FilmArray Respiratory Panel 2 plus (RP2plus)

DEN170017 · Biofire Diagnostics, LLC · PZF · Nov 24, 2017 · Pathology

Device Facts

• Record ID: DEN170017
• Device Name: FilmArray Respiratory Panel 2 plus (RP2plus)
• Applicant: Biofire Diagnostics, LLC
• Product Code: PZF · Pathology
• Decision Date: Nov 24, 2017
• Decision: DENG
• Submission Type: Direct
• Regulation: 21 CFR 866.4001
• Device Class: Class 2

Indications for Use

The FilmArray Respiratory Panel 2 plus (RP2plus) is a multiplexed nucleic acid test intended for use with FilmArray 2.0 or FilmArray Torch systems for the simultaneous qualitative detection and identification of nucleic acids from Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and multiple common viral and bacterial respiratory pathogens in nasopharyngeal swabs (NPS) obtained from individuals meeting MERS-CoV clinical and/or epidemiological criteria. Testing with the FilmArray RP2plus should not be performed unless the patient meets clinical and/or epidemiologic criteria for testing suspected MERS-CoV specimens. This includes: clinical signs and symptoms associated with MERS-CoV infection, contact with a probable or confirmed MERS-CoV case, history of travel to geographic locations where MERS-CoV cases were detected, or other epidemiological links for which MERS-CoV testing may be indicated. The FilmArray RP2plus identifies: Middle East Respiratory Syndrome Coronavirus (MERS-CoV) And the following viral and bacterial respiratory pathogen types and subtypes: 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, Mycoplasma pneumoniae. The detection and identification of specific viral and bacterial nucleic acids from MERS-CoV and other respiratory pathogens in individuals meeting MERS-CoV clinical and/or epidemiological criteria aids in the differential diagnosis of MERS-CoV infection, if used in conjunction with other clinical and epidemiological information in accordance with the guidelines provided by the appropriate public health authorities.

Device Story

Multiplexed nucleic acid test; uses FilmArray 2.0 or FilmArray Torch systems; inputs nasopharyngeal swabs (NPS); detects MERS-CoV, common viral/bacterial respiratory pathogens; aids differential diagnosis; results presumptive for MERS-CoV; requires confirmation by public health authorities; negative results do not rule out infection; used by healthcare professionals in clinical settings; output informs patient management and public health response.

Clinical Evidence

Prospective clinical study (N=1612) compared FilmArray RP2plus to FDA-cleared multiplexed respiratory panels and validated PCR/sequencing for B. parapertussis. Retrospective study (N=214) and contrived specimen study (N=50) supplemented data for low-prevalence analytes. MERS-CoV performance evaluated via retrospective clinical specimens (N=3) and contrived specimens (N=50). Results showed high PPA/NPA across analytes; MERS-CoV PPA 100%.

Technological Characteristics

Multiplexed nucleic acid test; uses FilmArray 2.0/Torch systems. Materials: plastic pouch with freeze-dried reagents, ceramic lysis beads, magnetic beads. Principle: nested multiplex RT-PCR followed by melt curve analysis. Energy: Peltier-based thermal control. Connectivity: Ethernet to PC. Software: automated interpretation of melt curves. Sterilization: N/A (single-use disposable pouch).

Indications for Use

Indicated for qualitative detection/identification of MERS-CoV and 21 common respiratory pathogens in nasopharyngeal swabs (NPS) from patients meeting MERS-CoV clinical/epidemiological criteria (e.g., symptoms, contact, travel).

Regulatory Classification

Identification

A multiplex respiratory panel to detect and identify emerging respiratory pathogen(s) and common respiratory pathogens in human clinical specimens is identified as an in vitro diagnostic device intended for the qualitative detection and identification of both emerging and common respiratory pathogens from individuals meeting specific emerging respiratory pathogen clinical and/or epidemiological criteria. For example, clinical signs and symptoms associated with infection of the emerging respiratory pathogen, contact with a probable or confirmed emerging respiratory pathogen case, history of travel to geographic locations where cases of the emerging respiratory pathogen were detected, or other epidemiological links for which testing of the emerging respiratory pathogen may be indicated. A device to detect and identify emerging respiratory pathogen(s) and common respiratory pathogens in human clinical specimens, and in turn to distinguish emerging respiratory pathogen(s) from common respiratory pathogens, is intended to aid in the differential diagnosis of the emerging respiratory pathogen infection, in conjunction with other clinical, epidemiologic, and laboratory data, in accordance with the guidelines provided by the appropriate public health authorities.

Special Controls

*Classification.* Class II (special controls). The special controls for this device are:(1) The intended use for the labeling required under § 809.10 of this chapter must include a description of what the device detects and measures, the specimen types, the results provided to the user, the clinical indications for which the test is to be used, the specific intended population(s), the testing location(s) where the device is to be used (if applicable), and other conditions of use as appropriate. (2) The labeling required under § 809.10 of this chapter must include: (i) A device description, including the parts that make up the device, ancillary reagents required but not provided, and an explanation of the methodology. (ii) Performance characteristics from analytical studies, including cut-off (if applicable), analytical sensitivity ( *i.e.,* limit of detection), inclusivity, reproducibility, interference, cross-reactivity, instrument carryover/cross-contamination (if applicable), and specimen stability.(iii) Detailed instructions for minimizing the risk of potential users' exposure to the emerging respiratory pathogen(s) that may be present in test specimens and those used as control materials. (iv) Detailed instructions for minimizing the risk of generating false positive test results due to carry-over contamination from positive test specimens and/or positive control materials. (v) A warning statement that the interpretation of test results requires experienced healthcare professionals who have training in principles and use of infectious disease diagnostics and reporting of results, in conjunction with the patient's medical history, clinical signs and symptoms, and the results of other diagnostic tests. (vi) A warning statement that culture should not be attempted in cases of positive results for an emerging respiratory pathogen unless a facility with an appropriate level of laboratory biosafety ( *e.g.,* BSL 3 and BSL 3+) is available to receive and culture specimens.(vii) A warning statement that device positive results for one or more common respiratory pathogens do not rule out bacterial infection, or co-infection with other common respiratory pathogens. (viii) A warning statement that respiratory pathogen(s) detected may not be the definite cause of disease. (ix) A warning statement that the use of additional laboratory testing ( *e.g.* bacterial culture, immunofluorescence, x-ray findings) and clinical presentation must be taken into consideration in order to obtain the final diagnosis of a respiratory infection.(x) A limiting statement that device negative results for the common respiratory pathogens do not preclude infection of a respiratory pathogen and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. (xi) A limiting statement that analyte targets ( *e.g.,* pathogen nucleic acid sequences or other molecular signatures) may persist in vivo, independent of organism viability. Detection of analyte target(s) does not imply that the corresponding pathogen(s) is infectious, nor is the causative agent(s) for clinical symptoms.(xii) A limiting statement that detection of pathogen nucleic acid sequences or other molecular signatures is dependent upon proper specimen collection, handling, transportation, storage and preparation. Failure to observe proper procedures in any one of these steps can lead to incorrect results. There is a risk of false negative values resulting from improperly collected, transported, or handled specimens. (xiii) A limiting statement that there is a risk of false positive values resulting from cross-contamination by target organisms, their nucleic acids or amplified product, or from non-specific signals in the assay. (xiv) A limiting statement that there is a risk of false negative results due to the presence of nucleic acid sequence variants in the pathogen targets of the device. (xv) A limiting statement that device performance was not established in immunocompromised patients. (xvi) A limiting statement that positive and negative predictive values are highly dependent on prevalence. The device performance was established during one or more specific respiratory seasons. The performance for some respiratory pathogens may vary depending on the prevalence and patient population tested. False positive test results are likely when prevalence of disease due to a particular respiratory pathogen is low or non-existent in a community. (xvii) In situations where the performance of the device was estimated based largely on testing pre-selected banked retrospective clinical specimens and/or contrived clinical specimen, a limiting statement that the estimated device performance of that specific pathogen or pathogen subtype may not reflect the performance or prevalence in the intended use population. (xviii) For devices with an intended use that includes detection of emerging respiratory pathogen(s), a limiting statement that testing with the device should not be performed unless the patient meets clinical and/or epidemiologic criteria for testing suspected specimens of the emerging respiratory pathogen. (xix) For devices with an intended use that includes detection of emerging respiratory pathogen(s), a limiting statement that positive results obtained with the device for the emerging respiratory pathogen are for the presumptive identification of that pathogen and that the definitive identification of the emerging respiratory pathogen requires additional testing and confirmation procedures in consultation with the appropriate public health authorities ( *e.g.,* local or state public health departments) for whom reporting is necessary.(xx) For devices with an intended use that includes detection of emerging respiratory pathogen(s), a limiting statement that negative results for the emerging respiratory pathogen, even in the context of device positive results for one or more of the common respiratory pathogens, do not preclude infection with the emerging respiratory pathogen and should not be used as the sole basis for patient management decisions. (xxi) For devices with an intended use that includes detection of emerging respiratory pathogen(s), a limiting statement that negative results for the emerging respiratory pathogen may be due to infection of the emerging respiratory pathogen at a specific respiratory tract location that may not be detected by a particular clinical specimen type. A negative result for the emerging respiratory pathogen in an asymptomatic individual does not rule out the possibility of future illness and does not demonstrate that the individual is not infectious. (xxii) For devices with an intended use that includes detection of emerging respiratory pathogen(s), a limiting statement that a nationally notifiable Rare Disease of Public Health Significance caused by an emerging respiratory pathogen must be reported, as appropriate, to public health authorities in accordance with local, state, and federal law. (3) Design verification and validation must include: (i) Performance results of an appropriate clinical study ( *e.g.,* a prospective clinical study) for each specimen type, and, if appropriate, results from additional characterized samples. The clinical study must be performed on a study population consistent with the intended use population and must compare the device performance to results obtained using FDA-accepted comparator methods or to expected negative results if the infection is not generally expected in the intended use population. Clinical specimens evaluated in the study must contain relevant organism concentrations applicable to the specimen type(s) and the targeted analyte(s). 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.(ii) For devices with an intended use that includes detection of emerging respiratory pathogen(s) for which an FDA recommended panel is available, design verification and validation must include the performance results of an analytical study testing an FDA recommended reference panel of characterized samples that contain the emerging respiratory pathogen. 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. (iii) An appropriate risk mitigation strategy, including a detailed description of all procedures and methods, for the post-market identification of genetic mutations and/or novel respiratory pathogen isolates or strains ( *e.g.,* regular review of published literature and annual in silico analysis of target sequences to detect possible mismatches. The required documentation for this device must also include all of the results, including any findings, from the application of this post-market mitigation strategy.(iv) For devices with an intended use that includes detection of multiple common respiratory pathogens, in addition to detecting emerging respiratory pathogen(s) in human clinical specimens, a detailed description of the identity, phylogenetic relationship, or other recognized characterization of the common respiratory pathogens that the device is designed to detect is addressed. Also, address in detail how the device results might be used in a diagnostic algorithm and other measures that might be needed for a laboratory diagnosis of respiratory tract infection. Perform 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. (v) A detailed device description, including the parts that make up the device, 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), 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 and appropriate.(vi) A detailed description of the device software, including software applications and hardware-based devices that incorporate software. (vii) For devices with an intended use that includes detection of Influenza A and Influenza B viruses and/or detection and differentiate between the Influenza A virus subtypes in human clinical specimens, in addition to detecting emerging respiratory pathogen(s), 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. Perform 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. (4) For devices with an intended use that includes detection of Influenza A and Influenza B viruses and/or detection and differentiate between the Influenza A virus subtypes in human clinical specimens, in addition to detecting emerging respiratory pathogen(s), the labeling required under § 809.10 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. When other Influenza A viruses are emerging, performance characteristics may vary. (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 required under § 809.10(b)(9) of this chapter must include a clear interpretation instruction for all valid and invalid output combinations, and recommendations for any required follow up 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 ( *e.g.,* 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.(5) The manufacturer must perform annual analytical reactivity testing of the device with contemporary influenza strains. This annual analytical reactivity testing must meet the following criteria: (i) The appropriate strains to be tested will be identified by FDA in consultation with the Centers for Disease Control and Prevention (CDC) and sourced from CDC or an FDA designated source. If the annual strains are not available from CDC, FDA will identify an alternative source for obtaining the requisite strains. (ii) The testing must be conducted according to a standardized protocol considered and determined by FDA to be acceptable and appropriate. (iii) By July 31 of each calendar year, the results of the last 3 years of annual analytical reactivity testing must be included as part of the device's labeling. If a device has not been on the market long enough for 3 years of annual analytical reactivity testing to have been conducted since the device received marketing authorization from FDA, then the results of every annual analytical reactivity testing since the device received marketing authorization from FDA must be included. The results must be presented as part of the device's labeling in a tabular format, which includes the detailed information for each virus tested as described in the certificate of authentication, either by: (A) Placing the results directly in the device's labeling required under § 809.10(b) of this chapter that physically accompanies the device in a separate section of the labeling where the analytical reactivity testing data can be found; or (B) In the device's label or in other labeling that physically 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 home page, as well as the primary part of the manufacturer's website that discusses the device, must provide a prominently placed hyperlink to the web page containing this information and must allow unrestricted viewing access. (6) If one of the actions listed at section 564(b)(1)(A)-(D) of the FD&C Act occurs with respect to an influenza viral strain, or if the Secretary of Health and Human Services (HHS) 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 viral samples in accordance with a standardized protocol considered and determined by FDA to be acceptable and appropriate. The procedure and location of testing may depend on the nature of the emerging virus. (ii) Within 60 days from the date that FDA notifies manufacturers that characterized 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 physically 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 physically 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 home page, as well as the primary part of the manufacturer's website that discusses the device, must provide a prominently placed hyperlink to the web page containing this information and must allow unrestricted viewing access.

Submission Summary (Full Text)

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## EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR FilmArray Respiratory Panel 2 plus (RP2plus)

## DECISION SUMMARY

## A. DEN Number:

DEN170017

#### B. Purpose for Submission:

De Novo request for evaluation of automatic class III designation for the FilmArray Respiratory Panel 2 plus (RP2plus).

#### C. Measurands:

The assay detects and identifies nucleic acids of the following respiratory pathogens: Middle East Respiratory Syndrome Coronavirus (MERS-CoV), 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 FilmArray 2.0 or FilmArray Torch systems for the simultaneous qualitative detection and identification of nucleic acids from Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and multiple common viral and bacterial respiratory pathogens (as identified above) in nasopharyngeal swabs (NPS) obtained from individuals meeting MERS-CoV clinical and/or epidemiological criteria (for example, clinical signs and symptoms associated with MERS-CoV infection, contact with a probable or confirmed MERS-CoV case. history of travel to geographic locations where MERS-CoV cases were detected, or other epidemiological links for which MERS-CoV testing may be indicated.

#### E. Applicant:

BioFire Diagnostics, LLC

#### F. Proprietary and Established Names: FilmArray Respiratory Panel 2 plus (RP2plus)

#### G. Regulatory Information:

- Regulation: 1. 21 CFR 866.4001
- 2. Classification: Class II (special controls)

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## 3. Product code(s):

PZF

- 4. Panel: 83- Microbiology
# H. Indications for Use:

# 1. Indications for Use:

The FilmArray Respiratory Panel 2 plus (RP2plus) is a multiplexed nucleic acid test intended for use with FilmArray 2.0 or FilmArray Torch systems for the simultaneous qualitative detection and identification of nucleic acids from Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and multiple common viral and bacterial respiratory pathogens in nasopharyngeal swabs (NPS) obtained from individuals meeting MERS-CoV clinical and/or epidemiological criteria.

Testing with the FilmArray RP2plus should not be performed unless the patient meets clinical and/or epidemiologic criteria for testing suspected MERS-CoV specimens. This includes: clinical signs and symptoms associated with MERS-CoV infection, contact with a probable or confirmed MERS-CoV case, history of travel to geographic locations where MERS-CoV cases were detected, or other epidemiological links for which MERS-CoV testing may be indicated.

The FilmArray RP2plus identifies:

- Middle East Respiratory Syndrome Coronavirus (MERS-CoV) ●
And the following viral and bacterial respiratory pathogen types and subtypes:

- 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 (IS 1001) ●

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- Bordetella pertussis (ptxP)
- . Chlamydia pneumoniae
- . Mycoplasma pneumoniae

The detection and identification of specific viral and bacterial nucleic acids from MERS-CoV and other respiratory pathogens in individuals meeting MERS-CoV clinical and/or epidemiological criteria aids in the differential diagnosis of MERS-CoV infection, if used in conjunction with other clinical and epidemiological information in accordance with the guidelines provided by the appropriate public health authorities.

FilmArray RP2plus MERS-CoV positive results are for the presumptive identification of MERS-CoV. The definitive identification of MERS-CoV requires additional testing and confirmation procedures in consultation with the appropriate public health authorities (e.g., local or state public health departments, etc.) for whom reporting is necessary. The diagnosis of MERS-CoV infection must be made based on history, signs, symptoms, exposure likelihood, and other laboratory evidence in addition to the identification of MERS-CoV.

FilmArray RP2plus MERS-CoV negative results, even in the context of a FilmArray RP2plus positive result for one or more of the common respiratory pathogens, do not preclude MERS-CoV infection and should not be used as the sole basis for patient management decisions. The levels of MERS-CoV that would be present in NPS specimens from individuals with early infection and from asymptomatic MERS-CoV carriers are not well understood. The FilmArray RP2plus MERS-CoV negative results may also be due to lower respiratory tract infection with MERS-CoV that may not be detected by an NPS specimen. In this context, collection of lower respiratory and serum specimens (if possible) for MERS-CoV testing using other laboratory tests is highly recommended in addition to testing for MERS-CoV RNA in NPS specimens (i.e., upper respiratory specimens) using the FilmArray RP2plus. A negative FilmArray RP2plus MERS-CoV result in an asymptomatic individual does not rule out the possibility of future illness and does not demonstrate that the individual is not infectious.

Viral culture should not be attempted in the cases of positive FilmArray RP2plus results for MERS-CoV unless a BSL 3 facility is available to receive and culture specimens.

Negative FilmArray RP2plus results in the setting of a respiratory illness may be due to infection with pathogens that are not detected by this test, or other pathogens that may not be detected by an NPS specimen. Positive FilmArray RP2plus results do not rule out coinfection with other organisms: the agent(s) detected by the FilmArray RP2plus may not be the definite cause of disease.

Due to the genetic similarity between Human Rhinovirus and Enterovirus, the FilmArrav RP2plus cannot reliably differentiate them. A positive FilmArray RP2plus Rhinovirus/Enterovirus result should be followed up using an alternate method (e.g., cell culture or sequence analysis) if differentiation is required.

Performance characteristics for Influenza A were established when Influenza A H1-2009,

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A H1, and A H3 were the predominant Influenza A viruses in circulation. Performance of detecting Influenza A may vary if other Influenza A strains are circulating or a novel Influenza A virus emerges. 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.

- 2. Special conditions for use statement(s): For prescription use only. For in vitro diagnostic use.
- 3. Special instrument requirements: FilmArray Respiratory Panel 2 plus (RP2plus) is performed on the FilmArray 2.0 or the FilmArray Torch systems.

## I. Device Description:

The FilmArray Respiratory Panel 2 plus (RP2plus) is designed to simultaneously detect and identify MERS-CoV and 21 different common pathogens (see the Indications for Use section) of respiratory tract infection from a single NPS specimen in a time frame (~45 minutes) that may allow the test results to be used as an aid in determining appropriate patient treatment and management. FilmArray RP2plus is compatible with BioFire Diagnostics' (BioFire) PCR-based in vitro diagnostic FilmArray 2.0 and FilmArray Torch systems for infectious disease testing. A specific software module (i.e., FilmArray RP2plus pouch module) is used to perform FilmArray RP2plus testing on these systems.

A test is initiated by loading Hydration Solution into one port of the FilmArray pouch and a NPS sample (in transport media) mixed with the provided Sample Buffer into the other port of the FilmArray RP2plus pouch and placing it in a FilmArray instrument. The FilmArray pouch contains all the reagents required for specimen testing and analysis in a freeze-dried format; the addition of Hydration Solution and Sample/Buffer Mix rehydrates the reagents. After the pouch is prepared, the FilmArray Software guides the user though the steps of placing the pouch into the instrument, scanning the pouch barcode, entering the sample identification, and initiating the run.

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

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Nucleic acid extraction occurs within the FilmArray pouch using mechanical and chemical lysis followed by purification using standard magnetic bead technology. After extracting and purifying nucleic acids from the unprocessed sample, the FilmArray performs a nested multiplex PCR that is executed in two stages. During the first stage, the FilmArray performs a single, large volume, highly multiplexed reverse transcription PCR (RT-PCR) reaction, PCR1. The products from first stage PCR are then diluted and combined with a fresh, primerfree master mix and a fluorescent double stranded DNA binding dye (LC Green Plus, BioFire Diagnostics, LLC). The solution is then distributed to each well of the array. Array wells contain sets of primers designed specifically to amplify sequences internal to the PCR products generated during the first stage PCR reaction. The 2nd stage PCR, or nested PCR, PCR2. is performed in singleplex fashion in each well of the array. At the conclusion of the 2nd stage PCR, the array is interrogated by melt curve analysis for the detection of signature amplicons denoting the presence of specific targets. A digital camera placed in front of the 2nd stage PCR captures fluorescent images of the PCR reactions and software interprets the data.

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

# Materials provided in each FilmArray RP2plus kit:

Each kit contains sufficient reagents to test 6 samples (6-test kit; RFIT-ASY-0137) or 30 samples (30-test kit; RFIT-ASY-0136):

- Individually-packaged FilmArray RP2plus pouches
- · Single-use (1.0 mL) Sample Buffer ampoules
- · Single-use pre-filled (1.5 mL) Hydration Injection Vials (blue)
- · Single-use Sample Injection Vials (red)
- Individually-packaged Transfer Pipettes

Materials required but not provided:

- 10% bleach solution
FilmArray system including:

- · FilmArray 2.0 or FilmArray Touch and accompanying software
- · FilmArray Pouch Loading Station

# Interpretation of Results

When PCR2 is complete, the FilmArray instrument performs a DNA melting analysis on the PCR products and measures the fluorescence signal generated in each well. The FilmArray Software then performs several analyses and assigns a final assay result. The steps in the analyses are described below.

- Analysis of melt curves
The FilmArray Software evaluates the DNA melt curve for each well of the PCR2 array to determine if a PCR product was present in that well. If the melt profile indicates the presence of a PCR product, then the analysis software calculates the melting temperature (Tm) of the curve and compares it against the expected Tm

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range for the assay. If the software determines that the Tm falls inside the assayspecific Tm range, the melt curve is called positive. If the software determines that the melt curve is not in the appropriate Tm range, the melt curve is called negative.

- · Analysis of replicates
Once melt curves have been identified, the software evaluates the three replicates for each assay to determine the assay result. For an assay to be called positive, at least two of the three associated melt curves must be called positive, and the Tm for at least two of the three positive melt curves must be similar (i.e., within 1°C). Assays that do not meet these criteria are called negative.

For the following organisms detected by the FilmArray RP2plus, the organism is reported as "Detected" if a single corresponding assay is positive.

- · Coronavirus 229E
- · Coronavirus HKU1
- · Coronavirus NL63
- · Coronavirus OC43
- Human Metapneumovirus
- Human Rhinovirus/Enterovirus
- · Influenza B
- · Parainfluenza Virus 1
- Parainfluenza Virus 2
- · Parainfluenza Virus 3
- · Parainfluenza Virus 4
- · Respiratory Syncytial Virus
- Bordetella parapertussis (IS 1001)
- Bordetella pertussis (ptxP)
- Chlamydia pneumoniae
- Mycoplasma pneumoniae

The test results for MERS-CoV, Adenovirus, and Influenza A (including subtyping) depend on the interpretation of results from more than one corresponding assay. Interpretation and actions for these results are provided below.

- MERS-CoV
The FilmArray RP2plus pouch contains two different assays for the detection of MERS-CoV. The FilmArray software interprets each of these assays independently and the results are combined as a final test result for the virus. Both assays must be positive for the test report result to be MERS-CoV "Detected". If only one assay is positive, the result is MERS-CoV "Equivocal" and the sample should be retested. If both the assays are negative, the test report result will be MERS-CoV "Not Detected".

- Adenovirus
The FilmArray RP2plus pouch contains five different assays (Adeno2, Adeno6, Adeno7.1, and Adeno8) for the detection of Adenovirus. The FilmArray Software interprets each of these assays independently and the results are combined as a final test result for the virus. If one or any combination of assays is positive, the

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test report result will be Adenovirus "Detected". If all assays are negative, the test report result will be Adenovirus "Not Detected".

- · Influenza A and Subtyping
The assays in the FilmArray RP2plus are designed to both detect Influenza A and to differentiate commonly occurring hemagglutinin subtypes. To accomplish this, the FilmArray RP2plus uses two Influenza A assays, FluA-pan-1 and FluA-pan-2, and three subtyping assays, FluA-H1-2, FluA-H1-2009, and FluA-H3, directed at the respective hemagglutinin gene. Each of the individual assays is interpreted independently and the test result reported for Influenza A is based on the combined results of the five assays as outlined in Table 1.

| Assay<br>Result | FluA-pan<br>Assays<br>(n=2) | FluA-H1-2 | FluA-H1-<br>2009 | FluA-H3 | Action |
|---------------------------------------|-----------------------------|------------|------------------|----------|----------------------------------------------------------------------------------|
| Influenza A Not Detected | Negative | Negative | Negative | Negative | |
| Influenza A H1 | ≥1 positive | Positive | Negative | Negative | None |
| Influenza A H3 | ≥1 positive | Negative | Negative | Positive | None |
| Influenza A H1-2009 | ≥1 positive | Any result | Positive | Negative | |
| Influenza A H1<br>Influenza A H3 | ≥1 positive | Positive | Negative | Positive | Multiple infections<br>are possible but<br>rare a, retest to<br>confirm result b |
| Influenza A H1-2009<br>Influenza A H3 | ≥1 positive | Any result | Positive | Positive | Multiple infections<br>are possible but<br>rare a, retest to<br>confirm result b |
| Influenza A (no subtype<br>detected) | 2 positive | Negative | Negative | Negative | Retest |
| Influenza A Equivocal | 1 positive | Negative | Negative | Negative | |
| Influenza A H1 Equivocal | Negative | Positive | Negative | Negative | Retest |
| Influenza A H3 Equivocal | Negative | Negative | Negative | Positive | Retest |
| Influenza A H1-2009<br>Equivocal | Negative | Any result | Positive | Negative | Retest |

Table 1: Possible Assay Results for Influenza A and the Corresponding Interpretation

a The FilmArray RP2plus can simultaneously detect multiple influenza viruses contained in multivalent vaccines.

b Repeated multiple positives should be further confirmed by other FDA cleared Influenza subtyping tests.

#### Influenza A (no subtype detected):

If both FluA-pan assays are positive, but none of the hemagglutinin subtyping assays are positive, then the interpretation is Influenza A (no subtype detected). This result could occur when the titer of the virus in the specimen is low and not detected by the subtyping assays. This result could also indicate the presence of a novel Influenza A strain. In both cases, the sample in question should be retest . If the retest provides a different result, test the sample a third time to ensure the accuracy of the result. If the retest provides the same result, then the function of the RP2 pouches should be verified by testing with appropriate external control materials (known positive samples for Influenza A H1. Influenza A H3 and Influenza A H1-2009), and a negative control should also be run to test for PCR-product contamination. If the FilmArray RP2plus accurately identifies the external and negative controls, contact the appropriate public health authorities for confirmatory testing.

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# FilmArray RP2plus Test Report

The FilmArray RP2plus test report is automatically displayed upon completion of a run and can be printed or saved as a PDF file. Each report contains a Run Summary, a Result Summary, and a Run Details section. An example of the test report is presented below:

| FilmArray<br>Respiratory Panel 2 plus | | | | | |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------|------------------------------|-------------------|--|
| | | | | www.BioFireDx.com | |
| Run Summary | | | | | |
| Sample ID: | RP2plus Example<br>Run Date:<br>06 Mar 2017 | | | | |
| Detected:<br>Equivocal: | 5:21 PM<br>Middle East Respiratory Syndrome Coronavirus (MERS-CoV)<br>Passed<br>Controls:<br>+ Influenza A | | | | |
| Result Summary | | | | | |
| | Viruses | | | | |
| Not Detected<br>Not Detected<br>Not Detected<br>Not Detected<br>Not Detected<br>Not Detected<br>Not Detected<br>* Equivocal<br>Not Detected<br>/ Detected<br>Not Detected<br>Not Detected<br>Not Detected<br>Not Detected<br>Not Detected | Adenovirus<br>Coronavirus 229E<br>Coronavirus HKU1<br>Coronavirus NL63<br>Coronavirus OC43<br>Human Metapneumovirus<br>Human Rhinovirus/Enterovirus<br>Influenza A<br>Influenza B<br>Middle East Respiratory Syndrome Coronavirus (MERS-CoV)<br>Parainfluenza Virus 1<br>Parainfluenza Virus 2<br>Parainfluenza Virus 3<br>Parainfluenza Virus 4<br>Respiratory Syncytial Virus | | | | |
| Bacteria | | | | | |
| Not Detected<br>Not Detected<br>Not Detected<br>Not Detected | Bordetella parapertussis (IS1001)<br>Bordetella pertussis (ptxP)<br>Chlamydia pneumoniae<br>Mycoplasma pneumoniae | | | | |
| Run Details | | | | | |
| Pouch:<br>Run Status:<br>Serial No .:<br>Lot No .: | RP2plus v1.0<br>Completed<br>06265525<br>161013E | Protocol:<br>Operator:<br>Instrument: | NPS2 v3.1<br>JDoe<br>TM8CCF3 | | |

## • Run Summary

The Run Summary section of the test report provides the Sample ID, time and date of the run, control results and an overall summary of the test results. Any organism with a "Detected" result will be listed in the corresponding field of the summary. If assays for all the organism were negative, then "None" will be displayed in the Detected field. All Influenza A equivocal results (refer to Table 2) will be displayed in the Equivocal field. Controls are listed as "Passed", "Failed" or "Invalid". Table 2 below provides additional information for each of the possible control field results.

#### Table 2: Interpretation of Controls Field on the FilmArray RP2plus Test Report

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| Control Result | Explanation | Action |
|----------------|-----------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Passed | The run was successfully completed<br>AND<br>Both pouch controls were successful. | None<br>Report the results provided on the test report |
| Failed | The run was successfully completed<br>BUT<br>At least one of the pouch controls (RNA<br>Process Control and/or PCR2 Control)<br>failed. | Repeat the test using a new pouch.<br>If the error persists, contact Technical Support for<br>further instruction. |
| Invalid | The controls are invalid because the run<br>did not complete.<br>(Typically this indicates a software or<br>hardware error). | Note any error codes displayed during the run and<br>the Run Status field in the Run Details section of<br>the report. Refer to the appropriate FilmArray<br>Operator's Manual or contact Technical Support<br>for further instruction.<br>Once the error is resolved, repeat the test or repeat<br>the test using another instrument. |

# • Results Summary

The Result Summary section of the test report lists the result for each target tested by the panel. Possible results for each organism except for Influenza A and subtyping are "Detected", "Not Detected", or "Invalid".

Table 3 below provides an explanation for each interpretation and any follow-up necessary to obtain a final result.

### Table 3: Reporting of Results and Required Actions

| Result | Explanation | Action |
|----------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------|
| Detected a | The run was successfully completed<br>AND<br>The pouch controls were successful (Passed)<br>AND<br>The assay(s) for the organism were POSITIVE<br>(i.e., met the requirements for a positive result described in the<br>Interpretation of Results section above) | Report results. |
| Not Detected | The run was successfully completed<br>AND<br>The pouch controls were successful (Passed)<br>AND<br>The assay(s) for the organism were NEGATIVE<br>(i.e., did not meet the requirements for a positive result described in<br>the Assay Interpretation section above) | Report results. |
| Equivocal<br>(Influenza A<br>and MERS-CoV<br>only) | The run was successfully completed<br>AND<br>The pouch controls were successful (Passed)<br>AND<br>The combination of positive and negative assay results for Influenza<br>A were inconclusive<br>(see Table 2) | Retest the original<br>specimen using a new<br>pouch and report the<br>results of the retest. |
| Invalid | The pouch controls were not successful (Failed)<br>OR<br>The run was not successful<br>(Run Status displayed as: Aborted, Incomplete, Instrument Error or<br>Software Error) | See Table 2 |

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

The Run Details section provides additional information about the run including: pouch information (type, lot number, and serial number), Run Status (Completed, Incomplete, Aborted, Instrument Error, Instrument Communication Error, or Software Error), the protocol that was used to perform the test, the identity of the operator that performed the test, and the instrument used to perform the test.

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

- · FDA guidance document issued on August 27, 2014, titled "Highly Multiplexed Microbiological/Medical Countermeasure In Vitro Nucleic Acid Based Diagnostic Devices"
- · FDA guidance document issued on October 9, 2009, titled "Class II Special Controls Guidance Document: Respiratory Viral Panel Multiplex Nucleic Acid Assay"
- · FDA guidance document issued on October 9, 2009, titled "Class II Special Controls Guidance Document: Testing for Detection and Differentiation of Influenza A Virus Subtypes Using Multiplex Assays"
- · FDA guidance document issued on October 9, 2009, titled "Class II Special Controls Guidance Document: Testing for Human Metapneumovirus (hMPV) Using Nucleic Acid Assays"
- · FDA guidance document issued on March 13, 2007, titled "Statistical Guidance on Reporting Results from Studies Evaluating Diagnostic Tests"
- · FDA guidance document issued on July 15, 2011, titled "Establishing the Performance Characteristics of In Vitro Diagnostic Devices for the Detection or Detection and Differentiation of Influenza Viruses"
- · FDA guidance document issued on April 25, 2005, titled "Guidance on Informed Consent for In Vitro Diagnostic Device Studies Using Leftover Human Specimens that are Not Individually Identifiable"
- · FDA guidance document issued on May 11, 2005, titled "Guidance for the Content of Premarket Submissions for Software Contained in Medical Devices"
- · FDA guidance document issued on September 9, 1999, titled "Off-The-Shelf Software Use in Medical Devices"
- · FDA guidance document issued on January 11, 2002, titled "General Principle of Software Validation"
- · FDA guidance document issued on January 1, 2000, titled "Guidance for Industry and FDA on Alternative to Certain Prescription Device Labeling Requirements'
- · Interference Testing in Clinical Chemistry; Clinical and Laboratory Standards Institute (CLSI) Approved Guideline - Second Addition, EP7-A2 (2005)
- 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) Approved Guideline, MM3-A2 (February 2006)

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- · Evaluation of Stability of In Vitro Diagnostic Reagents; Clinical and Laboratory Standards Institute (CLSI) Approved Guideline, EP25-A (September 2009)
# K. Test Principle:

The FilmArray instrument, software, and pouch work together to perform sample lysis and purification, amplification, and detection of nucleic acid.

One of the primary functions of a FilmArray instrument is to drive the various steps in the testing process by moving liquids to appropriate locations within the pouch. Liquids are moved from the fitment to the blisters of the pouch by means of (b) (4) in the instrument piston block which press on the syringe-like plunging devices in the pouch fitment. There are 12 pistons that operate in a specified sequence to deliver reagents to the appropriate blisters in the pouch when they are needed.

Within the pouch, liquids are moved by using bladders and hard seals to exert pressure on the exterior of the pouch, such that the instrument is never in contact with the liquids contained in the pouch. The bladders are inflatable elastomeric membranes used to 'squish' the pouch blisters, thus forcing the liquid out of the compressed blister and along any connecting channels. The hard-seals are piston driven actuators used to direct the flow of liquids from the blisters by pinching shut the channels and blocking flow while pinched.

Thermal interactions between the pouch and instrument play a crucial role in the amplification of target nucleic acids. Temperature control is driven by a pair of numerically controlled Peltier devices; solid-state thermal control instruments fitted with calibrated temperature sensors and protective circuitry. These Peltier devices heat and cool to perform the first and second stage PCR reactions and carefully control the temperature across the array during DNA melting. The instrument uses a blue LED to illuminate the second stage PCR array and a digital camera to record fluorescence generated in the second stage PCR. The optical system is designed to detect the fluorescence signal generated during DNA melting.

The instrument communicates with the computer and the FilmArray software using Ethernet cables. The software provides instructions to the instrument to control each of the steps described above.

A detailed explanation of specific steps in the testing process is provided below:

- 1. Sample Lysis and Purification
Nucleic acid purification occurs in the first four blisters of the pouch using magnetic bead technology.

## a. Sample Lysis

Prior to loading the sample into the pouch, nucleases are inactivated by mixing the sample with a denaturing buffer (FilmArray Sample Buffer). The sample/sample buffer mixture is then loaded into the pouch via the injection port and pouch vacuum pulls the

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liquid into the sample well of the pouch fitment.

During the addition of the sample/sample buffer mixture, the template for the RNA Process Control is rehydrated and introduced into the reaction mixture. The RNA Process Control targets an mRNA of Schizosaccharomyces pombe, which is freezedried into the sample well of each pouch. The S. pombe is processed in parallel with the patient sample through each step of the test including nucleic acid extraction, reverse transcription (RT), PCR1, PCR2, and DNA melting. A positive result for the RNA Process Control indicates that all steps in the test are functioning properly.

The instrument activates a piston located above the fitment to move the sample/sample buffer mixture from the fitment into the trapezoidal sample lysis blister and then heatseals the fitment to prevent sample loss. The sample lysis blister contains ceramic beads. The instrument then activates the bead beater assembly which rotates a metal bar that strikes the pouch for 60 seconds to lyse organisms in the sample by creating high speed impacts between the sample and beads (bead beating). At the conclusion of the bead beating process, cells and organisms are lysed and the cell contents, including the nucleic acids, are released into the reaction mixture.

## b. Nucleic Acid Isolation

The instrument inflates the appropriate bladder and moves the lysed sample into the magnetic bead blister. Here, the liberated nucleic acids are captured by adsorption to silica- magnetic beads.

The instrument then uses a retractable magnet positioned adjacent to the blister to hold the beads against the inside of the blister while they are washed to remove proteins, cell debris, and other potential PCR inhibitors. The instrument moves the wash buffer from the fitment into the appropriate blisters by exerting pressure on pistons located above the fitment.

After the washes are completed, an elution buffer is moved from the fitment to the appropriate blister resulting in the nucleic acids being released from the beads. The instrument then moves the purified nucleic acid solution to the 1st stage PCR blister while the beads and other waste products are pushed back to the trapezoidal blister.

# 2. Reverse Transcription and 1st Stage Multiplex PCR

In the 1st stage PCR blister, liquid containing the purified nucleic acid rehydrates a freezedried reagent pellet containing all the outer primers. A PCR master mix, containing all components needed for PCR and reverse transcription (RT), is moved from the fitment to an adioining blister. A Peltier device is in contact with these two blisters and the instrument performs a "hot-start" PCR and RT by preheating the blisters containing the purified sample and the PCR master mix. Once the appropriate temperature is reached, the contents of the two blisters are mixed and the RT step and thermo-cycling is initiated.

Since the FilmArray RP2plus includes RNA viruses, an RT step is needed to convert the viral RNA into cDNA that can be amplified by PCR. Both the RT and the first stage of the

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nested PCR reaction are performed using the same outer primers and master mix.

- 3. Dilution, 2nd Stage PCR and DNA Melt Analysis
Following completion of the RT and 1st stage PCR steps, a second singleplex PCR is carried out. To accomplish this, the instrument dilutes the products of the 1st stage PCR with fresh PCR master mix containing a double stranded DNA binding dye (LC Green Plus, BioFire Diagnostics, LLC). This solution is distributed over the 2nd stage PCR array, where it rehydrates the dried primers in each well. The individual wells in the array contain primers for different assays (each assay is present in triplicate wells of the array) that target specific nucleic acid sequences from each of the pathogens or control templates. The primers in the PCR2 array are "nested" or internal to the specific PCR products of the 1st stage multiplex reaction. A second Peltier device is responsible for driving the PCR2 reaction and for controlling temperature during DNA melting. The product of the 2nd stage PCR is visualized with the fluorescent LC Green Plus dye. At the conclusion of PCR2, the temperature of the array is gradually increased and the fluorescence in each well is monitored and analyzed to generate a melt curve. The instrument then transfers images and temperature measurements to the FilmArray software for analysis.

The PCR2 array also contains a control assay, called the PCR2 Control, which is comprised of a specific set of PCR2 assay primers along with the corresponding template pre-spotted into three specific wells of the array. Failure of the PCR2 Control invalidates the run and indicates a test failure that is specific to the PCR2 step of the testing process.

## 4. Data Analysis and Result Reporting

The temperature at which a specific PCR product melts (melting temperature or Tm) is consistent and predictable, and the FilmArray software automatically evaluates the results from replicate wells of each assay for the detection of amplicons with a specific Tm, which denotes the presence of specific bacterial or viral targets. The FilmArray software uses the following steps to interpret the melt curve data generated from each FilmArray RP2plus assay:

# a. Analysis of Melt Curves

First, the FilmArray RP2plus Melt Detector performs a set of 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 associated assay. If the software determines that the melt is positive and the melt curve falls inside the assay's specific melt range, then the 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.

# b. Analysis of Replicates

Next, the analysis software evaluates the replicates for each assay (target and control) to determine if the assay is positive or negative. To be called positive, at least two of the three wells associated with an assay must have a positive melt curve and the Tm for

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the positive curves must be similar (i.e., within 1°C). Assays with replicates that do not meet these criteria are called negative.

- c. Analysis of Controls
Results for control assays are compared to their expected values and assigned a single pass or fail result for each control. Pouch-specific rules define how control failures affect interpretations. The default rule specifies that any control failure invalidates the entire run. For the FilmArray RP2plus, failure of the RNA Process Control or the PCR2 Control is interpreted as a control failure and all target assays (regardless of the assay result) are assigned a test result of invalid.

- d. Interpretation of Assay Results
Once the results for the individual assays are determined, the software applies interpretation rules to determine the final test results. For many organisms, the target is determined to be present or absent if a single associated assay is positive or negative, respectively. For these organisms, the final test result is either "Detected" (for positive results), "Not Detected" (for negative results) or "Invalid" (when either control fails or the run fails). The FilmArray RP2plus also includes test results that rely on the results of multiple assays. See the Interpretation of Results section for more information on interpreting these test results.

# L. Performance Characteristics:

# 1. Analytical performance:

- a. Reproducibility
A reproducibility study was conducted at three testing sites on a combination of FilmArray 2.0 and FilmArray Torch systems. The study incorporated a range of potential variation introduced by site (three testing sites), day (five different days), operator (at least two per site), system, instrument or Torch module (at least three per site/sample), and pouch lot (at least three).

A total of four contrived NPS samples containing known quantities of various RP2plus analytes (Table 4 below) were prepared in simulated NPS in VTM sample matrix . The contrived samples contained combinations of 12 different FilmArray RP2plus analytes2, each at three different concentrations, Negative, Low Positive (1×LoD), and Moderate Positive (3×LoD). The negative data were acquired from samples not spiked with a particular analyte (i.e., negative data for the analytes in Sample#1 and #2 were obtained from Sample#3 and #4, and vice-versa).

⁴ Note: The simulated NPS in VTM sample matrix and the natural NPS in VTM sample matrix were demonstrated to be equivalent to FilmArray RP test detectability of analytical studies in support of the original FDA-clearance of the FilmArray RP test. Refer to K103175, K110764, and K120267 for additional details of the analytical studies.

² Note: Single-spiked and multi-spiked specimens were demonstrated to FilmArray RP test detectability of analytes in analytical studies in support of the original FDA-clearance of the FilmArray RP test. Refer to K103175, K110764. and K120267 for additional details of the analytical studies.

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| Organism | Strain/Sero<br>type | Source/ ID | Limit of Detection<br>(LoD) Concentration | Sample #1<br>Concentration<br>(1xLoD) | Sample #2<br>Concentration<br>(3xLoD) |
|-----------------------------------|-----------------------------|----------------------------|-------------------------------------------------|-------------------------------------------------|-------------------------------------------------|
| Coronavirus<br>OC43 | OC43 | ATCC VR-<br>759 | 5.6E+02 Copies/mL<br>(3.0E+01 TCID50/mL) | 5.6E+02 Copies/mL<br>(3.0E+01<br>TCID50/mL) | 1.7E+03 Copies/mL<br>(9.0E+01<br>TCID50/mL) |
| Parainfluenza<br>virus 2 | Type 2 | Zeptometrix<br>0810015CF | 3.0E+01 Copies/mL<br>(5.0E-01 TCID50/mL) | 3.0E+01 Copies/mL<br>(5.0E-01 TCID50/mL) | 9.0E+01 Copies/mL<br>(1.5E+00<br>TCID50/mL) |
| Adenovirus C2 | Species C<br>Serotype 2 | ATCC VR-<br>846 | 3.7E+01 Copies/mL<br>(2.0E+00 TCID50/mL) | 3.7E+01 Copies/mL<br>(2.0E+00<br>TCID50/mL) | 1.1E+02 Copies/mL<br>(6.0E+00<br>TCID50/mL) |
| Influenza A<br>H3N2 | A/Port<br>Chalmers/1/<br>73 | ATCC VR-<br>810 | 2.1E+01 Copies/mL<br>(1.0E-01 TCID50/mL) | 2.1E+01 Copies/mL<br>(1.0E-01 TCID50/mL) | 6.3E+01 Copies/mL<br>(3.0E-01 TCID50/mL) |
| Rhinovirus | Type 1A | Zeptometrix<br>0810012CFN | 3.8E+01 Copies/mL<br>(1.0E-01 TCID50/mL) | 3.8E+01 Copies/mL<br>(1.0E-01 TCID50/mL) | 1.1E+02 Copies/mL<br>(3.0E-01 TCID50/mL) |
| Bordetella<br>parapertussis | A747 | Zeptometrix<br>081461 | 5.8E+01 IS1001<br>Copies/mL<br>(4.1E+01 CFU/mL) | 5.8E+01 IS1001<br>Copies/mL<br>(4.1E+01 CFU/mL) | 1.7E+02 IS1001<br>Copies/mL<br>(1.2E+02 CFU/mL) |
| Organism | Strain/Sero<br>type | Source/ ID | Limit of Detection<br>(LoD) | Sample #3<br>(1xLoD) | Sample #4<br>(3xLoD) |
| Chlamydia<br>pneumoniae | TW183 | ATCC VR-92 | 6.6E+01 Copies/mL<br>(1.0E-01 TCID50/mL) | 6.6E+01 Copies/mL<br>(1.0E-01 TCID50/mL) | 2.0E+02 Copies/mL<br>(3.0E-01 TCID50/mL) |
| Influenza B | B/FL/04/06 | Zeptometrix<br>0810255CF a | 3.4E+01 Copies/mL<br>(5.0E+00 TCID50/mL) | 3.4E+01 Copies/mL<br>(5.0E+00<br>TCID50/mL) | 1.0E+02 Copies/mL<br>(1.5E+01<br>TCID50/mL) |
| Parainfluenza<br>virus 4 | Type 4a | Zeptometrix<br>0810060CF | 1.6E+03 Copies/mL<br>(5.0E+01 TCID50/mL) | 1.6E+03 Copies/mL<br>(5.0E+01<br>TCID50/mL) | 4.8E+03 Copies/mL<br>(1.5E+02<br>TCID50/mL) |
| Human<br>Metapneumo<br>virus | Type 16, A1<br>IA10-2003 | Zeptometrix<br>0810161CF | 1.2E+03 Copies/mL<br>(1.0E+01 TCID50/mL) | 1.2E+03 Copies/mL<br>(1.0E+01<br>TCID50/mL) | 3.6E+03 Copies/mL<br>(3.0E+01<br>TCID50/mL) |
| Respiratory<br>Syncytial<br>Virus | Type A | Zeptometrix<br>0810040ACF | 9.0E+00 Copies/mL<br>(2.0E-02 TCID50/mL) | 9.0E+00 Copies/mL<br>(2.0E-02 TCID50/mL) | 2.7E+01 Copies/mL<br>(6.0E-02 TCID50/mL) |
| Bordetella<br>pertussis b | A639 | Zeptometrix<br>0801459 | 1.0E+03 CFU/mL | 1.0E+03 CFU/mL | 3.0E+03 CFU/mL |

Table 4: Reproducibility Test Panel for the FilmArray RP2nlus

ª Formerly Zeptometrix 0810037CF.

b For B. pertussis, the FilmArray RP2plus amplifies a single-copy target and commercially available qPCR assays typically target the multi-copy IS481 sequences, therefore RP2plus testing was performed based on the CFU/mL and an equivalent Copies/mL was not determined for this analyte.

> Once prepared, each sample of the reproducibility study panel was tested with the FilmArray RP2plus to confirm it contained the intended analytes at the intended concentration and then divided into single-use aliquots (400 µL) and stored frozen (≤ -70°C) until the day of testing.

> After being distributed to the sites, six replicates of each sample were tested on five different days on various FilmArray Torch modules (Site A, Site C) or FilmArray 2.0

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instruments (Site B. Site C). Sites A and C were configured with at least three Torch modules per sample, while sites B and C were configured to utilize at least three different FilmArray 2.0 instruments per sample. The Site C tested a total of 12 replicates of each sample per day, with six replicates tested on the Torch system and six replicates tested on the FilmArray 2.0 system.

Daily testing was performed by at least two different operators per system and site, and three different pouch lots were used on rotating days so that data from all variables were distributed between reagent lots. For any required retest per the Instructions for Use, another aliquot of the same sample was tested on the same day by the same operator using the same system, instrument or Torch module, and pouch lot. Results of the valid retest were used as the final test result for the sample aliquot.

In total, 120 data points per sample (over a total of 480 valid runs) were obtained, with 60 data points per sample per system (i.e., FilmArray 2.0 and FilmArray Torch systems), 30 data points per sample per Site A and B, and 60 data points at Site C.

Over the course of the reproducibility study, a total of 15 different FilmArray 2.0 instruments and 19 different FilmArray Torch modules (four Torch bases) were used by seven operators at three sites. Valid results were obtained from 480 out of the 489 runs that were initiated (480/489, 98.2%). The majority of invalid runs (8/9) were associated with a Control failure, while one invalid run was due to an instrument error (Table 5 below).

| | Runs | Control Failure<br>(Percentage) | Instrument Errors<br>(Percentage) | Software Errors<br>(Percentage) |
|-----------------|------|---------------------------------|-----------------------------------|---------------------------------|
| FilmArray 2.0 | 247 | 6<br>(2.4%) | 1<br>(0.4%) | 0<br>(0.0%) |
| FilmArray Torch | 242 | 2<br>(0.8%) | 0<br>(0.0%) | 0<br>(0.0%) |
| Total | 489 | 8 a<br>(1.6%) | 1<br>(0.2%) | 0<br>(0.0%) |

Table 5: Performance of the FilmArray Systems and RP2plus Controls during the Reproducibility Study

" Seven control failures occurred on pouch lot 349116, while one occurred on pouch lot 347416.

A summary of the reproducibility study results (percent (%) agreement with the expected Detected or Not Detected result) for each analyte (by site and system) is provided in Table 6 below.

| | | | Table 6: Reproducibility of FilmArray RP2plus Results on FilmArray Torch and FilmArray 2.0 Systems | |
|--|--|--|----------------------------------------------------------------------------------------------------|--|
| | | | | |

| Analyte | Concentration<br>Tested | Expected<br>Result | Agreement with Expected Result<br>FilmArray Torch | | | Agreement with Expected Result<br>FilmArray 2.0 | | | All<br>Sites/Systems<br>(95% CI) | |
|-------------------------------------|-------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------|---------------------------------------------------|-----------------|-------------------------|-------------------------------------------------|-----------------|-------------------------|---------------------------------------|---------------------------------------|
| | | | Site A | Site C | System<br>Sub-Total | Site B | Site C | System<br>Sub-Total | | |
| Viruses | | | | | | | | | | |
| MERS-CoV | None<br>(no analyte) | Not<br>Detected | 120/120<br>100% | 120/120<br>100% | 240/240<br>100% | 120/120<br>100% | 120/120<br>100% | 240/240<br>100% | 480/480<br>100%<br>(99.2%-100%) | |
| | Concentration | Expected<br>Result | Agreement with Expected Result | | | | | | | |
| | | | FilmArray Torch | | | FilmArray 2.0 | | | All | |
| Analyte | Tested | | Site A | Site C | System<br>Sub-<br>Total | Site B | Site C | System<br>Sub-<br>Total | Sites/Systems<br>(95% CI) | |
| | Moderate Positive<br>(3× LoD)<br>1.1E+02<br>Copies/mL<br>(6.0E+00<br>TCID50/mL) | Detected | 30/30<br>100% | 29/30<br>96.7% | 59/60<br>98.3% | 29/30<br>96.7% | 30/30<br>100% | 59/60<br>98.3% | 118/120<br>98.3%<br>(94.1%-<br>99.8%) | |
| Adenovirus | Low Positive<br>(1× LoD)<br>3.7E+01<br>Copies/mL<br>(2.0E+00<br>TCID50/mL) | Detected | 30/30<br>100% | 30/30<br>100% | 60/60<br>100% | 30/30<br>100% | 29/30<br>96.7% | 59/60<br>98.3% | 119/120<br>99.2%<br>(95.4%-100%) | |
| | None<br>(no analyte) | Not<br>Detected | 60/60<br>100% | 60/60<br>100% | 120/120<br>100% | 60/60<br>100% | 60/60<br>100% | 120/120<br>100% | 240/240<br>100%<br>(98.5%-100%) | |
| Coronavirus<br>229E | None<br>(no analyte) | Not<br>Detected | 120/120<br>100% | 120/120<br>100% | 240/240<br>100% | 120/120<br>100% | 120/120<br>100% | 240/240<br>100% | 480/480<br>100%<br>(99.2%-100%) | |
| Coronavirus<br>HKU1 | None<br>(no analyte) | Not<br>Detected | 120/120<br>100% | 120/120<br>100% | 240/240<br>100% | 120/120<br>100% | 120/120<br>100% | 240/240<br>100% | 480/480<br>100%<br>(99.2%-100%) | |
| | Moderate Positive<br>(3× LoD)<br>1.7E+03<br>Copies/mL<br>(9.0E+01<br>TCID50/mL) | Detected | 29/30<br>96.7% | 29/30<br>96.7% | 58/60<br>96.7% | 29/30<br>96.7% | 30/30<br>100% | 59/60<br>98.3% | 117/120<br>97.5%<br>(92.9%-<br>99.5%) | |
| Coronavirus<br>OC43 | Low Positive<br>(1× LoD)<br>5.6E+02<br>Copies/mL<br>(3.0E+01<br>TCID50/mL) | Detected | 30/30<br>100% | 30/30<br>100% | 60/60<br>100% | 30/30<br>100% | 27/30<br>90.0% | 57/60<br>95.0% | 117/120<br>97.5%<br>(92.9%-<br>99.5%) | |
| | None<br>(no analyte) | Not<br>Detected | 60/60<br>100% | 60/60<br>100% | 120/120<br>100% | 60/60<br>100% | 60/60<br>100% | 120/120<br>100% | 240/240<br>100%<br>(98.5%-100%) | |
| Coronavirus<br>NL63 | None<br>(no analyte) | Not<br>Detected | 120/120<br>100% | 120/120<br>100% | 240/240<br>100% | 120/120<br>100% | 120/120<br>100% | 240/240<br>100% | 480/480<br>100%<br>(99.2%-100%) | |
| Human<br>Metapneumo<br>virus | Moderate Positive<br>(3× LoD)<br>3.6E+03<br>Copies/mL<br>(3.0E+01<br>TCID50/mL) | Detected | 30/30<br>100% | 30/30<br>100% | 60/60<br>100% | 30/30<br>100% | 30/30<br>100% | 60/60<br>100% | 120/120<br>100%<br>(97.0%-100%) | |
| | Concentration | Expected<br>Result | FilmArray Torch | | | FilmArray 2.0 | | | All | |
| | | | Agreement with Expected Result | | | | | | Sites/Systems<br>(95% CI) | |
| Analyte | Tested | | Site A | Site C | System<br>Sub-<br>Total | Site B | Site C | System<br>Sub-<br>Total | | |
| | Low Positive<br>(1× LoD)<br>1.2E+03<br>Copies/mL<br>(1.0E+01<br>TCID50/mL) | Detected | 30/30<br>100% | 30/30<br>100% | 60/60<br>100% | 28/30<br>93.3% | 30/30<br>100% | 58/60<br>96.7% | 118/120<br>98.3%<br>(94.1%-<br>99.8%) | |
| | None<br>(no analyte) | Not<br>Detected | 60/60<br>100% | 60/60<br>100% | 120/120<br>100% | 60/60<br>100% | 60/60<br>100% | 120/120<br>100% | 240/240<br>100%<br>(98.5%-100%) | |
| | Moderate Positive<br>(3× LoD)<br>1.1E+02<br>Copies/mL<br>(3.0E-01<br>TCID50/mL) | Detected | 30/30<br>100% | 30/30<br>100% | 60/60<br>100% | 28/30<br>93.3% | 30/30<br>100% | 58/60<br>96.7% | 118/120<br>98.3%<br>(94.1%-<br>99.8%) | |
| Human<br>Rhinovirus/<br>Enterovirus | Low Positive<br>(1× LoD)<br>3.8E+01<br>Copies/mL<br>(1.0E-01<br>TCID50/mL) | Detected | 30/30<br>100% | 30/30<br>100% | 60/60<br>100% | 30/30<br>100% | 30/30<br>100% | 60/60<br>100% | 120/120<br>100%<br>(97.0%-100%) | |
| | None<br>(no analyte) | Not<br>Detected | 60/60<br>100% | 60/60<br>100% | 120/120<br>100% | 60/60<br>100% | 60/60<br>100% | 120/120<br>100% | 240/240<br>100%<br>(98.5%-100%) | |
| | Moderate Positive<br>(3× LoD)<br>6.3E+01<br>Copies/mL<br>(3.0E-01<br>TCID50/mL) | Detected | 30/30<br>100% | 30/30<br>100% | 60/60<br>100% | 29/30<br>96.7% | 30/30<br>100% | 59/60<br>98.3% | 119/120<br>99.2%<br>(95.4%-100%) | |
| Influenza A<br>H3 | Low Positive<br>(1× LoD)<br>2.1E+01<br>Copies/mL<br>(1.0E-01<br>TCID50/mL) | Detected | 30/30<br>100% | 30/30<br>100% | 60/60<br>100% | 30/30<br>100% | 30/30<br>100% | 60/60<br>100% | 120/120<br>100%<br>(97.0%-100%) | |
| | None<br>(no analyte) | Not<br>Detected | 60/60<br>100% | 60/60<br>100% | 120/120<br>100% | 60/60<br>100% | 60/60<br>100% | 120/120<br>100% | 240/240<br>100%<br>(98.5%-100%) | |
| Influenza A<br>H1-2009 | None<br>(no analyte) | Not<br>Detected | 120/12<br>0<br>100% | 120/120<br>100% | 240/240<br>100% | 120/120<br>100% | 120/120<br>100% | 240/240<br>100% | 480/480<br>100%<br>(99.2%-100%) | |
| Influenza A<br>H1 | None<br>(no analyte) | Not<br>Detected | 120/12<br>0<br>100% | 120/120<br>100% | 240/240<br>100% | 120/120<br>100% | 120/120<br>100% | 240/240<br>100% | 480/480<br>100%<br>(99.2%-100%) | |
| | Concentration<br>Tested | Expected<br>Result | FilmArray Torch | | | FilmArray 2.0 | | | All<br>Sites/Systems<br>(95% CI) | |
| Analyte | | | Site A | Site C | System<br>Sub-Total | Site B | Site C | System<br>Sub-Total | | |
| Influenza B | Moderate Positive<br>(3× LoD)<br>1.0E+02<br>Copies/mL<br>(1.5E+01<br>TCID50/mL) | Detected | 30/30<br>100% | 30/30<br>100% | 60/60<br>100% | 30/30<br>100% | 30/30<br>100% | 60/60<br>100% | 120/120<br>100%<br>(97.0%-100%) | |
| | Low Positive<br>(1× LoD)<br>3.4E+01<br>Copies/mL<br>(5.0E+00<br>TCID50/mL) | Detected | 30/30<br>100% | 30/30<br>100% | 60/60<br>100% | 30/30<br>100% | 30/30<br>100% | 60/60<br>100% | 120/120<br>100%<br>(97.0%-100%) | |
| | None<br>(no analyte) | Not<br>Detected | 60/60<br>100% | 60/60<br>100% | 120/120<br>100% | 60/60<br>100% | 60/60<br>100% | 120/120<br>100% | 240/240<br>100%<br>(98.5%-100%) | |
| Parainfluenza<br>Virus 1 | None<br>(no analyte) | Not<br>Detected | 120/12<br>0<br>100% | 120/120<br>100% | 240/240<br>100% | 120/120<br>100% | 120/120<br>100% | 240/240<br>100% | 480/480<br>100%<br>(99.2%-100%) | |
| Parainfluenza<br>Virus 2 | Moderate Positive<br>(3× LoD)<br>9.0E+01<br>Copies/mL<br>(1.5E+00<br>TCID50/mL) | Detected | 30/30<br>100% | 29/30<br>96.7% | 59/60<br>98.3% | 29/30<br>96.7% | 30/30<br>100% | 59/60<br>98.3% | 118/120<br>98.3%<br>(94.1%-<br>99.8%) | |
| | Low Positive<br>(1× LoD)<br>3.0E+01<br>Copies/mL<br>(5.0E-01<br>TCID50/mL) | Detected | 30/30<br>100% | 29/30<br>96.7% | 59/60<br>98.3% | 30/30<br>100% | 27/30<br>90.0% | 57/60<br>95.0% | 116/120<br>96.7%<br>(91.7%-<br>99.1%) | |
| | None<br>(no analyte) | Not<br>Detected | 60/60<br>100% | 60/60<br>100% | 120/120<br>100% | 60/60<br>100% | 60/60<br>100% | 120/120<br>100% | 240/240<br>100%<br>(98.5%-100%) | |
| Parainfluenza<br>Virus 3 | None<br>(no analyte) | Not<br>Detected | 120/12<br>0<br>100% | 120/120<br>100% | 240/240<br>100% | 120/120<br>100% | 120/120<br>100% | 240/240<br>100% | 480/480<br>100%<br>(99.2%-100%) | |
| Parainfluenza<br>Virus 4 | Moderate Positive<br>(3× LoD)<br>4.8E+03<br>Copies/mL<br>(1.5E+02<br>TCID50/mL) | Detected | 30/30<br>100% | 30/30<br>100% | 60/60<br>100% | 30/30<br>100% | 30/30<br>100% | 60/60<br>100% | 120/120<br>100%<br>(97.0%-100%) | |
| | Low Positive<br>(1× LoD)<br>1.6E+03<br>Copies/mL<br>(5.0E+01<br>TCID50/mL) | Detected | 30/30<br>100% | 29/30<br>96.7% | 59/60<br>98.3% | 29/30<br>96.7% | 30/30<br>100% | 59/60<br>98.3% | 118/120<br>98.3%<br>(94.1%-<br>99.8%) | |
| | Concentration | Expected<br>Result | Agreement with Expected Result | | | | | | | |
| | | | | FilmArray Torch | | FilmArray 2.0 | | | All | |
| Analyte | Tested | | Site A | Site C | System<br>Sub-<br>Total | Site B…