
Parainfluenza Virus 1	4	3x	Multiple strains of PIV1
Parainfluenza Virus 2	2	3x	Multiple strains of PIV2	None
Parainfluenza Virus 3	3	3x	Multiple strains of PIV3
Parainfluenza Virus 4	4	3x	Subtypes: A and B
Respiratory Syncytial Virus	7	Up to
10xh	Subtypes: A and B	None
Bacteria
Bordetella parapertussis (IS1001)	6	3x	Strains of B. parapertussis and B. bronchiseptica containing IS1001i	Nonej
Bordetella pertussis (ptxP)	9	3x	Strains of B. pertussis	None
Chlamydia pneumoniae	4	3x	Strains of C. pneumoniae	None
Mycoplasma pneumoniae	9	3x	Strains of M. pneumoniae	None

Table 27. Summary of BioFire RP2.1 Analytical Reactivity Testing and in silico Analysis®

44

ª Includes limitations observed in tosting by in silico analyses. Data for Alenovins, Sever Acute Respiratory
Syndrome Coronavirus 2 (SARS-CoV-2), and C. presincesting with

45

b In silico analysis of available sequences predicts that the BioFire RP2.1 will react with all currently characterized serotypes of Adenovirus, including B55, C57, several species D serotypes and G52, which were not tested.
6 The reactivity assessment for SARS-CoV-2 also included fifty clinical specimens from the 2020 pandemic collected in three geographic regions of the United States.
4 In silico analysis of available sequences predicts that the HRV/EV assay with all currently characterized species and serotypes of Human Rhinovirus and Enterovirus, including Polioviruses.
6 Influenza A H1N1 isolates ATCC VR-95 (H1N1 PR/8/1934) and swine variant Hsw1N1, ATCC VR-897 (HswN1 ANew Jersey/8/76) were detected as Influenza A H1 at 10×LoD. All other H1N1 isolates were detected as Influenza A H1 at 1× or 3× LoD.
f Influenza A H1N1pdm09 isolate BEI NR-44345 (Hong Kong/H090-761-V1(0)/2009) was detected as Influenza A H1-2009 at 10× LoD. All other H1N1pdm09 isolates tested were detected as Influenza A H1-2009 at 1× or 3× LoD.
& All human and avian isolates (or genomic RNA) tested were reported as either Influenza A (no subtyped detected) at 3× LoD. Three strains reported as Influenza A Equivocal at the 3× LoD concentration were reported as Influenza A (no subtype detected) when tested at higher concentrations of 10× LoD (H2N2 strains, BEI NR-2775) or 100× LoD (Avian H2N3 Mallard/Alberta/9/2003). Avian H10N7 (Chicken/Germany/N/49; BEI NR-2765) was only reported as Influenza A Equivocal, even at a concentration equal to 100× LoD.
b Respiratory Syncytial Virus, subtype B, ATCC VR-1580 (Washington DC/1962) was detected at 10× LoD. All other RSV isolates tested (subtypes A and B) were detected at 1× or 3× LoD.
i Reactivity with IS 001 sequences in B. bronchiseptica represents the intended reactivity of the analyte will be inaccurately reported as B. parapertussis. The assay is not expected to react with IS 1001-like sequences in B. holmesii.

Analytical Specificity/Cross-reactivity: f.

The potential for cross-reactivity between all BioFire RP2.1 assays (including SARS-CoV-2) and various on-panel or off-panel organisms that may be present in clinical respiratory specimens was evaluated with a combination of empirical testing and in silico analysis. The isolates tested represent all organisms evaluated on the previousgeneration respiratory panel (i.e., BioFire FilmArray Respiratory Panel 2 (RP2)) as well as additional isolates selected to assess the specificity of the novel SARS-CoV-2 assays.

Over 25 on-panel organisms (Table 29) were tested at high concentrations to assess the potential for intra-panel cross-reactivity. For off-panel testing, organisms were selected that represent normal respiratory flora and pathogens that may be present in the respiratory tract, as well as near-neighbors or species genetically related to the organisms detected by the panel. The off-panel isolates included more than 65 bacterial, viral, and fungal species, including common causes of pharyngitis and upper or lower respiratory infection as well as other SARS or SARS-like coronaviruses (Table 30). On-panel and off-panel isolates were tested at the highest concentration possible (generally >1.0E+07 units/mL for bacteria and fungi and ≥1.0E+05 units/mL for viruses). These concentrations are equal to or greater than those tested on the FilmArray RP2. Each isolate was tested in triplicate (once on each of the three different pouch/reagent lots) according to standard testing procedure.

Most bacterial and fungal isolates were grown in-house using traditional culture methods and quantified in CFU/mL (plate counting or turbidity standards) or cells/mL (optical density (OD600) reading). Some difficult-to-culture organisms, such as obligate intracellular bacteria, were cultured by an outside source laboratory and documented, with a Certificate of Analysis, in standard quantification units (CFU/mL or cells/mL), infectivity units (TCID50 or IFU), or other measures such as color changing units (CCU) or cell counts (i.e. nuclei/mL).

46

Viruses were cultured by outside source laboratories and provided as culture fluids quantified in TCID50/mL or other relevant units (provided on a Certificate of Analysis from the culture collection). Five coronaviruses (SARS-CoV, SARS-CoV-2, MERS-CoV, and two bat SARS-like CoVs) were cultured and tested in a contracted biosafety level 3 laboratory. Two viruses (bocavirus and CoV HKU1) that are not amenable to in vitro culture were acquired for testing as clinical specimens and quantified in RNA copies/mL.

Organisms that could not be acquired as intact organism cultures were tested as purified preparations of genomic DNA (gDNA) and reported in units of genomic equivalents per mL (GE/ mL) or were evaluated via directed in silico analysis of publicly available whole genome sequences.

Any samples generating unexpected results were retested to determine if the results were reproducible and/or investigated to rule in or rule out contamination, isolate misidentification, or cross-reactivity. Confirmed cross-reactivities or limitations identified in this and previous studies are included in the descriptions for the BioFire RP2.1.

Over 90 organisms were tested with the BioFire RP2.1, and only those few crossreactivities previously described for the BioFire RP2 assays were identified in the testing (e.g. Bordetella vertussis ptxP assay cross-reactivity with the ptxP pseudogene in other Bordetella species; see Table 32). In silico evaluation of the SARS-CoV-2 assays did indicate a risk of cross-reactivity with closely related SARS-like coronaviruses of bat and pangolin origin, although these viruses are not predicted to be present in human clinical specimens.

All known or predicted risks of cross-reactivity for the BioFire RP2.1 are summarized in Table 31. No additional cross-reactivities were identified in this study for previously existing panel assays or for the novel SARS-CoV-2 assays.

Organism	Isolate ID	Concentration Tested	Cross-Reactivity Detected
Bacteria
Bordetella parapertussis	Zeptometrix 0801462	6.43E+09 CFU/mL	Bordetella pertussis (ptxp)a
Bordetella pertussis	ATCC 9797	5.50E+09 CFU/mL	Human
Rhinovirus/Enterovirusb
Chlamydia pneumoniae	ATCC 53592	1.93E+07 IFU/mL	None
Mycoplasma pneumoniae	Zeptometrix 0801579	2.65E+07 CCU/mL	None
Viruses
Adenovirus	7A (species B)	Zeptometrix 0810021CF	1.02E+07 TCID50/mL	None
	1 (species C)	Zeptometrix 0810050CF	2.26E+07 TCID50/mL	None
	4 (species E)	ATCC VR-1572	1.58E+06 TCID50/mL	None

Table 28. On-Panel Organisms Tested for Evaluation of BioFire RP2.1 Analytical Specificity

47

| Organism | Isolate ID | Concentration
Tested | Cross-Reactivity
Detected |
|-----------------------------------------------------------------|------------------------|---------------------------|------------------------------|
| Coronavirus 229E | Zeptometrix 0810229CF | 1.13E+05 TCID50/mL | None |
| Coronavirus HKU1 | Clinical specimen | 8.94E+06 RNA
copies/mL | None |
| Coronavirus NL63 | Zeptometrix 0810228CF | 2.34E+05 TCID50/mL | None |
| Coronavirus OC43 | Zeptometrix 0810024CF | 6.37E+06 TCID50/mL | None |
| Severe Acute Respiratory Syndrome
Coronavirus 2 (SARS-CoV-2) | USA-WA1/2020 | 2.4E+09 copies/mL | None |
| Human Metapneumovirus | Zeptometrix 0810159CF | 1.05E+06 TCID50/mL | None |
| Human Rhinovirus
(Type 1A) | Zeptometrix 0810012CFN | 8.40E+05 TCID50/mL | None |
| Enterovirus (D68) | ATCC VR-1823 | 1.58E+07 TCID50/mL | None |
| Influenza A H1N1
(A1/FM/1/47) | ATCC VR-97 | 1.58E+08 CEID50/mL | None |
| Influenza A Hsw N1
(A/NewJersey/8/76) | ATCC VR-897 | 8.89E+06 CEID50/mL | Influenza A H1-2009 c |
| Influenza A (H1N1) pdm09
(Michigan/45/15) | Zeptometrix 0810538CF | 9.40E+04 TCID50/mL | None |
| Influenza A H3N2
(A/Alice) | ATCC VR-776 | 3.33E+08 CEID50/mL | None |
| Influenza B
(Massachusetts/2/12) | Zeptometrix 0810239CF | 9.55E+05 TCID50/mL | None |
| Parainfluenza Virus 1 | Zeptometrix 0810014CF | 6.80E+07 TCID50/mL | None d |
| Parainfluenza Virus 2 | Zeptometrix 0810357CF | 4.57E+06 TCID50/mL | None d |
| Parainfluenza Virus 3 | ATCC VR-93 | 6.80E+07 TCID50/mL | None |
| Parainfluenza Virus 4 | ATCC VR-1377 | 4.17E+04 TCID50/mL | None |
| Respiratory Syncytial Virus | Zeptometrix 0810040ACF | 7.00E+05 TCID50/mL | None |

ª Bordetella pertussis (ptxP) assay may amplify pertussis toxin pseudogene sequences from some strains of B. parapertussis at high concentration (>1.2E+09 CFU/mL).

b Human Rhinovirus Enterovinus assay may amplify non-target sequences from Bordetella species (B. parapertussis, and B. bronchiseptica) at a concentration >4.5E+07 CFU/mL.

6 The H1 hemagglutinin (HA) gene of Influenza A H1N1 strains of swine origin (prior to 2009) will be amplified by the H1 assay (Influenza A H1 Detected). However, some strains/sequences of swine origin may also be amplified by the H1-2009 assay (Influenza A H1-2009 Detected) at higher concentrations. Testing of this strain at 8.89E+06 CEIDsoinL generated an Influenza A H1 Detected result in 1/3 replicates and an Influenza A H1-2009 Detected in 2/3 replicates.

d Parainfluenza Virus 3 (PIV3) was detected in 2/5 replicates of the Parainfluenza Virus 1 (PIV1) isolate tested and in 2/4 replicates of the Parainfluenza 2 isolate tested. Sequencing of the amplicons generated match PIV3 sequences, indicating contamination of both the PIV1 and PIV2 isolate stocks with PIV3 nucleic acid.

	Table 29. Off-Panel Organisms Tested for Evaluation of BioFire RP2.1 Analytical Specificity
--	---------------------------------------------------------------------------------------------

| Organism Detected/Predicted | |
|------------------------- | Bacteria | Acinetobacter calcoaceticus | Arcanobacterium haemolyticum | Bacillus anthracis | Bordetella avium | Bordetella bronchiseptica | Organism Detected/Predicted | |
| Bordetella hinzii | Bordetella holmesii | Burkholderia cepacia | Chlamydia trachomatis | Chlaymdia psittaci | Corynebacterium diphtheriae | Corynebacterium striatum | Coxiella burnetii | Escherichia coli | Fusobacterium necrophorum | Haemophilus influenzae | Klebsiella (Enterobacter) aerogenes | Klebsiella oxytoca | Klebsiella pneumoniae | Lactobacillus acidophilus | Lactobacillus plantarum | Legionella (Fluoribacter)
bozemanae | Legionella (Fluoribacter) dumoffii | Legionella feeleii | Legionella longbeachae | Legionella (Tatlockia) micdadei | Legionella pneumophila | Leptospira interrogans (genomic DNA) | Moraxella catarrhalis | Mycobacterium tuberculosis (avirulent strain) | Mycoplasma genitalium (genomic DNA) | Mycoplasma hominis | Mycoplasma orale | Neisseria elongata | Neisseria gonorrhoeae | Neisseria meningitidis | Proteus mirabilis | Pseudomonas aeruginosa | Serratia marcescens | Staphylococcus aureus (MRSA) | Staphylococcus epidermidis | Stenotrophomonas maltophilia | Streptococcus agalactiae | Streptococcus dysgalactiae | Organism Detected/Predicted | |
| Streptococcus pneumoniae | Streptococcus pyogenes | Streptococcus salivarius | Ureaplasma urealyticum | Viruses (SARS-CoV-2 | Bat SARS-like Coronavirus (Recombinant with
SARS Urbani RBD) | 3.15E+06 TCID50/mL | Bat SARS-like Coronavirus HKU5 (Recombinant with
SARS Urbani SE) | 1.95E+06 TCID50/mL | Severe Acute Respiratory Coronavirus (SARS) | BEI NR-18925
Urbani strain | Viruses | Bocavirus | Cytomegalovirus (CMV) | Epstein-Barr Virus (EBV) | Herpes Simplex Virus 1 (HSV1) | Herpes Simplex Virus 2 (HSV2) | Human Herpes Virus 6 (HHV6) | Human Parechovirus (HPeV) | Influenza C | Measles Virus | Mumps | Fungi | Aspergillus flavus | Aspergillus fumigatus | Blastomyces dermatitidis (genomic DNA) | Candida albicans | Cryptococcus neoformans | Histoplasma capsulatum | Pneumocystis jirovecii (carinii) | Isolate ID | Concentration Tested | Cross-Reactivity
--------------------------------|-------------------------------------------------------|----------------------|----------------------------------------|------|
| | | | |
| ATCC 23055 | 5.15E+09 CFU/mL | None | |
| ATCC 9345 | 5.70E+09 CFU/mL | None | |
| Evaluated in silico | | None | |
| ATCC 35086 | 1.88E+09 cells/mL | None | |
| ATCC 10580 | 2.09E+09 cells/mL | Bordetella pertussis (ptxp) a | |
| Isolate ID | Concentration Tested | Cross-Reactivity
| ATCC 51783 | 4.30E+06 CFU/mL | None | |
| ATCC 700052 | 3.15E+07 CFU/mL | None | |
| ATCC 17762 | 5.04E+09 CFU/mL | None | |
| Zeptometrix 0801775 | 1.67E+08 IFU/mL | None | |
| Evaluated in silico | Evaluated in silico | None | |
| Zeptometrix 0801882 | 7.47E+08 CFU/mL | None | |
| ATCC BAA-1293 | 5.20E+09 CFU/mL | None | |
| Evaluated in silico | Evaluated in silico | None | |
| CDC AR Bank #0538 | 5.53E+09 CFU/mL | None | |
| ATCC 27852 | 1.33E+08 cells/mL | None | |
| ATCC 33391 | 5.85E+09 CFU/mL | None | |
| CDC AR Bank #0074 | 6.83E+09 CFU/mL | None | |
| JMI 7818 | 5.60E+09 CFU/mL | None | |
| NCTC 13465 | 1.75E+08 CFU/mL | None | |
| Zeptometrix 0801540 | 1.60E+08 CFU/mL | None | |
| Zeptometrix 0801507 | 1.20E+09 CFU/mL | None | |
| ATCC 33217 | 3.24E+09 cells/mL | None | |
| ATCC 33279 | 2.65E+09 cells/mL | None | |
| ATCC 35849 | 1.49E+09 cells/mL | None | |
| Zeptometrix 0801577 | 1.93E+08 CFU/mL | None | |
| Zeptometrix 0801576 | 1.80E+09 CFU/mL | None | |
| Zeptometrix 0801530 | 1.75E+09 CFU/mL | None b | |
| ATCC BAA-1198D-5
| 7.89E+08 GE/mL | None | |
| ATCC 8176 | 5.73E+09 CFU/mL | None | |
| Zeptometrix 0801660
| 9.07E+06 CFU/mL | None | |
| ATCC 33530D
| 8.40E+07 GE/mL | None | |
| Zeptometrix 0804011 | 2.11E+09 CCU/mL | None | |
| ATCC 19524 | 1.00E+07 CCU/mL | None | |
| Zeptometrix 0801510 | 1.99E+08 CFU/mL | None c | |
| ATCC 19424 | 2.31E+09 CFU/mL | None | |
| ATCC 13090 | 1.99E+09 CFU/mL | None | |
| ATCC 12453 | 5.60E+09 CFU/mL | None | |
| ATCC 27853 | 4.33E+09 CFU/mL | None | |
| JMI 697 | 4.75E+09 CFU/mL | None | |
| ATCC 10832 | 1.88E+08 CFU/mL | None | |
| ATCC 29887 | 4.95E+09 CFU/mL | None | |
| ATCC 700475 | 4.93E+09 CFU/mL | None | |
| ATCC 13813 | 5.45E+09 CFU/mL | None | |
| ATCC 43078 | 5.70E+09 CFU/mL | None | |
| Isolate ID | Concentration Tested | Cross-Reactivity
| ATCC BAA-341 | 5.20E+09 CFU/mL | None | |
| ATCC 19615 | 5.46E+07 CFU/mL | None | |
| ATCC 13419 | 4.92E+09 CFU/mL | None | |
| ATCC 27618 | 1.00E+08 CCU/mL | None | |
Related Coronaviruses) | | | | |
| BEI NR-44009
| None | |
| BEI NR-48814
| None | |
Syndrome
| 5.3E+09 copies/mL | None | |
| | | | |
| Clinical specimen | 1.40E+08 copies/mL | None | |
| Zeptometrix 0810003CF | 7.67E+06 TCID50/mL | None | |
| Zeptometrix 0810008CF | 3.65E+07 copies/mL | None | |
| ATCC VR-1778 | 3.30E+08 copies/mL | None | |
| Zeptometrix 0810217CF | 1.30E+07 TCID50/mL | None | |
| Zeptometrix 0810072CF | 4.11E+08 copies/mL | None | |
| Zeptometrix 0810147CF | 2.26E+07 TCID50/mL | None | |
| Evaluated in silico | | None | |
| Zeptometrix 0810025CF | 1.63E+05 TCID50/mL | None | |
| Zeptometrix 0810079CF | 4.83E+05 units/mL | None | |
| | | | |
| Zeptometrix 0801598 | 1.15E+08 CFU/mL | None | |
| Zeptometrix 0801716 | 5.47E+07 CFU/mL | None | |
| ATCC 26199D-2
| 7.05E+07 GE/mL | None | |
| ATCC 10231 | 1.19E+06 CFU/mL | None | |
| ATCC MYA-4564 | 6.00E+07 CFU/mL | None | |
| Evaluated in silico | | | None |
| ATCC PRA-159 | 6.67E+07 nuclei/mL | None | |

48

49

B Bordetella pertussis (ptxP) assay may amplify pertussis toxin pseudogene sequences from some strains of B. bronchiseptica at high concentration (≥1.2E+09 CFU/mL).

b Bordetella parapertussis (IS1001) was detected in 3/3 replicates. Amplification with an alternate B. parapertussis/151001 PCR assay confirmed the presence of IS1001 nucleic acid in the L. pneumophila stock (contamination).

& Mycoplasma pneumoniae was detected in 3/3 replication with an alternate M. pneumoniae PCR assay (gyrB gene) confirmed the presence M. pneumoniae nucleic acids in the N. elongata stock (contamination).

50

Table 30. Predicted and Observed Cross-Reactivity of the BioFire RP2.1

Cross-reactive Organism(s)/Sequence(s)	BioFire RP2.1 Result	Description
Bat coronavirus RaTG132
(accession# MN996532)
Pangolin coronavirus3
(accession# MT08407)
Bat SARS-like coronavirus
(accession# MG772933 and MG772934)	Severe Acute Respiratory Syndrome
Coronavirus 2
(SARS-CoV-2)	The SARS-CoV-2 assays can amplify a small selection
of sequences from closely related Sarbecoviruses
isolated from bats and pangolin. The SARSCOV2-2
assay is predicted to cross-react with all four
sequences, while the SARSCOV2-1 assay will likely
only cross-react with the bat coronavirus RaTG13.
Non-pertussis Bordetella species
(e.g. Bordetella parapertussis, Bordetella
bronchisepticaa)	Bordetella pertussis (ptxP)b	The Bordetella pertussis (ptxP) assay can amplify
pertussis toxin pseudogene sequences in B.
bronchiseptica and B. parapertussis, primarily when
present at high concentrations (≥1.2E+09 CFU/mL).
Bordetella bronchisepticaa
(with IS1001 sequences)	Bordetella parapertussis (IS1001)	Some strains of B. bronchiseptica carry IS1001
insertion sequences identical to those carried by B.
parapertussis. These sequences will be efficiently
amplified by the IS1001 assay and reported by BioFire
RP2.1 as Bordetella parapertussis (IS1001).
Bordetella pertussis
Bordetella parapertussis
Bordetella bronchiseptica	Human Rhinovirus/Enterovirusd	The Human Rhinovirus/Enterovirus assay may amplify
off-target sequences found in strains of B. pertussis, B.
bronchiseptica, and B. parapertussis when present at
high concentration. Cross-reactivity with B. pertussis
was observed at a concentration of 4.5E+07 CFU/mL
or higher.
Influenza A H1N1
(swine origin)	Influenza A H1-2009e	The Influenza A H1-2009 assay may react with H1
hemagglutinin gene sequences from viruses of swine
origin.
BioFire RP2.1 will report either Influenza A H1 or
Influenza A H1-2009, depending on the strain and
concentration in the sample.

a B. bronchiseptica infection is rare in humans and more common in domesticated animals ('kennel cough').

Cross-reactivity between the Bordetella pertussis (ptxP) assay and B. parapertussis will be reported as a co-detection (Bordetella parapertussis (IS 1001) Detected and Bordetella pertussis (ptxP) Detected); while cross-reactivity with most strains of B. bronchiseptica (that do not carry IS 1001) will be reported only as Bordetella pertussis (pt.rP) Detected.

& Cross-reactivity with B. parapertussis and B. bronchiseptica is predicted based on in silico analysis but was not observed when tested at a concentration of 1.2E+09 CFU/mL.

4 Cross-reactivity between the Human Rhinovirus assays and B. pertussis or B. parapertussis will be reported as a co-detection (Bordetella pertussis (pts.P) Detected and Human Rhinovirus Detected or Bordetella parabertussis (IS1001) Detected and Human Rhinovirus Detected); while cross-reactivity with most strains of B. bronchiseptica (that do not carry IS 1001) will be reported (falsely) only as Human Rhinovirus Detected.
6 Swine origin Hsw1N1 (ANew Jersey/8/1976 ; ATCC VR-897) was detected as either Influenza A H1-2009 at a concentration of 8.9E+06 CEID50/mL.

Select organisms that could not be acquired for empirical testing were evaluated via in silico analysis to assess the potential for cross-reactivity between the novel SARS-CoV-2 assay primers and the organism whole genome sequences. The isolates evaluated include medically relevant select agents (Bacillus anthracis and

2 Peng Zhou et al., "A Pneunonia Outbreak Associated with a New Coronavirus of Probable Bat Origin," Nature 579, no. 7798 (March 2020): 270-73, https://doi.org/10.1038/s41586-020-2012-7.

Rachele Caglian et al., "Computational Inference of Selection Underlying the Novel Coronavinus, SARS-CoV-2," Journal of Virology, April 1, 2020, JVI.00411-20, jvi;JVI.00411-20v1, https://doi.org/10.1128/JVI.00411-20.

51

Coxiella burnetii), a BSL3 fungal respiratory pathogen (Histoplasma capsulatum), a rare zoonotic fungal pathogen (Chlamydia psittaci), and a relatively underdiagnosed cause of influenza infection (Influenza C virus).

Analysis involved retrieval of at least three representative whole genome sequences for each organism from the publicly available GenBank database and subsequent BLAST analysis to identify regions with greater than 80% homology to the SARS-CoV-2 assay primers. Homology was evaluated for both outer and inner assay primers, although only reactivity with inner primers is essential to generate a crossreactive result using the nested FilmArray system. When regions with >80% homology to at least one inner assay primer were identified, the sequences were further assessed to determine the potential for bidirectional amplification and generation of a detectable amplicon sequence (80% homology to individual outer or inner primer sequences were identified (see Table 32), no primer binding sites were located in an orientation to allow for bi-directional amplification of complementary strands nor within sufficient proximity to generate an appropriately sized, detectable amplicon. As a result, no risk of cross-reactivity was identified for the organisms evaluated.

	Table 31. In silico Evaluation of Potential Cross-Reactivity Between SARS-CoV-2 Assay Primers and Select Off-Panel
Organisms

Percent homology under SARS-CoV-2 assay outer forward (IF), inner reverse (IR), and outer forward (OF)
primers. Results for outer reaction primers are shaded grey. Sequences with >80% homology to an inner are shaded
green. Sequences with no homology to assay primers are denoted NH (no homology).

| Organism | GenBank Accession
No. | SARSCoV2-1 Assay
Primers | | | | SARSCoV2-2 Assay Primers | | | | Predicted
Cross-reactivity |
|---------------------------|--------------------------|-----------------------------|----|----|----|--------------------------|-------|-------|-----|-------------------------------|
| | | OF | IF | IR | OR | OF | IF | IR | OR | |
| Bacillus
anthracis | NC_005945.1 | NH | NH | NH | NH | 85% | 83% | NH | 81% | None |
| | CP012728.1 | NH | NH | NH | NH | 85% | 83% | NH | 81% | |
| | NZ_KN050648.1 | NH | NH | NH | NH | 85% | 83% | NH | 81% | |
| Chlamydia
psittaci | NC_020248.1 | NH | NH | NH | NH | 85% | NH | NH | 81% | None |
| | NC_015470.1 | NH | NH | NH | NH | 85% | NH | NH | 81% | |
| | NZ_KE355746.1 | NH | NH | NH | NH | 85% | NH | NH | 81% | |
| Coxiella
burnetii | NC_002971 | 81% | NH | NH | NH | 80% | NH | NH | NH | None |
| | CP000733 | 81% | NH | NH | NH | 80% | NH | NH | NH | |
| | CP007555 | 81% | NH | NH | NH | 80% | NH | NH | NH | |
| | CP018150 | 81% | NH | NH | NH | 80% | NH | NH | NH | |
| | HG825990 | 81% | NH | NH | NH | 80% | NH | NH | NH | |
| | NC_004704 | NH | NH | NH | NH | 80% | NH | NH | NH | |
| | CP000735 | NH | NH | NH | NH | 80% | NH | NH | NH | |
| | CP000914 | NH | NH | NH | NH | 80% | NH | NH | NH | |
| Histoplasma
capsulatum | ABBT00000000 | 81% | NH | NH | NH | 80% | 83.3% | 81.8% | 81% | None |
| | AAJI00000000 | 81% | NH | NH | NH | 80% | 83.3% | 81.8% | 81% | |

52

| Organism | GenBank Accession
No. | SARSCoV2-1 Assay Primers | | | | SARSCoV2-2 Assay Primers | | | | Predicted
Cross-reactivity |
|----------------------|------------------------------|--------------------------|----|----|----|--------------------------|-------|-------|-----|-------------------------------|
| | | OF | IF | IR | OR | OF | IF | IR | OR | |
| | ABBS000000000 | 81% | NH | NH | NH | 80% | 83.3% | 81.8% | 81% | |
| | ABRJ0000000000 | 81% | NH | NH | NH | 80% | 83.3% | 81.8% | 81% | |
| | ABRK000000000 | 81% | NH | NH | NH | 80% | 83.3% | 81.8% | 81% | |
| Influenza C
Virus | 1419 Sequences
Evaluateda | NH | NH | NH | NH | NH | NH | NH | NH | None |

a GenBank accession numbers evaluated for Influenza C cross-reactivity analysis were provided separately.

g. Assay cut-off:

Not applicable

h. Interfering Substances:
Testing for possible interference from select substances that may be present in nasopharyngeal swabs (NPS) was performed with the BioFire RP2.1. The majority of interference data was collected in the interference study performed with the predecessor panel, BioFire FilmArray Respiratory Panel 2 (RP2). Both the BioFire RP2.1 and BioFire RP2 test the same sample type using the same pouch chemistry and cycling conditions. The only difference between the panels is the addition of the SARS-CoV-2 assays; therefore, the previous BioFire RP2 interference study results remain relevant and are applied to the BioFire RP2.1 as appropriate.

Substances evaluated for interference generally were categorized as-endogenous substances (i.e., biological substances naturally in NPS samples), competing microorganisms (i.e., pathogens or natural flora tested at high concentrations to evaluate polymicrobial NPS specimens), exogenous substances (i.e., non-native substances in NPS samples), or technique-specific substances (i.e., substances introduced during sample processing, collection, or testing).

The endogenous and technique-specific substances, as well as competing microorganisms, evaluated on the BioFire RP2.1 in this study were selected to assess the risk of possible interference with the SARS-CoV-2 assays as well as the other panel assays. Substances were also selected to reproduce a subset of the data collected in the BioFire RP2 study in order to verify that the data collected with one panel could be applied to the other.

Each substance was added to contrived samples containing representative organisms at concentrations near (2-3x) LoD (Table 33). The concentration of substance added to the samples was equal to or greater than the highest level expected to be in NPS specimens. The organisms in the sample included the new analyte (SARS-COV-2), two analytes included in the RP2 interference study (Adenovirus and Bordetella parapertussis), and three additional analytes not previously evaluated in the RP2 interference study (Coronavirus NL63, Influenza A H1N1pdm09, and Respiratory

53

Syncytial Virus). The test sample was composed to represent the types of analytes detected by the panel, including bacteria and enveloped or non-enveloped viruses with DNA and RNA genomes.

| Organism | Description | Source | 3x LoD
(Sample)
Concentration |
|---------------------------------------------------------------------|-------------------------------------------------------------------|-----------------------------------------------|-------------------------------------|
| Severe Acute Respiratory
Syndrome Coronavirus 2 (SARS-
CoV-2) | New analyte for RP2.1
RNA genome (+ strand)
enveloped virus | ATCC VR-1986HK
(heat inactivated) | 1.5E+03
copies/mL |
| Adenovirus (Type 2) | DNA genome (double-stranded)
non-enveloped virus | WHO International
Standard
NIBSC 16/324 | 9.0E+03 IU/mL |
| Coronavirus NL63 | RNA genome (+ strand)
enveloped virus | BEI NR-470 | 7.5E-01
TCID50/mL |
| Influenza A H1N1pdm09 (H1-
2009) | RNA genome (- strand)
enveloped virus | Zeptometrix
0810109CFN | 1.5E+00
TCID50/mL |
| Respiratory Syncytial Virus | RNA genome (+ strand)
enveloped virus | Zeptometrix 810040ACF | 6.0E-02
TCID50/mL |
| Bordetella parapertussis | Gram-negative bacterium
DNA genome | Zeptometrix 0801461 | 1.8E+02
IS1001
copies/mL |

Table 32. Contrived Sample Composition for BioFire RP2.1 Interference Testing

Testing near LoD was to identify the effects of even minor interference on analyte detection. A control sample with no substance (positive control) was tested on each day of evaluation to demonstrate the expected detection without any potential interference. Each potential interferent was also added to a negative sample (substance only negative control) and tested in tandem with the corresponding positive sample to serve as a control for the substance alone (e.g. detection of an onpanel competing microorganism). Each type of sample (positive control, negative control, and sample with interfering substance) was tested in triplicate, with one replicate on each of three different pouch lots. The pouch control and analyte results from the spiked sample with potential interferents and negative control samples were compared to the results from the positive control sample replicates to evaluate whether there was interference in detection.

In addition, in silico analysis of the SARS-CoV-2 assays during assay development identified some closely related coronaviruses with >80% homology under one or more assay primers. Of those, three (Bat CoV KY770858, Bat CoV GU190215 and SARS-like coronavirus KR559017) show the indicated homology to only the outer forward primer and are, therefore, not expected to react with the SARS-CoV-2 assays nor present a risk of interference. However, four sequences from the proposed precursors to SARS-CoV-2 (Bat CoV RTG13 MN996532, Pangolin CoV MT084071, and Bat SARS-like coronavirus MG772933 and MG772934) are predicted to cross-react with one or more of the BioFire RP2.1 SARS-CoV-2 assays. These viruses could not be acquired for interference testing. However, none of these

54

viruses have been isolated from human infections. Therefore, the risk of interference with the presence of these coronaviruses in polymicrobial infections with SARS-CoV-2 appears to be negligible.

The combination of BioFire RP2.1 and BioFire RP2 interference testing evaluated a total of forty-three substances (Table 34). The notable results from testing are summarized as follows-a total of 41 pouch runs were performed on both FilmArray Torch and FilmArray 2.0 systems and all completed with valid results (i.e., no errors). Overall, testing with potentially interfering substances at high, "worst-case scenario" concentrations (Table 35) demonstrated no interference with pouch controls or detection of panel analytes. The only exception was with bleach, resulting in missed detection of various panel analytes likely due to damaged nucleic acids (this was also observed in the previous BioFire RP2 panel). A general warning to avoid contact between samples and bleach is noted.

Endogenous substance interference testing with the BioFire RP2.1 confirmed that presence of blood and human genomic DNA in samples had no effect on detection of low-level SARS-CoV-2 or other analytes, consistent with results obtained with these substances in the BioFire RP2 evaluation. In addition, no interference with SARS-CoV-2 detection was observed when testing a clinically relevant concentration of human peripheral blood mononuclear cells (PBMCs).

Competing microorganism testing with the BioFire RP2.1 showed that there was no effect with a high concentration of another human beta-coronavirus that can cause respiratory illness (Coronavirus OC43), nor a high concentration of a bacterium representing normal flora of the respiratory tract (Streptococcus salivarius). During evaluation of the potential Streptococcus salivarius interferent, one spiked substance replicate vielded a no detection with the Respiratory Syncvtial Virus analyte. However, the lack of detection was not reproducible in the two repeated replicates on the same pouch lot.

For technique-specific substance testing, no interference was observed when samples were prepared and tested in PrimeStore Molecular Transport Medium (MTM).

Table 33. Results from the Evaluation of Potentially Interfering Substance Effects on Analyte Detection - FilmArray RP2 on BioFire RP2.1

Substances tested with the BioFire RP2.1 are in bold fort. Results for substances tested only with the BioFire FilmArray RP2 previously are applied to BioFire RP2.1

Substance Tested	Concentration Tested	Result
Endogenous Substances
Human Whole Blood	10% v/v	No Interference
Human Mucus (Sputum)	1 swab/mL sample	No Interference
Human Genomic DNA	20 ng/µL	No Interference
Human Peripheral Blood Mononuclear Cells (PBMCs)	1.0E+03 cells/µL	No Interference
Competitive Microorganisms
Coronavirus 229E	1.7E+04 TCID50/mL	No Interference
Substance Tested	Concentration Tested	Result
Coronavirus OC43 (betacoronavirus)	9.6E+05 TCID50/mL	No Interference
Adenovirus A12	8.9E+05 TCID50/mL	No Interference
Parainfluenza Virus 3	6.6E+05 TCID50/mL	No Interference
Bordetella pertussis	5.8E+08 CFU/mL	No Interference
Enterovirus D68	1.6E+07 TCID50/mL	No Interference
Echovirus 6	1.0E+07 TCID50/mL	No Interference
Respiratory Syncytial Virus	4.2E+04 TCID50/mL	No Interference
Staphylococcus aureus	2.5E+07 CFU/mL	No Interference
Streptococcus pneumoniae	1.7E+07 CFU/mL	No Interference
Streptococcus salivarius	2.5E+09 CFU/mL	No Interference
Haemophilus influenzae	6.2E+07 CFU/mL	No Interference
Candida albicans	1.0E+06 CFU/mL	No Interference
Herpes Simplex Virus 1	1.6E+06 TCID50/mL	No Interference
Cytomegalovirus	1.2E+06 TCID50/mL	No Interference
Exogenous Substances ª
Tobramycin (systemic antibiotic)	0.6 mg/mL	No Interference
Mupirocin
(active ingredient in anti-bacterial ointment)	2% w/v	No Interference
Saline Nasal Spray with Preservatives
(0.65% NaCl, Phenylcarbinol, Benzalkonium chloride)	1% v/v	No Interference
Nasal Decongestant Spray
(Oxymetazoline HCl 0.05%, Benzalkonium chloride, phosphate)	1% v/v	No Interference
Analgesic ointment (Vicks®VapoRub®)	1% w/v	No Interference
Petroleum Jelly (Vaseline®)	1% w/v	No Interference
Snuff (Tobacco)	1% w/v	No Interference
Disinfecting/Cleaning Substances
Bleach	1% and 2% v/v
[up to 1024 ppm chlorine]	Interference b
Disinfecting wipes (ammonium chloride)	½ in²	No Interference
Ethanol	7% v/v	No Interference
DNAZap (Ambion AM9891G & AM9892G)	1% v/v	No Interference
RNaseZap (Ambion AM9782)	1% v/v	No Interference
Specimen Collection Materials
Rayon Swabs (Copan 168C)	N/A	No Interference
Nylon Flocked Swabs (Copan 553C)	N/A	No Interference
Polyester Swabs (Copan 175KS01)	N/A	No Interference
Calcium Alginate Swabs (Puritan 25-801 A 50)	N/A	No Interference
M4 Transport Medium (Remel)	100%	No Interference
M4-RT Transport Medium (Remel)	100%	No Interference
M5 Transport Medium (Remel)	100%	No Interference
M6 Transport Medium (Remel)	100%	No Interference
Universal Viral Transport vial (BD)	100%	No Interference
PrimeStore Molecular Transport Medium	70% v/v	No Interference
Sigma-Virocult Viral Collection and Transport System	100%	No Interference
Substance Tested	Concentration Tested	Result
(Swab and Transport Medium)
Copan ESwab Sample Collection and Delivery System
(Swab and Liquid Amies Medium)	100%	No Interference

55

56

4 Nasal vaccines (e.g. FluMist) were not evaluated, but are predicted to be reactive with the BioFire RP2.1 Influenza A (subtype) and Influenza B assays.

b Not Detected results were reported for several analytes after incubation of the sample with 2% bleach for 10 minutes or ovenight. It was concluded that interference resulted prinarily from damage to the organisms hucleic acids in the sample, rather than inhibition or interference with pouch function(s).

i. Carry-Over Contamination:

A formal carry-over study in support of this regulatory submission for the BioFire RP2.1 was not performed since carry-over studies with high positive samples followed by negative samples have been performed for other FDA-cleared FilmArray Panels that are similar to the RP2.1 (i.e., BioFire FilmArray RP, BCID, and GI) for both the FilmArray 2.0 and the FilmArray Torch systems, and no significant carryover has been observed.

2. Comparison studies:

a. FDA SARS-CoV-2 Reference Panel Testing:

Quality assessments such as evaluating the BioFire RP2.1 device with the FDA SARS-CoV-2 reference panel, provide additional information on the relative sensitivity and specificity of the included SARS-CoV-2 assays. An evaluation of SARS-CoV-2 sensitivity and MERS-CoV cross-reactivity was performed using reference material, blinded samples, and a standard protocol provided by FDA. The study included a range finding study and a confirmatory study for LoD. Blinded sample testing was used to establish specificity and to confirm the LoD.

The results are summarized in the following table-

Table 34. Summary of LoD Confirmation Result using the FDA SARS-CoV-2 Reference Panel

Reference Materials
Provided by FDA	Specimen Type	LoD Concentration	Cross-Reactivity
SARS-CoV-2	NPS in
transport medium	6.0E+03 NDU/mLa	N/A
MERS-CoV	NPS in
transport medium	N/A	Not Detected

4 NDUmL = RNA NAAT detectable units/mL

b. Matrix comparison:
Not applicable

57

3. Clinical studies:

Prospective Clinical Study

A clinical evaluation of the BioFire RP2.1 panel was performed with prospectively collected NPS specimens. Specimens that were residual NPS in transport media left over from standard of care testing for SARS-CoV-2, and those that were either held at room temperature for