Who is the manufacturer of BioFire Respiratory Panel 2.1 (RP2.1)?

Biofire Diagnostics, LLC filed the De Novo classification request for BioFire Respiratory Panel 2.1 (RP2.1) (DEN200031).

What is the FDA product code for BioFire Respiratory Panel 2.1 (RP2.1)?

QOF. It is classified under 21 CFR 866.3981 as a Class 2 device in the Microbiology panel.

What is the regulatory pathway for BioFire Respiratory Panel 2.1 (RP2.1)?

De Novo classification (submission type: Direct).

Who can help prepare an FDA De Novo submission like BioFire Respiratory Panel 2.1 (RP2.1)?

Innolitics — a medical-device software consultancy — provides regulatory and engineering support for FDA De Novo submissions. See https://innolitics.com/services/regulatory/ or contact https://innolitics.com/contact.

BioFire Respiratory Panel 2.1 (RP2.1)

DEN200031 · Biofire Diagnostics, LLC · QOF · Mar 17, 2021 · Microbiology

Device Facts

Record ID	DEN200031
Device Name	BioFire Respiratory Panel 2.1 (RP2.1)
Applicant	Biofire Diagnostics, LLC
Product Code	QOF · Microbiology
Decision Date	Mar 17, 2021
Decision	DENG
Submission Type	Direct
Regulation	21 CFR 866.3981
Device Class	Class 2

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

Device Story

PCR-based multiplexed nucleic acid test; utilizes BioFire FilmArray 2.0 or Torch systems. Input: nasopharyngeal swabs (NPS). Process: automated nucleic acid extraction, amplification, and detection of viral/bacterial targets. Output: qualitative detection/identification of respiratory pathogens. Used in clinical settings; operated by laboratory personnel. Results aid diagnosis in conjunction with clinical/epidemiological data; not sole basis for patient management. Benefits: rapid, simultaneous identification of multiple respiratory pathogens to inform clinical decision-making.

Clinical Evidence

Prospective clinical study (N=524) compared RP2.1 to a composite comparator of three FDA EUA tests. SARS-CoV-2 PPA was 98.4% (61/62) and NPA was 98.9% (457/462). Retrospective study (N=98) showed 98% PPA and 100% NPA for SARS-CoV-2. Contrived clinical specimen study (N=50) showed 100% PPA and NPA. Clinical comparison study (N=210) demonstrated high concordance between RP2.1 and RP2 for shared analytes.

Technological Characteristics

PCR-based multiplexed nucleic acid test. Operates on BioFire FilmArray 2.0 or Torch systems. Automated extraction, amplification, and detection. Software-controlled analysis. Requires FDA-cleared/exempt specimen collection devices. Special controls mandate detailed documentation of analytical performance (LoD, inclusivity, cross-reactivity, interference, precision) and clinical study protocols.

Indications for Use

Indicated for individuals suspected of respiratory tract infections, including COVID-19, to qualitatively detect and identify multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (NPS).

Regulatory Classification

Identification

A device to detect and identify nucleic acid targets in respiratory specimens from microbial agents that cause the SARS-CoV-2 respiratory infection and other microbial agents when in a multi-target test is an in vitro diagnostic device intended for the detection and identification of SARS-CoV-2 and other microbial agents when in a multi-target test in human clinical respiratory specimens from patients suspected of respiratory infection who are at risk for exposure or who may have been exposed to these agents. The device is intended to aid in the diagnosis of respiratory infection in conjunction with other clinical, epidemiologic, and laboratory data or other risk factors.

Special Controls

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

Related Devices

K143080 — FilmArray Respiratory Panel (RP) for use with Multi-instrument FilmArray System · Biofire Diagnostics, LLC · Feb 17, 2015
K211079 — BioFire COVID-19 Test 2 · Biofire Defense, LLC · Nov 1, 2021

Submission Summary (Full Text)

{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 - 2. Classification: Class II (special controls) {1}------------------------------------------------ # 3. Product code(s): QOF - 4. 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. - 3. 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 0801461) | 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 (<5% Not Detected results when testing at or above LoD). No pattern in Not Detected results was observed in the study variables (site, system, day instrument/modules, operator, or reagent lot). - b. Linearity/assay reportable range: Not applicable - c. Traceability, Stability, Expected values (controls, calibrators, or methods): # Controls Two process controls are included in each pouch: #### RNA Process Control The RNA Process Control assay targets an RNA transcript from the yeast Schizosaccharomyces pombe. The yeast is present in the pouch in a freeze-dried form and becomes rehydrated when sample is loaded. The control material is carried through all stages of the test process, including lysis, nucleic acid purification, reverse transcription, PCR1, dilution, PCR2, and DNA melting. A positive control result indicates that all steps carried out in the FilmArray RP2.1 pouch were successful. #### PCR2 Control The PCR2 Control assay detects a DNA target that is dried in the array along with the corresponding primers. A positive result indicates that PCR2 was successful. {15}------------------------------------------------ Both control assays must be positive for the test run to pass. If the controls fail, the sample should be retested using a new pouch. The FilmArray Software automatically fails the run if the melting temperature for either the RNA Process Control or the PCR2 Control is outside of an acceptable range. The following is also described in the product package insert regarding to external controls: External controls should be used in accordance with laboratory protocols and the appropriate accrediting organization requirements, as applicable. Transport media can be used as an external negative control. Previously characterized positive samples or negative samples spiked with well-characterized organisms can be used as external positive controls. Commercial external control materials may be available from other manufacturers; these should be used in accordance with the manufacturers' instructions and appropriate accrediting organization requirements, as applicable. ## Calibrators This device does not contain calibrators. ### Specimen Stability The BioFire RP2.1 test requires approximately 0.3mL of NPS specimen, collected according to standard technique and placed in transport media or saline. Samples in medium should be tested as soon as possible, but they may be stored at room temperature (approximately 23°C) for up to four hours, under refrigeration (approximately 4°C) for up to three days, or frozen (<-15 °C or <-70°C) for up to 30 davs. Detailed documentation concerning NPS sample storage and transport was provided in the original FilmArray RP submissions (K103175, K110764, K120267) for NPS specimens stored in viral transport media. The BioFire RP2 and the BioFire RP2.1 utilize this same sample type and test principles and the additional organisms detected (B. parapertussis and SARS-CoV-2) are biologically similar to others detected by the FilmArray RP (i.e., a representative bacteria and virus). Therefore, the original FilmArray RP specimen stability study data are applicable to the BioFire RP2 and BioFire RP2.1 panels for samples stored in viral transport media. However, for establishing sample stability and storage conditions for NPS specimens in saline, an additional study was performed to validate claims. For the study, natural NPS in saline matrix was prepared by eluting two NPS specimens collected from a single anonymous, asymptomatic volunteer in 6mL of 0.9% saline. Contrived organism mixes (Table 3) were prepared using analyte {16}------------------------------------------------ negative individual donor natural NPS in saline matrices. For each contrived mix, a total of ten unique donor NPS in saline matrices were individually spiked with organisms corresponding to the RP2.1 panel to a final concentration based on the LoD (up to 5x). LoD of the analytes were determined in a separate limit of detection for saline samples study as described in the Limit of Detection. Immediately following sample preparation (TO), ten replicates (one from each donor) were tested to serve as a no storage control and to establish the expected Detected and Not Detected results. | Sample Mix | Organisma | Strain/Isolate/ Serotype | Concentration Testedb | Units | Concentration Relative to LoDc | | |---------------|--------------------------------------------------------------------------|------------------------------------|--------------------------|---------------------------------|-----------------------------------|--| | | Adenovirus Species C | Serotype 2 WHO Int Std | 1.5E+04 (1.5E+04) | IU/mLd (copies/mL)e | 5x | | | | Coronavirus NL63 | NL63 | 1.3E+00 (2.7E+02) | TCID50/mL (copies/mL) | 5x | | | | Influenza A H1N1pdm09 (H1-2009) | A/SwineNY/ 3/2009 | 2.6E+00 (1.7E+03) | TCID50/mL (copies/mL) | 5x | | | M1 | Bordetella parapertussis | A747 | 2.1E+02 (3.0E+02) | CFU/mL (IS1001 copies/mL) | 5x | | | | Parainfluenza Virus 3 | Type 3 | 8.1E+01 (1.9E+02) | TCID50/mL (copies/mL) | 5x | | | | Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) | USA-WA1/2020 (heat inactivated) | 2.5E+03h | copies/mL | 5x | | | | Coronavirus 229E | 229E | 2.0E+00 (3.3E+02) | TCID50/mL (copies/mL) | 5x | | | | Human Metapneumovirus | Type 16, IA10-2003, A1 | 5.0E+01 | TCID50/mLi | 5x | | | | Human Rhinovirus | 1A | 3.8E+00 (1.9E+01) | TCID50/mL (copies/mL) | 5x | | | M4 | Influenza A H3N2 (H3)j | Hong Kong/4801/14 | 1.3E+00 (1.1E+00) | TCID50/mL (copies/mL) | 5x | | | | Parainfluenza Virus 4 | Type 4a | 2.5E+02 (8.0E+03) | TCID50/mL (copies/mL) | 5x | | | | Mycoplasma pneumoniae | M129 | 6.3E+00k (4.7E+02)k | CCU/mL copies/mL | 60xk | | | | Middle East Respiratory Syndrome Coronavirus | EMC/2012 (heat inactivated) | 6.7E+02 | copies/mL | 5x | | | | Coronavirus OC43 | OC43 | 7.1E-02 (2.8E+02) | TCID50/mL (copies/mL) | 5x | | | | Enterovirus | D68 | 6.0E+02 (5.2E+01) | TCID50/mL (copies/mL) | 2xl | | | M5 | Influenza A H3N2 (H3)j | Hong Kong/4801/14 | 1.4E+00 (1.1E+00) | TCID50/mL (copies/mL) | 5x | | | | Parainfluenza Virus 2 | Type 2 | 2.5E+00 (1.5E+02) | TCID50/mL (copies/mL) | 5x | | | | Chlamydia pneumoniae | AR-39 | 9.0E+01 (6.7E+02) | IFU/mL (copies/mL) | 5x | | | Sample Mix | Organisma | Strain/Isolate/ Serotype | Concentration Testedb | Units | Concentration Relative to LoDc | | | M6 | Coronavirus HKU1 | Clinical NPS Specimen 53727 | 1.0E+04h | RNA copies/mL | 5× | | | | Influenza A H1N1 (H1) | A/New Caledonia/20/99 | 5.0E+03 (7.0E+02) | TCID50/mL (copies/mL) | 5× | | | | Influenza B | B/FL/04/06 | 2.5E+01 (1.7E+02) | TCID50/mL (copies/mL) | 5× | | | | Parainfluenza Virus 1 | Type 1 | 1.6E+00 (5.0E+02g) | TCID50/mL (copies/mL) | 5× | | | | Bordetella pertussis | A639 | 2.0E+03 | CFU/mL | 2×1 | | | | Respiratory Syncytial Virusn | Type A | 2.1E-01 (9.0E+01) | TCID50/mL (copies/mL) | 10× | | Table 3. Organism composition of each contrived sample mix for stability studies with saline samples {17}------------------------------------------------ Following testing at T0. individual sample aliquots were prepared from each contrived donor mix and these were stored at ambient (25°C), refrigerated (8°C), or frozen ( <- 15°C) temperatures for the durations indicated in Table 4. Note, the T0 time point was collected on different days for different analytes but the frozen time point for all analytes was collected on the same day. This resulted in analytes in the different sample mixes to be tested for at least 30 days after the no storage time point. At each time point, ten replicates (one from each donor) evenly distributed between the FilmArray 2.0 and FilmArray Torch instruments were tested. The reported results of Detected (D), Equivocal (E), and Not Detected (ND) were evaluated for each analyte across the storage conditions and compared to the results observed at TO. 0140 (b) (4) | Storage Condition | | Sample Size | |-----------------------|-------------------------|-------------| | Temperature | Time | | | N/A | No Storage Control (T0) | 10 | | Ambient (25°C) | 4 hours | 10 | | Refrigerated (8°C) | 2 days | 10 | | | 3 days | 10 | | Frozen (≤-15°C) | ≥30 days | 10 | | | Total | 50 | Table 4. Storage conditions and sample size to be tested at each condition. A valid result (i.e., all internal pouch controls passing) was required for each pouch tested. Pouches with invalid results due to a control failure, instrument error, or software error were retested until a valid result was obtained. Only results from the valid pouches were considered in subsequent analyses. Samples with Influenza A/subtype Equivocal or Influenza A (no subtype detected) results were retested according to the intended result interpretation algorithm (see Table 1). {18}------------------------------------------------ Any observed trending across conditions (i.e., time- and/or temperature-dependent shift in assay parameters) would be indicative of possible impact on sample stability. The below table provides a summary of the saline sample stability study. As indicated in the summary table, some observations across different analytes and storage conditions did not meet the expected Detected results. Some of the missed detections did not correspond to the same donor samples and they were distributed such that it appears no trend was observed (e.g., an unexpected Not Detected result for T0 but Detected result for later time points and other storage conditions, etc.). However, for three analytes detected by the RP2.1 device (RSV, Parainfluenza Virus 2, and Mycoplasma Pneumoniae) an additional study was conducted to clarify any possible negative trends. Table 5. Summary of analyte detection results observed for samples tested at TO (no storage control), ambient, refrigerated, and frozen conditions. Results are reported Detected results (D) in samples that contained the relevant analyte and as expected Not Detected (ND) results in samples that did not contain the relevant analyte. | Organism | Source ID | Concentration Testeda | No Storage Control T0 | | Ambient (25°C) 4 hours | | Refrigerated (8°C) 2 days | | Refrigerated (8°C) 3 days | | Frozen (≤-15°C) ≤30 daysb | | |---------------------------|-----------------------------------|--------------------------|-----------------------------|-------|------------------------------|-------|---------------------------------|-------|---------------------------------|-------|---------------------------------|-------| | | | | D | ND | D | ND | D | ND | D | ND | D | ND | | Adenovirus C (WHO IS)c | NIBSC 16/324 | $1.5E+04$ IU/mLd | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | | Coronavirus 229E | ATCC VR-740 | $3.3E+02$ copies/mL | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | | Coronavirus HKU1 | Clinical NPS Specimen 53727 | $1.0E+04$ copies/mL | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | | Coronavirus NL63 | BEI NR-470 | $2.7E+02$ copies/mL | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | | Coronavirus OC43 | Zeptometrix 0810024CF | $2.8E+02$ copies/mL | 10/10 | 30/30 | 10/10 | 30/30 | 9/10 | 30/30 | 10/10 | 30/30 | 9/10 | 30/30 | | Human Metapneumovirus | Zeptometrix 0810161CF | $5.0E+01$ TCID50/mL | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | | Human Enterovirus | ATCC VR-1823 | $2.6E+02$ copies/mLe | 10/10 | | 10/10 | | 10/10 | | 10/10 | | 10/10 | | | Human Rhinovirus | Zeptometrix 0810012CFN | $1.9E+01$ copies/mL | 10/10 | 20/20 | 10/10 | 20/20 | 9/10 | 20/20 | 9/10 | 20/20 | 8/10 | 20/20 | | Influenza A H1N1 | Zeptometrix 0810036CF | $7.0E+02$ copies/mL | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | | Influenza A H1N1- 2009 | Zeptometrix 0810249CFf | $1.7E+03$ copies/mL | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | | Influenza A H3N2h | Zeptometrix 0810526CF | $1.1E+00$ copies/mL | 10/10 | 20/20 | 10/10 | 20/20 | 10/10 | 20/20 | 10/10 | 20/20 | 10/10 | 20/20 | | Influenza B | Zeptometrix 0810255CF | $1.7E+02$ copies/mL | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | | Parainfluenza Virus 1 | Zeptometrix 0810014CF | $5.0E+02$ copies/mL | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | 10/10 | 30/30 | {19}------------------------------------------------ | | | Concentration | No Storage Control T0 | | Ambient (25°C) 4 hours | | Refrigerated (8°C) 2 days | | Refrigerated (8°C) 3 days | | Frozen (≤-15°C) <30 daysb | | |----------------------------------------------------------|--------------------------|-----------------------|-----------------------------|-------|------------------------------|-------|---------------------------------|-------|---------------------------------|-------|---------------------------------|-------| | Organism | Source ID | Testeda | D | ND | D | ND | D…

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