FilmArray Respiratory Panel 2 plus (RP2plus)

• K Number: DEN170017
• Device Name: FilmArray Respiratory Panel 2 plus (RP2plus)
• Manufacturer: BioFire Diagnostics, LLC
• Date Cleared: 2017-11-24 ( 253 days)
• Product Code: PZF
• Regulation Number: 866.4001

Intended 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) ● - 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, 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.

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. 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.

Predicate Devices

K103175, K110764, K120267

Reference Devices

K103175, K110764, K120267

AI/ML (Artificial Intelligence / Machine Learning)

Yes
The document explicitly mentions that the "Melt Detector" algorithm was "tuned against a large data set comprising typical and atypical melting curves (i.e., training data set) with expert annotation (positive or negative calls)" during development. This process of training an algorithm on annotated data to improve its performance is a core characteristic of machine learning.

Therapeutic

No
Explanation: This device is an in vitro diagnostic test designed for the qualitative detection and identification of nucleic acids from specific respiratory pathogens. It is used to aid in diagnosis, not to provide therapy or treatment.

Diagnostic

Yes

The device is a multiplexed nucleic acid test intended for the simultaneous qualitative detection and identification of nucleic acids from MERS-CoV and multiple respiratory pathogens, which directly aids in the differential diagnosis of infections.

SaMD (Software as a Medical Device)

No

The device description explicitly states that the FilmArray RP2plus is a multiplexed nucleic acid test intended for use with FilmArray 2.0 or FilmArray Torch systems, which are described as PCR-based in vitro diagnostic systems containing hardware components like inflatable bladders, seal points, pneumatic pistons, and Peltier devices. While software is used for interpretation, the device itself is a combination of hardware and software.

IVD (In Vitro Diagnostic)

Based on the provided information, yes, this device is an IVD (In Vitro Diagnostic).

Here's why:

• Intended Use: The document explicitly states the device 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)..." This describes a test performed in vitro (outside the body) on a biological sample (NPS) to diagnose or aid in the diagnosis of a disease (respiratory infections, specifically MERS-CoV).
• Device Description: The description details the process of using the device, which involves handling a biological sample (NPS), using reagents within a pouch, and performing nucleic acid extraction and PCR amplification. This is consistent with the methodology of an in vitro diagnostic test.
• Performance Studies: The document includes detailed performance studies (Reproducibility, Analytical Reactivity, Analytical Specificity, Detection Limit, Interfering Substances, Clinical Studies) which are standard requirements for demonstrating the performance of an IVD.
• Reference Devices: The mention of "Reference Device(s)" with K numbers (which are FDA premarket notification numbers for medical devices, including IVDs) further indicates that this device is being compared to other legally marketed IVDs.

Therefore, the FilmArray Respiratory Panel 2 plus (RP2plus) clearly fits the definition and characteristics of an In Vitro Diagnostic device.

PCCP (Predetermined Change Control Plan) Authorized

No
The document lists PCCP as a general regulatory provision (section 515C(b)(1)) within the special controls for this device type, but it does not explicitly state that the FDA has reviewed, approved, or cleared a PCCP for this specific device.

Overview

Intended Use / 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) ●

• 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 FilmArray 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, 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.

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

PZF

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.

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.

Mentions image processing

Yes

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

nasopharyngeal swabs (NPS)

Indicated Patient Age Range

Not Found

Intended User / Care Setting

clinical laboratories

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

To maximize the sensitivity and specificity of the Melt Detector, the algorithm was tuned against a large data set comprising typical and atypical melting curves (i.e., training data set) with expert annotation (positive or negative calls) during the development of the original FilmArray RP.

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

Not Found

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

1. Analytical Performance: Reproducibility Study

• Study Type: Reproducibility study
• Sample Size: 480 valid runs. 120 data points per sample, with 60 data points per sample per system (FilmArray 2.0 and FilmArray Torch systems), 30 data points per sample for Sites A and B, and 60 data points at Site C.
• Key Results:
  • Overall system performance reproducibility (% agreement with expected results) ranged from 93.3% to 100% across various analytes and concentrations.
  • MERS-CoV, Coronavirus 229E, HKU1, NL63, Influenza A H1, H1-2009, H3, Influenza B, PIV1, PIV3, PIV4, and Chlamydia pneumoniae generally showed 100% agreement at both 1xLoD and 3xLoD concentrations, or 100% for Not Detected.
  • Some analytes had slightly lower agreement at 1xLoD, such as Bordetella parapertussis (88.3% for Torch, 98.3% for FilmArray 2.0), Parainfluenza Virus 2 (98.3% for Torch, 95.0% for FilmArray 2.0), and Respiratory Syncytial Virus (98.3% for Torch, 98.3% for FilmArray 2.0).
  • The reproducibility (standard deviation) of melting temperature (Tm) for the amplification products was evaluated, with a Tm standard deviation of ± 0.5°C or less observed within and between the FilmArray 2.0 and FilmArray Torch systems.

2. Analytical Performance: Limit of Detection (LoD) Study

• Study Type: LoD estimation and confirmation studies using contrived samples.
• Sample Size: For confirmation, 40 replicates (20 per system) tested at 1xLoD and 40 replicates (20 per system) at 0.1xLoD for each analyte.
• Key Results:
  • LoD confirmation criteria were met for each analyte on both FilmArray 2.0 and FilmArray Torch systems at the same concentration.
  • Detection rates at 1xLoD were typically 95% or greater, and detection rates at 0.1xLoD were less than 95%.
  • Confirmed LoD concentrations are provided for all 22 analytes, in Copies/mL or CFU/mL, with corresponding TCID50/mL where applicable.

3. Analytical Performance: Analytical Reactivity (Inclusivity) Study

• Study Type: In silico analysis and empirical testing of unique strains/isolates.
• Sample Size: Various numbers of isolates/strains for each analyte (e.g., >25 Adenovirus isolates, 25 Human Rhinovirus/Enterovirus species and serotypes, several isolates of Influenza A subtypes).
• Key Results:
  • All isolates wet tested were amplified and detected at concentrations within 10xLoD.
  • In silico analysis for MERS-CoV assays (MERS1 and MERS2) showed no evidence of human-identified sequence variants contributing to altered reactivity.
  • Expected outcomes (e.g., Influenza A (no subtype detected) or Equivocal) were observed for zoonotic and recombinant influenza A viruses of non-H1, H1-2009, or H3 hemagglutinin subtypes.

4. Analytical Performance: Analytical Specificity/Cross-Reactivity Evaluation

• Study Type: Challenging the system with high concentrations of on-panel and off-panel organisms (wet testing). In silico analysis also performed.
• Sample Size: 23 on-panel analytes and 50 off-panel bacteria, viruses, and fungi.
• Key Results:
  • The redesigned Coronavirus OC43 assay showed no inaccurate Coronavirus OC43 Detected results even with high concentrations of Coronavirus HKU1.
  • Cross-reactivity between Bordetella pertussis (ptxP) assay and non-pertussis Bordetella strains (e.g., B. parapertussis, B. bronchiseptica) carrying a pertussis toxin pseudogene was not observed at tested high concentrations, but mentioned as possible.
  • Some reactivity of the H1-2009 assay with historical/novel H1N1 strains of swine origin was demonstrated, potentially leading to reporting as Influenza A H1 or H1-2009.
  • Non-specific amplification by the Human Rhinovirus/Enterovirus (HRV/EV) assay was confirmed for B. pertussis at high concentrations (>=4.5E+07 CFU/mL).
  • Only one non-specific interaction observed with off-panel organisms: Bordetella pertussis (ptxP) Detected in one of three replicates of B. bronchiseptica at 1.2E+09 CFU/mL.
  • Cross-reactivity with B. bronchiseptica by the Bordetella parapertussis (IS1001) assay, misidentifying it as B. parapertussis, is noted.
  • Overall, the likelihood, risk, and impact of non-specific interactions are predicted to be minor, occurring almost exclusively at very high organism levels.

5. Analytical Performance: Interfering Substances Study

• Study Type: Evaluation of potential inhibitory effects of substances commonly found in nasopharyngeal specimens or introduced during collection/handling.
• Sample Size: 39 different endogenous and exogenous substances, competing microorganisms, specimen collection materials, and disinfecting agents.
• Key Results:
  • No interference observed from biological substances (e.g., blood) or high levels of competing microorganisms.
  • No interference from various transport media types or swabs.
  • No effect from saline, decongestants, or ointments.
  • Exposure to bleach prior to testing could damage organisms/nucleic acids, leading to inaccurate false negative results (lack of detection).

6. Comparator Studies: MERS-CoV Proficiency Test Panel

• Study Type: Blinded comparative testing of a MERS-CoV proficiency panel from QCMD.
• Sample Size: MERS-CoV proficiency panel with MERS-CoV samples and common coronaviruses.
• Key Results: The analytical sensitivity of FilmArray RP2plus detecting MERS-CoV appeared to be at least equivalent to the CDC Novel Coronavirus 2012 Real-time RT-PCR Assay and the RealStar MERS-CoV RT-PCR Kit U.S. (both EUA authorized).

7. Clinical Studies: Prospective Clinical Study

• Study Type: Multi-center prospective study at three US sites.
• Sample Size: 1612 specimens included in final data analysis (out of 1635 acquired).
• Comparator: FDA-cleared multiplexed respiratory pathogens panel and two analytically-validated PCR followed by bidirectional sequencing assays for B. parapertussis.
• Key Results:
  • Overall success rate for initial specimen tests: 99.3%.
  • Positive Percent Agreement (PPA) / Negative Percent Agreement (NPA) (Overall):
    • MERS-CoV: PPA 0/0 (N/A), NPA 100% (1612/1612)
    • Adenovirus: PPA 94.6% (70/74), NPA 96.9% (1490/1538)
    • CoV-229E: PPA 91.7% (11/12), NPA 99.7% (1595/1600)
    • CoV-HKU1: PPA 100% (43/43), NPA 99.2% (1557/1569)
    • CoV-NL63: PPA 100% (40/40), NPA 99.4% (1562/1572)
    • CoV-OC43: PPA 80.5% (33/41), NPA 99.7% (1566/1571)
    • hMPV: PPA 97.3% (73/75), NPA 99.5% (1529/1537)
    • HRV/EV: PPA 97.5% (425/436), NPA 93.5% (1099/1176)
    • FluA: PPA 100% (78/78), NPA 100% (1531/1531)
    • FluA H1: PPA 0/0 (N/A), NPA 100% (1609/1609)
    • FluA H1-2009: PPA 100% (74/74), NPA 100% (1535/1535)
    • FluA H3: PPA 100% (4/4), NPA 100% (1605/1605)
    • FluB: PPA 100% (14/14), NPA 99.9% (1596/1598)
    • PIV1: PPA 100% (9/9), NPA 99.9% (1602/1603)
    • PIV2: PPA 97.9% (46/47), NPA 99.5% (1557/1565)
    • PIV3: PPA 95.6% (43/45), NPA 99.4% (1557/1567)
    • PIV4: PPA 100% (9/9), NPA 99.6% (1596/1603)
    • RSV: PPA 99.4% (175/176), NPA 98.3% (1412/1436)
    • B. parapertussis (IS1001): PPA 85.7% (6/7), NPA 100% (1605/1605)
    • B. pertussis (ptxP): PPA 66.7% (2/3), NPA 99.9% (1608/1609)
    • C. pneumoniae: PPA 100% (5/5), NPA 99.9% (1606/1607)
    • M. pneumoniae: PPA 95.8% (23/24), NPA 99.7% (1583/1588)
  • Mixed Infection Analysis: 245 specimens with multiple organism detections (15.2% of all specimens). 50.6% were concordant with comparator methods.

8. Clinical Studies: Retrospective Clinical Study - Common Respiratory Pathogens

• Study Type: Evaluation of preselected archived retrospective specimens.
• Sample Size: 214 specimens included in performance analysis (out of 217 initially received).
• Comparator: Same as prospective study: FDA-cleared multiplexed respiratory pathogens panel and PCR followed by bidirectional sequencing for B. parapertussis.
• Key Results:
  • PPA / NPA (Overall):
    • MERS-CoV: PPA 0/0 (N/A), NPA 100% (214/214)
    • Adenovirus: PPA 0/0 (N/A), NPA 97.4% (189/194)
    • CoV-229E: PPA 100% (15/15), NPA 100% (175/175)
    • CoV-HKU1: PPA 0/0 (N/A), NPA 100% (194/194)
    • CoV-NL63: PPA 100% (2/2), NPA 100% (192/192)
    • CoV-OC43: PPA 0/0 (N/A), NPA 100% (194/194)
    • hMPV: PPA 100% (1/1), NPA 99.5% (192/193)
    • HRV/EV: PPA 94.7% (18/19), NPA 96.0% (168/175)
    • Influenza A: PPA 100% (22/22), NPA 100% (172/172)
    • Influenza A H1: PPA 100% (3/3), NPA 100% (191/191)
    • Influenza A 2009-H1: PPA 100% (1/1), NPA 100% (193/193)
    • Influenza A H3: PPA 100% (18/18), NPA 100% (176/176)
    • Influenza B: PPA 100% (16/16), NPA 100% (177/177)
    • PIV1: PPA 100% (16/16), NPA 100% (178/178)
    • PIV2: PPA 100% (16/16), NPA 100% (177/177)
    • PIV3: PPA 100% (17/17), NPA 98.9% (175/177)
    • PIV4: PPA 100% (17/17), NPA 98.3% (174/177)
    • RSV: PPA 100% (2/2), NPA 99.5% (191/192)
    • B. parapertussis (IS1001): PPA 100% (16/16), NPA 100% (4/4)
    • B. pertussis (ptxP): PPA 96.2% (25/26), NPA 98.8% (160/162)
    • C. pneumoniae: PPA 100% (17/17), NPA 100% (176/176)
    • M. pneumoniae: PPA 100% (16/16), NPA 98.8% (171/173)

9. Clinical Studies: Retrospective Clinical Study - MERS-CoV

• Study Type: Evaluation of archived retrospective NPS in VTM specimens from confirmed MERS-CoV cases.
• Sample Size: 3 specimens.
• Key Results: Positive Percent Agreement (PPA) of FilmArray RP2plus results with previous test results for MERS-CoV was 100% (3/3).

10. Clinical Studies: Testing Contrived Clinical Specimens

• Study Type: Evaluation of contrived specimens (spiked with Influenza A H1 and MERS-CoV) in negative NPS specimens.
• Sample Size: 50 contrived MERS-CoV positive specimens and 50 un-spiked negative specimens. 48 contrived Influenza A H1 positive specimens and 50 un-spiked negative specimens.
• Key Results:
  • MERS-CoV: Overall PPA 100% (50/50) across various LoD multiples; NPA 100% (50/50).
  • Influenza A H1: Overall PPA 97.9% (47/48) across various LoD multiples; NPA 100% (50/50).

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

Key metrics reported are Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA).

Prospective Clinical Study Performance (Overall)

• MERS-CoV:
  • PPA: 0/0 (0%)
  • NPA: 1612/1612 (100%)
• Adenovirus:
  • PPA: 70/74 (94.6%)
  • NPA: 1490/1538 (96.9%)
• CoV-229E:
  • PPA: 11/12 (91.7%)
  • NPA: 1595/1600 (99.7%)
• CoV-HKU1:
  • PPA: 43/43 (100%)
  • NPA: 1557/1569 (99.2%)
• CoV-NL63:
  • PPA: 40/40 (100%)
  • NPA: 1562/1572 (99.4%)
• CoV-OC43:
  • PPA: 33/41 (80.5%)
  • NPA: 1566/1571 (99.7%)
• hMPV:
  • PPA: 73/75 (97.3%)
  • NPA: 1529/1537 (99.5%)
• HRV/EV:
  • PPA: 425/436 (97.5%)
  • NPA: 1099/1176 (93.5%)
• FluA:
  • PPA: 78/78 (100%)
  • NPA: 1531/1531 (100%)
• FluA H1:
  • PPA: 0/0 (0%)
  • NPA: 1609/1609 (100%)
• FluA H1-2009:
  • PPA: 74/74 (100%)
  • NPA: 1535/1535 (100%)
• FluA H3:
  • PPA: 4/4 (100%)
  • NPA: 1605/1605 (100%)
• FluB:
  • PPA: 14/14 (100%)
  • NPA: 1596/1598 (99.9%)
• PIV1:
  • PPA: 9/9 (100%)
  • NPA: 1602/1603 (99.9%)
• PIV2:
  • PPA: 46/47 (97.9%)
  • NPA: 1557/1565 (99.5%)
• PIV3:
  • PPA: 43/45 (95.6%)
  • NPA: 1557/1567 (99.4%)
• PIV4:
  • PPA: 9/9 (100%)
  • NPA: 1596/1603 (99.6%)
• RSV:
  • PPA: 175/176 (99.4%)
  • NPA: 1412/1436 (98.3%)
• B. parapertussis (IS1001):
  • PPA: 6/7 (85.7%)
  • NPA: 1605/1605 (100%)
• B. pertussis (ptxP):
  • PPA: 2/3 (66.7%)
  • NPA: 1608/1609 (99.9%)
• C. pneumoniae:
  • PPA: 5/5 (100%)
  • NPA: 1606/1607 (99.9%)
• M. pneumoniae:
  • PPA: 23/24 (95.8%)
  • NPA: 1583/1588 (99.7%)

Retrospective Clinical Study (Common Respiratory Pathogens) Performance (Overall)

• MERS-CoV:
  • PPA: 0/0 (0%)
  • NPA: 214/214 (100%)
• Adenovirus:
  • PPA: 0/0 (0%)
  • NPA: 189/194 (97.4%)
• CoV-229E:
  • PPA: 15/15 (100%)
  • NPA: 175/175 (100%)
• CoV-HKU1:
  • PPA: 0/0 (0%)
  • NPA: 194/194 (100%)
• CoV-NL63:
  • PPA: 2/2 (100%)
  • NPA: 192/192 (100%)
• CoV-OC43:
  • PPA: 0/0 (0%)
  • NPA: 194/194 (100%)
• hMPV:
  • PPA: 1/1 (100%)
  • NPA: 192/193 (99.5%)
• HRV/EV:
  • PPA: 18/19 (94.7%)
  • NPA: 168/175 (96.0%)
• Influenza A:
  • PPA: 22/22 (100%)
  • NPA: 172/172 (100%)
• Influenza A H1:
  • PPA: 3/3 (100%)
  • NPA: 191/191 (100%)
• Influenza A 2009-H1:
  • PPA: 1/1 (100%)
  • NPA: 193/193 (100%)
• Influenza A H3:
  • PPA: 18/18 (100%)
  • NPA: 176/176 (100%)
• Influenza B:
  • PPA: 16/16 (100%)
  • NPA: 177/177 (100%)
• Parainfluenza Virus 1:
  • PPA: 16/16 (100%)
  • NPA: 178/178 (100%)
• Parainfluenza Virus 2:
  • PPA: 16/16 (100%)
  • NPA: 177/177 (100%)
• Parainfluenza Virus 3:
  • PPA: 17/17 (100%)
  • NPA: 175/177 (98.9%)
• Parainfluenza Virus 4:
  • PPA: 17/17 (100%)
  • NPA: 174/177 (98.3%)
• RSV:
  • PPA: 2/2 (100%)
  • NPA: 191/192 (99.5%)
• Bordetella parapertussis (IS1001):
  • PPA: 16/16 (100%)
  • NPA: 4/4 (100%)
• Bordetella pertussis (ptxP):
  • PPA: 25/26 (96.2%)
  • NPA: 160/162 (98.8%)
• Chlamydia pneumoniae:
  • PPA: 17/17 (100%)
  • NPA: 176/176 (100%)
• Mycoplasma pneumoniae:
  • PPA: 16/16 (100%)
  • NPA: 171/173 (98.8%)

Retrospective Clinical Study - MERS-CoV

• MERS-CoV: PPA: 3/3 (100%)

Testing Contrived Clinical Specimens

• MERS-CoV:
  • PPA: 50/50 (100%)
  • NPA: 50/50 (100%)
• Influenza A H1:
  • PPA: 47/48 (97.9%)
  • NPA: 50/50 (100%)

Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.

Not Found

Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.

K103175, K110764, K120267

Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).

Not Found

Regulation Number and Section

N/A

Summary

0

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.

4

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

5

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

6

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
Result | FluA-pan
Assays
(n=2) | FluA-H1-2 | FluA-H1-
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
Influenza A H3 | ≥1 positive | Positive | Negative | Positive | Multiple infections
are possible but
rare a, retest to
confirm result b |
| Influenza A H1-2009
Influenza A H3 | ≥1 positive | Any result | Positive | Positive | Multiple infections
are possible but
rare a, retest to
confirm result b |
| Influenza A (no subtype
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
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.

7

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

Respiratory Panel 2 plus
				www.BioFireDx.com
Run Summary
Sample ID:	RP2plus Example
Run Date:
06 Mar 2017
Detected:
Equivocal:	5:21 PM
Middle East Respiratory Syndrome Coronavirus (MERS-CoV)
Passed
Controls:

• Influenza A | | | | |
  | Result Summary | | | | | |
  | | Viruses | | | | |
  | Not Detected
  Not Detected
  Not Detected
  Not Detected
  Not Detected
  Not Detected
  Not Detected

• Equivocal
  Not Detected
  / Detected
  Not Detected
  Not Detected
  Not Detected
  Not Detected
  Not Detected | Adenovirus
  Coronavirus 229E
  Coronavirus HKU1
  Coronavirus NL63
  Coronavirus OC43
  Human Metapneumovirus
  Human Rhinovirus/Enterovirus
  Influenza A
  Influenza B
  Middle East Respiratory Syndrome Coronavirus (MERS-CoV)
  Parainfluenza Virus 1
  Parainfluenza Virus 2
  Parainfluenza Virus 3
  Parainfluenza Virus 4
  Respiratory Syncytial Virus | | | | |
  | Bacteria | | | | | |
  | Not Detected
  Not Detected
  Not Detected
  Not Detected | Bordetella parapertussis (IS1001)
  Bordetella pertussis (ptxP)
  Chlamydia pneumoniae
  Mycoplasma pneumoniae | | | | |
  | Run Details | | | | | |
  | Pouch:
  Run Status:
  Serial No .:
  Lot No .: | RP2plus v1.0
  Completed
  06265525
  161013E | Protocol:
  Operator:
  Instrument: | NPS2 v3.1
  JDoe
  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
AND
Both pouch controls were successful.	None
Report the results provided on the test report
Failed	The run was successfully completed
BUT
At least one of the pouch controls (RNA
Process Control and/or PCR2 Control)
failed.	Repeat the test using a new pouch.
If the error persists, contact Technical Support for
further instruction.
Invalid	The controls are invalid because the run
did not complete.
(Typically this indicates a software or
hardware error).	Note any error codes displayed during the run and
the Run Status field in the Run Details section of
the report. Refer to the appropriate FilmArray
Operator's Manual or contact Technical Support
for further instruction.
Once the error is resolved, repeat the test or repeat
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
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 described in the
Interpretation of Results section above)	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 described in
the Assay Interpretation section above)	Report results.
Equivocal
(Influenza A
and MERS-CoV
only)	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
(see Table 2)	Retest the original
specimen using a new
pouch and report the
results 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 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)

10

• · 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

11

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

12

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

13

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.

14

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

15

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
(Percentage) | Instrument Errors
(Percentage) | Software Errors
(Percentage) |
|-----------------|------|---------------------------------|-----------------------------------|---------------------------------|
| FilmArray 2.0 | 247 | 6
(2.4%) | 1
(0.4%) | 0
(0.0%) |
| FilmArray Torch | 242 | 2
(0.8%) | 0
(0.0%) | 0
(0.0%) |
| Total | 489 | 8 a
(1.6%) | 1
(0.2%) | 0
(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
Tested | Expected
Result | Agreement with Expected Result
FilmArray Torch | | | Agreement with Expected Result
FilmArray 2.0 | | | All
Sites/Systems
(95% CI) | |
|-------------------------------------|-------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------|---------------------------------------------------|-----------------|-------------------------|-------------------------------------------------|-----------------|-------------------------|---------------------------------------|---------------------------------------|
| | | | Site A | Site C | System
Sub-Total | Site B | Site C | System
Sub-Total | | |
| Viruses | | | | | | | | | | |
| MERS-CoV | None
(no analyte) | Not
Detected | 120/120
100% | 120/120
100% | 240/240
100% | 120/120
100% | 120/120
100% | 240/240
100% | 480/480
100%
(99.2%-100%) | |
| | Concentration | Expected
Result | Agreement with Expected Result | | | | | | | |
| | | | FilmArray Torch | | | FilmArray 2.0 | | | All | |
| Analyte | Tested | | Site A | Site C | System
Sub-
Total | Site B | Site C | System
Sub-
Total | Sites/Systems
(95% CI) | |
| | Moderate Positive
(3× LoD)
1.1E+02
Copies/mL
(6.0E+00
TCID50/mL) | Detected | 30/30
100% | 29/30
96.7% | 59/60
98.3% | 29/30
96.7% | 30/30
100% | 59/60
98.3% | 118/120
98.3%
(94.1%-
99.8%) | |
| Adenovirus | Low Positive
(1× LoD)
3.7E+01
Copies/mL
(2.0E+00
TCID50/mL) | Detected | 30/30
100% | 30/30
100% | 60/60
100% | 30/30
100% | 29/30
96.7% | 59/60
98.3% | 119/120
99.2%
(95.4%-100%) | |
| | None
(no analyte) | Not
Detected | 60/60
100% | 60/60
100% | 120/120
100% | 60/60
100% | 60/60
100% | 120/120
100% | 240/240
100%
(98.5%-100%) | |
| Coronavirus
229E | None
(no analyte) | Not
Detected | 120/120
100% | 120/120
100% | 240/240
100% | 120/120
100% | 120/120
100% | 240/240
100% | 480/480
100%
(99.2%-100%) | |
| Coronavirus
HKU1 | None
(no analyte) | Not
Detected | 120/120
100% | 120/120
100% | 240/240
100% | 120/120
100% | 120/120
100% | 240/240
100% | 480/480
100%
(99.2%-100%) | |
| | Moderate Positive
(3× LoD)
1.7E+03
Copies/mL
(9.0E+01
TCID50/mL) | Detected | 29/30
96.7% | 29/30
96.7% | 58/60
96.7% | 29/30
96.7% | 30/30
100% | 59/60
98.3% | 117/120
97.5%
(92.9%-
99.5%) | |
| Coronavirus
OC43 | Low Positive
(1× LoD)
5.6E+02
Copies/mL
(3.0E+01
TCID50/mL) | Detected | 30/30
100% | 30/30
100% | 60/60
100% | 30/30
100% | 27/30
90.0% | 57/60
95.0% | 117/120
97.5%
(92.9%-
99.5%) | |
| | None
(no analyte) | Not
Detected | 60/60
100% | 60/60
100% | 120/120
100% | 60/60
100% | 60/60
100% | 120/120
100% | 240/240
100%
(98.5%-100%) | |
| Coronavirus
NL63 | None
(no analyte) | Not
Detected | 120/120
100% | 120/120
100% | 240/240
100% | 120/120
100% | 120/120
100% | 240/240
100% | 480/480
100%
(99.2%-100%) | |
| Human
Metapneumo
virus | Moderate Positive
(3× LoD)
3.6E+03
Copies/mL
(3.0E+01
TCID50/mL) | Detected | 30/30
100% | 30/30
100% | 60/60
100% | 30/30
100% | 30/30
100% | 60/60
100% | 120/120
100%
(97.0%-100%) | |
| | Concentration | Expected
Result | FilmArray Torch | | | FilmArray 2.0 | | | All | |
| | | | Agreement with Expected Result | | | | | | Sites/Systems
(95% CI) | |
| Analyte | Tested | | Site A | Site C | System
Sub-
Total | Site B | Site C | System
Sub-
Total | | |
| | Low Positive
(1× LoD)
1.2E+03
Copies/mL
(1.0E+01
TCID50/mL) | Detected | 30/30
100% | 30/30
100% | 60/60
100% | 28/30
93.3% | 30/30
100% | 58/60
96.7% | 118/120
98.3%
(94.1%-
99.8%) | |
| | None
(no analyte) | Not
Detected | 60/60
100% | 60/60
100% | 120/120
100% | 60/60
100% | 60/60
100% | 120/120
100% | 240/240
100%
(98.5%-100%) | |
| | Moderate Positive
(3× LoD)
1.1E+02
Copies/mL
(3.0E-01
TCID50/mL) | Detected | 30/30
100% | 30/30
100% | 60/60
100% | 28/30
93.3% | 30/30
100% | 58/60
96.7% | 118/120
98.3%
(94.1%-
99.8%) | |
| Human
Rhinovirus/
Enterovirus | Low Positive
(1× LoD)
3.8E+01
Copies/mL
(1.0E-01
TCID50/mL) | Detected | 30/30
100% | 30/30
100% | 60/60
100% | 30/30
100% | 30/30
100% | 60/60
100% | 120/120
100%
(97.0%-100%) | |
| | None
(no analyte) | Not
Detected | 60/60
100% | 60/60
100% | 120/120
100% | 60/60
100% | 60/60
100% | 120/120
100% | 240/240
100%
(98.5%-100%) | |
| | Moderate Positive
(3× LoD)
6.3E+01
Copies/mL
(3.0E-01
TCID50/mL) | Detected | 30/30
100% | 30/30
100% | 60/60
100% | 29/30
96.7% | 30/30
100% | 59/60
98.3% | 119/120
99.2%
(95.4%-100%) | |
| Influenza A
H3 | Low Positive
(1× LoD)
2.1E+01
Copies/mL
(1.0E-01
TCID50/mL) | Detected | 30/30
100% | 30/30
100% | 60/60
100% | 30/30
100% | 30/30
100% | 60/60
100% | 120/120
100%
(97.0%-100%) | |
| | None
(no analyte) | Not
Detected | 60/60
100% | 60/60
100% | 120/120
100% | 60/60
100% | 60/60
100% | 120/120
100% | 240/240
100%
(98.5%-100%) | |
| Influenza A
H1-2009 | None
(no analyte) | Not
Detected | 120/12
0
100% | 120/120
100% | 240/240
100% | 120/120
100% | 120/120
100% | 240/240
100% | 480/480
100%
(99.2%-100%) | |
| Influenza A
H1 | None
(no analyte) | Not
Detected | 120/12
0
100% | 120/120
100% | 240/240
100% | 120/120
100% | 120/120
100% | 240/240
100% | 480/480
100%
(99.2%-100%) | |
| | Concentration
Tested | Expected
Result | FilmArray Torch | | | FilmArray 2.0 | | | All
Sites/Systems
(95% CI) | |
| Analyte | | | Site A | Site C | System
Sub-Total | Site B | Site C | System
Sub-Total | | |
| Influenza B | Moderate Positive
(3× LoD)
1.0E+02
Copies/mL
(1.5E+01
TCID50/mL) | Detected | 30/30
100% | 30/30
100% | 60/60
100% | 30/30
100% | 30/30
100% | 60/60
100% | 120/120
100%
(97.0%-100%) | |
| | Low Positive
(1× LoD)
3.4E+01
Copies/mL
(5.0E+00
TCID50/mL) | Detected | 30/30
100% | 30/30
100% | 60/60
100% | 30/30
100% | 30/30
100% | 60/60
100% | 120/120
100%
(97.0%-100%) | |
| | None
(no analyte) | Not
Detected | 60/60
100% | 60/60
100% | 120/120
100% | 60/60
100% | 60/60
100% | 120/120
100% | 240/240
100%
(98.5%-100%) | |
| Parainfluenza
Virus 1 | None
(no analyte) | Not
Detected | 120/12
0
100% | 120/120
100% | 240/240
100% | 120/120
100% | 120/120
100% | 240/240
100% | 480/480
100%
(99.2%-100%) | |
| Parainfluenza
Virus 2 | Moderate Positive
(3× LoD)
9.0E+01
Copies/mL
(1.5E+00
TCID50/mL) | Detected | 30/30
100% | 29/30
96.7% | 59/60
98.3% | 29/30
96.7% | 30/30
100% | 59/60
98.3% | 118/120
98.3%
(94.1%-
99.8%) | |
| | Low Positive
(1× LoD)
3.0E+01
Copies/mL
(5.0E-01
TCID50/mL) | Detected | 30/30
100% | 29/30
96.7% | 59/60
98.3% | 30/30
100% | 27/30
90.0% | 57/60
95.0% | 116/120
96.7%
(91.7%-
99.1%) | |
| | None
(no analyte) | Not
Detected | 60/60
100% | 60/60
100% | 120/120
100% | 60/60
100% | 60/60
100% | 120/120
100% | 240/240
100%
(98.5%-100%) | |
| Parainfluenza
Virus 3 | None
(no analyte) | Not
Detected | 120/12
0
100% | 120/120
100% | 240/240
100% | 120/120
100% | 120/120
100% | 240/240
100% | 480/480
100%
(99.2%-100%) | |
| Parainfluenza
Virus 4 | Moderate Positive
(3× LoD)
4.8E+03
Copies/mL
(1.5E+02
TCID50/mL) | Detected | 30/30
100% | 30/30
100% | 60/60
100% | 30/30
100% | 30/30
100% | 60/60
100% | 120/120
100%
(97.0%-100%) | |
| | Low Positive
(1× LoD)
1.6E+03
Copies/mL
(5.0E+01
TCID50/mL) | Detected | 30/30
100% | 29/30
96.7% | 59/60
98.3% | 29/30
96.7% | 30/30
100% | 59/60
98.3% | 118/120
98.3%
(94.1%-
99.8%) | |
| | Concentration | Expected
Result | Agreement with Expected Result | | | | | | | |
| | | | | FilmArray Torch | | FilmArray 2.0 | | | All | |
| Analyte | Tested | | Site A | Site C | System
Sub-
Total | Site B | Site C | System
Sub-
Total | Sites/Systems
(95% CI) | |
| | None
(no analyte) | Not
Detected | 60/60
100% | 60/60
100% | 120/120
100% | 60/60
100% | 60/60
100% | 120/120
100% | 240/240
100%
(98.5%-100%) | |
| Respiratory
Syncytial
Virus | Moderate Positive
(3× LoD)
2.7E+01
Copies/mL
(6.0E-02
TCID50/mL) | Detected | 30/30
100% | 30/30
100% | 60/60
100% | 30/30
100% | 30/30
100% | 60/60
100% | 120/120
100%
(97.0%-100%) | |
| | Low Positive
(1× LoD)
9.0E+00
Copies/mL
(2.0E-02
TCID50/mL) | Detected | 29/30
96.7% | 30/30
100% | 59/60
98.3% | 30/30
100% | 29/30
96.7% | 59/60
98.3% | 118/120
98.3%
(94.1%-
99.8%) | |
| | None
(no analyte) | Not
Detected | 60/60
100% | 60/60
100% | 120/120
100% | 60/60
100% | 60/60
100% | 120/120
100% | 240/240
100%
(98.5%-100%) | |
| | | | | Bacteria | | | | | | |
| Bordetella
parapertussis | Moderate Positive
(3× LoD)
1.7E+02 IS1001
Copies/mL
(1.2E+02
CFU/mL) | Detected | 30/30
100% | 30/30
100% | 60/60
100% | 29/30
96.7% | 30/30
100% | 59/60
98.3% | 119/120
99.2%
(95.4%-100%) | |
| | Low Positive
(1× LoD)
5.8E+01 IS1001
Copies/mL
(4.1E+01
CFU/mL) | Detected | 24/30 a
80.0% | 29/30
96.7% | 53/60a
88.3% | 29/30
96.7% | 30/30
100% | 59/60
98.3% | 112/120
93.3%
(87.3%-
97.1%) | |
| | None
(no analyte) | Not
Detected | 60/60
100% | 60/60
100% | 120/120
100% | 60/60
100% | 60/60
100% | 120/120
100% | 240/240
100%
(98.5%-100%) | |
| Bordetella
pertussis | Moderate Positive
(3× LoD)
3.0E+03 CFU/mL | Detected | 30/30
100% | 30/30
100% | 60/60
100% | 30/30
100% | 30/30
100% | 60/60
100% | 120/120
100%
(97.0%-100%) | |
| | Low Positive
(1× LoD)
1.0E+03 CFU/mL | Detected | 28/30
93.3% | 30/30
100% | 58/60
96.7% | 30/30
100% | 30/30
100% | 60/60
100% | 118/120
98.3%
(94.1%-
99.8%) | |
| | None
(no analyte) | Not
Detected | 60/60
100% | 60/60
100% | 120/120
100% | 60/60
100% | 60/60
100% | 120/120
100% | 240/240
100%
(98.5%-100%) | |
| | Analyte | Concentration
Tested | Expected
Result | FilmArray Torch | | | FilmArray 2.0 | | | All
Sites/Systems
(95% CI) |
| | | | | Site A | Site C | System
Sub-
Total | Site B | Site C | System
Sub-
Total | |
| | Chlamydia
pneumoniae | Moderate Positive
(3× LoD)
$2.0E+02$
Copies/mL
(3.0E-01
TCID50/mL) | Detected | 30/30
100% | 30/30
100% | 60/60
100% | 30/30
100% | 30/30
100% | 60/60
100% | 120/120
100%
(97.0%-100%) |
| | | Low Positive
(1× LoD)
$6.6E+01$
Copies/mL
(1.0E-01
TCID50/mL) | Detected | 28/30
93.3% | 30/30
100% | 58/60
96.7% | 29/30
96.7% | 30/30
100% | 59/60
98.3% | 117/120
97.5%
(92.9%-
99.5%) |
| | | None
(no analyte) | Not
Detected | 60/60
100% | 60/60
100% | 120/120
100% | 60/60
100% | 60/60
100% | 120/120
100% | 240/240
100%
(98.5%-100%) |
| | Mycoplasma
pneumoniae | None
(no analyte) | Not
Detected | 120/120
100% | 120/120
100% | 240/240
100% | 120/120
100% | 120/120
100% | 240/240
100% | 480/480
100%
(99.2%-100%) |
| | Overall Agreement with the Expected Result | | | 9,562/9,600 | | | | | | |
| | All Analytes and All Test Levels | | | 99.6% | | | | | | |
| | (95% Confidence Interval) | | | (99.5% - 99.7%) | | | | | | |

16

17

18

19

20

ª Data from Site A were further reviewed by the unique site-specific variables including test day, Torch module, and operator. No correlation could be found between the Not Detected results and any one of these variables. The Not Detected results at Site A were found to be statistically non-significant (p>0.05 by Fisher's exact test) and therefore do not appear to indicate a site- or system-dependent variance in precision of the FilmArray RP2plus Bordetella parapertusis (IS/100) results.

The reproducibility (standard deviation) of melting temperature (Tm) for the amplification products generated by the FilmArray RP2plus was also evaluated, with a Tm standard deviation for each assay of ± 0.5℃ 3 or less observed within and between the FilmArray 2.0 and FilmArray Torch systems (Table 7 below).

		Tm Observed (°C)ª
		FilmArray Torch		FilmArray 2.0
Analyte	FilmArray
RP2 Assay	Site A
Mean	Site A
StDev	Site C
Mean	Site C
StDev	Site B
Mean	Site B
StDev	Site C
Mean	Site C
StDev	All Sites/Systems
Mean	All Sites/Systems
StDev
Controls	Yeast RNA	82.3	± 0.3	82.1	±0.2	82.2	±0.3	82.0	± 0.2	82.1	±0.3
	PCR2	76.1	± 0.2	75.9	± 0.2	76.0	± 0.2	75.8	± 0.2	75.9	±0.2
VIRUSES
Adenovirus	Adeno2	89.0	± 0.2	88.8	±0.1	88.8	± 0.3	88.7	± 0.2	88.8	±0.2
	Adeno6	89.6	± 0.2	89.4	±0.2	89.4	±0.3	89.2	± 0.2	89.4	±0.3
Coronavirus OC43	CoV-OC43-	80.7	± 0.2	80.6	±0.1	80.6	±0.3	80.5	± 0.2	80.6	±0.2

Table 7: Reproducibility of Tm (ºC) For Select FilmArray Torch and FilmArray Torch and FilmArray 2.0 Systems

3 Note: The Tm window for an assay is determined based on a mathematical model of known sequences and empirical data. An observed standard deviation in Tm of 0.5℃ or less is believed to be adequately ensure that Tm variability contributed by the system (rather than amplicon sequence) will not lead to inaccurate results.

21

		Tm Observed (°C)ª
		FilmArray Torch		Site C		FilmArray 2.0		Site C		All Sites/Systems
	FilmArray	Mean	StDev	Mean	StDev	Mean	StDev	Mean	StDev	Mean	StDev
Analyte	RP2 Assay
	2
Human	hMPV	78.2	±0.3	78.0	±0.2	78.0	±0.3	77.8	±0.2	78.0	±0.3
Metapneumovirus
Rhinovirus	HRV/EV	84.3	±0.2	84.2	±0.2	84.3	±0.3	84.1	±0.2	84.2	±0.2
	FluA-pan1	84.2	±0.2	84.0	±0.1	84.0	±0.3	83.8	±0.2	84.0	±0.2
Influenza A H3N2	FluA-pan2	78.9	±0.2	78.9	±0.1	78.9	±0.2	78.8	±0.2	78.9	±0.2
	FluA-H3	82.1	±0.2	81.9	±0.2	82.0	±0.3	81.9	±0.2	82.0	±0.2
Influenza B	FluB	80.4	±0.3	80.3	±0.2	80.4	±0.2	80.2	±0.2	80.3	±0.2
Parainfluenza virus 2	PIV2	83.2	±0.2	83.1	±0.2	83.2	±0.2	83.0	±0.2	83.1	±0.2
Parainfluenza virus 4	PIV4	77.1	±0.2	77.0	±0.3	77.2	±0.3	77.0	±0.2	77.1	±0.3
Respiratory Syncytial
Virus	RSV	81.2	±0.2	81.1	±0.2	81.1	±0.2	81.0	±0.2	81.1	±0.2
BACTERIA
Bordetella	IS1001	87.7	±0.2	87.6	±0.2	87.6	±0.3	87.5	±0.2	87.6	±0.2
parapertussis
Bordetella pertussis	ptxP	88.6	±0.2	88.5	±0.2	88.5	±0.3	88.2	±0.2	88.4	±0.3
Chlamydia
pneumoniae	Cpne	79.6	±0.3	79.5	±0.2	79.5	±0.3	79.3	±0.2	79.5	±0.3

1 Mean Tm values are calculated from a combination of Tm values obtained at the 3× LoD and 1× LoD concentrations.

• b. Linearity/assay Reportable Range: Not Applicable
• c. Traceability, Stability, Expected Values (controls, calibrators, or methods):

Assay 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 RP2plus pouch were successful.

PCR2 Control

The PCR2 Control assay detects a DNA target that is dried into wells of the array along with the corresponding primers. A positive result indicates that PCR2 was successful.

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 (Tm) for either the RNA Process Control or the PCR2 Control is outside of an acceptable

22

range (i.e., 80.3-84.3°C for the RNA Process Control and 73.8-77.8°C for the PCR2 Control). If required by local, state, or accrediting organization quality control requirements, users can monitor the system by trending Tm values for the control assays and maintaining records according to standard laboratory quality control practices.

External Controls

External controls are not provided with the FilmArray RP2plus. However, five frozen (-70°C) external control mixes (ECMs) (see Table 8 below) were prepared and provided to the clinical study sites for testing during the prospective clinical trial and the clinical study testing contrived specimens. FilmArray operators were required to complete a valid ECM run (correct results obtained) on each day of clinical specimen testing (tested on a rotating basis).

External Control Mixes	Expected Calls
ECM 1	Adenovirus, Coronavirus 229E, Human Metapneumovirus, Influenza A H1,
	Influenza B. and Mycoplasma pneumoniae
	Coronavirus NL63, Influenza A H3, Parainfluenza Virus 3, Respiratory
ECM 2	Syncytial Virus and Bordetella pertussis (ptxP)
	MERS-CoV, Coronavirus HKU1, Influenza A H1-2009, Parainfluenza Virus 4,
ECM 3	and Bordetella parapertussis (IS1001)
	Coronavirus OC43. Human Rhinovirus/Enterovirus. Parainfluenza Virus 1.
ECM 4	Parainfluenza Virus 2 and Chlamydia pneumoniae
ECM 5	Negative (Not Detected)

Table 8: External Control Mixes (ECMs) Utilized in the Clinical Evaluations

Since the completion of the prospective clinical trial and the clinical study testing contrived specimens, Maine Molecular Quality Controls Inc. (MMQCI) has developed an external quality control panel specifically for the FilmArray RP2 and FilmArray RP2plus assays that consists of ready-to-use single tubes of negative control (matrix only) and positive control composed of synthetic RNA specific for all target analytes by the FilmArray RP2plus assay. Three clinical study sites, previously involved in the FilmArray RP2plus clinical evaluation, performed testing with three lots of this control material using three lots of the FilmArray RP2plus pouches. The expected results were obtained for all 180 control tests performed (i.e., 90/90 negative controls and 90/90 positive controls). These data were provided to MMQCI for inclusion in a 510(k) submission to the FDA.

The sponsor included the following recommending in the product package insert regarding testing external controls:

Good laboratory practice recommends running external positive and negative controls regularly. 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.

Alternatively, Maine Molecular Quality Controls, Inc. provides an external positive

23

and negative assayed quality control panel designed to monitor the performance of in vitro laboratory nucleic acid testing procedures for the qualitative detection of Adenovirus, Coronavirus, Human Metapneumovirus, Human Rhinovirus/ Enterovirus, Influenza A, Influenza A subtype H1, Influenza A subtype H1-2009, Influenza A subtype H3. Influenza B. Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Parainfluenza Virus, Respiratory Syncytial Virus, Bordetella parapertussis, Bordetella pertussis, Chlamydia pneumoniae, and Mycoplasma pneumoniae by BioFire's FilmArray RP2 and RP2plus assays on the FilmArray 2.0 or the FilmArray Torch Systems. The FilmArray RP2/RP2plus Control Panel is composed of synthetic nucleic acid specifically designed for and intended to be used solely with the FilmArray RP2 and FilmArray RP2plus assays. This material is composed of synthetic nucleic acid specific for all analytes targeted by the FilmArray RP2 and the FilmArray RP2plus assays, including a MERS-CoV synthetic nucleic acid of less than 500bp. The material is provided as a liquid in a stabilizing matrix. To use the product, the operator opens the tube and uses the Transfer Pipette to deliver the same volume of material as in the actual test, and otherwise runs the test according to protocol. This control is shipped and stored at -20°C. This product is not intended to replace manufacturer internal controls provided with the test system.

The MMQCI external control material is available for purchase directly from: Maine Molecular Ouality Controls, Inc. 23 Mill Brook Road, Saco, Maine 04072 Phone: [phone] http://www.mmqci.com FilmArray® RP2/RP2plus Control Panel M315

External controls should be used in accordance with the appropriate accrediting organization requirements, as applicable. It is ultimately the responsibility of each laboratory to determine the frequency of external control testing with the FilmArray Respiratory Panel 2 plus as part of the laboratory's Quality Control program.

Specimen Stability

FilmArray RP2plus testing requires approximately 0.3 mL of NPS in Viral Transport Medium (VTM) specimen, collected according to standard technique. Samples in VTM should be tested as soon as possible, though 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 (1.2E+09 CFU/mL).

4 Non-specific amplification by the HRV/EV assay, generating a Human Rhinovirus/Enterovirus Detected result. was observed when testing B. pertussis at high concentration (i.e., ≥4.5E+07 CFU/mL). Similar cross-reactivity is predicted to be possible with B. parapertussis, but was not observed when tested up to 1.2E+09 CFU/mL.

8 Heat-inactivated viral culture obtained through BEI Resources, NIAID, NIH: Middle East Respiratory

Syndrome Coronavirus (MERS-CoV), EMC/2012, Heat-Inactivated, NR-50171.

Two clinical NPS specimens known to contain high concentrations of

37

Coronavirus HKU1 (i.e., up to 8.9E+08 Copies/mL or >4.0E+05× LoD) were tested and no inaccurate Coronavirus OC43 Detected results were reported. These data support the conclusion that the intra-panel crossreactivity observed between the Coronavirus OC43 assay contained in the FilmArray RP and high concentrations of Coronavirus HKU1 (>1.0E+08 Copies/mL) has been eliminated by the redesigned Coronavirus OC43 assay (CoV-OC43-2) contained in the FilmArray RP2plus.

Based on sequence analysis and experience with the same assay in the FilmArray RP, cross-reactivity was expected between the assay for the detection of Bordetella pertussis (ptxP) and non-pertussis Bordetella strains that carry a pertussis toxin pseudogene (e.g. B. parapertussis and the offpanel species B. bronchiseptica) when present at concentrations >1.0E+06 CFU/mL. However, reaction conditions for the FilmArray RP2plus were determined to be less permissive for this non-specific interaction and the predicted cross-reactivity was not observed when testing on-panel B. parapertussis at a much higher concentration (i.e., >1.2E+09 CFU/mL). (Note: also, see B. bronchiseptica testing in off-panel testing section below). Though not observed, the potential for cross-reactivity with B. parapertussis will be described in the labeling as possible, but in the FilmArray RP2plus, the true organism (B. parapertussis) will also be identified.

The FilmArray RP2plus includes assays to distinguish classical human Influenza A H1 and the pandemic H1-2009 variant derived from swine. However, due to sequence similarity, some reactivity of the H1-2009 assay may be observed with historical and/or novel H1N1 strains of swine origin. This was demonstrated by testing a swine origin influenza A Hsw1N1 isolate from the 1970s (A/New Jersey/8/1976) which was detected and reported as Influenza A H1 in one replicate and as Influenza A H1-2009 in two replicates when tested at a high concentration of 8.9E+06 CEIDs0/mL.

Finally, the FilmArray RP2plus assay for detection of Human Rhinovirus/Enterovirus (HRV/EV) contains a primer pair that, despite several mismatches, is able to amplify sequences of the oxidoreductase gene found in Bordetella pertussis as well as selected published B. parapertussis and B. bronchiseptica sequences. The predicted non-specific amplification was confirmed when testing B. pertussis at a concentration of 4.5E+07 CFU/mL and higher. The cross-reactivity was not observed when testing B. parapertussis (or the off-panel organism B. bronchiseptica, see below) at a concentration of 1.2E+09 CFU/mL, but it may still be possible. Bordetella infections are less common than the "common cold' caused by Rhinoviruses (and Enteroviruses), so not only should this cross-reactivity be observed rarely, the presumably more significant Bordetella pertussis infection will be appropriately identified for treatment, with possible extra care for a concurrent Rhinovirus infection associated with the non-specific result.

Off-Panel Organisms Testing

38

In silico analysis of assay specificity was supplemented with wet testing of 50 off-panel bacteria, viruses, and fungi at high concentrations (typically ≥1.0E+06 CFU/ml for bacteria and fungi and ≥1.0E+05 TCID50/mL for viruses). The organisms tested are shown in Table 23 and results are discussed below.

Table 23: Off-panel Organisms Tested by the FilmArray RP2plus for Evaluation of Analytical
Specificity (Organisms with the potential for non-specific amplification by a FilmArray RP2plus assay are shown in
bold type)

Bacteria	Bacteria	Bacteria	Viruses
Acinetobacter calcoaceticus	Enterobacter aerogenes	Neisseria gonorrhoeae	Bocavirus
Bordetella avium	Escherichia coli	Neisseria meningitidis	Cytomegalovirus (CMV)
Bordetella bronchiseptica a,b,c	Haemophilus influenzae	Proteus mirabilis	Epstein-Barr Virus (EBV)
Bordetella hinzii	Klebsiella oxytoca	Pseudomonas aeruginosa	Herpes Simplex Virus 1
Bordetella holmesii	Klebsiella pneumoniae	Serratia marcescens	Measles Virus
Legionella bozemanii	Lactobacillus acidophilus	Staphylococcus aureus	Mumps
Legionella dumofii	Lactobacillus plantarum	Staphylococcus epidermidis	Severe Acute Respiratory
Syndrome (SARS) Virus
recombinant d
Legionella feeleii	Moraxella catarrhalis	Stenotrophomonas maltophilia
Legionella longbeacheae	Mycoplasma genitalium	Streptococcus pneumoniae	Fungi/Yeast
Legionella micdadei	Mycoplasma hominis	Streptococcus agalactiae	Candida albicans
Legionella pneumophila	Mycoplasma orale	Streptococcus pyogenes	Cryptococcus neoformans
Chlamydia trachomatis	Mycobacterium tuberculosis	Streptococcus salivarus	Aspergillus fumigatus
Corynebacterium diphteriae	Neisseria elongata	Ureaplasma urealyticum	Aspergillus flavus

ª Non-specific amplification of pertussis toxin pseudogene sequences by the pt:P assay is possible at very high concentrations (≥1.2E+09 CFU/mL), generating a Bordetella pertussis (ptxP) Detected result.

6 Non-specific amplification by the HRV/EV assay, generating a Human Rhinovirus/Enterovirus Detected result, is predicted at high concentrations but was not observed when tested up to 1.2E+09 CFU/mL.

& Strains of Bordetella bronchiseptica that carry IS1001 insertion sequences will be amplified by the IS1001 assay as intended, but misidentified by the FilmArray RP2 as Bordetella parapertussis (1S1001).

d SARS NR-18925, Recombinant Infectious Clone of Urbani Strain (icSARS-CoV) from BEI resources.

The only non-specific interaction observed in testing of off-panel organisms was a Bordetella pertussis (ptxP) Detected result in one of three replicates of B. bronchiseptica tested at a concentration of 1.2E+09 CFU/mL. This cross-reactivity has been observed at lower concentrations in the FilmArray RP and was predicted based on the presence of pertussis toxin pseudogene sequences in this species. Though not observed, Human Rhinovirus/Enterovirus assay cross-reactivity with some sequences of B. bronchiseptica is predicted by in silico analyses. In addition, certain strains of B. bronchiseptica are known to carry the IS1001 insertion sequences that are most common to B. parapertussis. In these cases, the FilmArray RP2 assay for detection of Bordetella parapertussis (IS1001) will amplify the correct targeted sequence, but will misidentify the organism as Bordetella parapertussis rather than B. bronchiseptica. As B.

39

bronchiseptica is a very rare human pathogen (more commonly associated with veterinary cases of canine "kennel cough"), the probability of inaccurate Bordetella pertussis (ptxP) results or inaccurate Bordetella parapertussis (IS1001) results due to cross-reactivity with B. bronchiseptica is low.

Analytical Specificity Evaluation Conclusion

Analytical specificity testing has demonstrated that the majority of the FilmArray RP2plus assays are highly specific for the organisms they are designed to detect. Cross-reactivity that was identified will occur almost exclusively at high organism levels (i.e., >1.0E+07 units/mL) and most events are associated with near-neighbor species that carry the same genes or highly similar sequences as the targeted organism. Overall, the likelihood, risk, and impact of the FilmArray RP2plus non-specific interactions are predicted to be minor. All identified FilmArray RP2plus cross-reactivity cases are indicated in the product Instructions for Use, as a precaution to minimize misinterpretation of results. A summary is provided in Table 24 below.

Cross-reactive Organism	FilmArray RP2plus Result	Description
Non-pertussis Bordetella
species (e.g., Bordetella
parapertussis or B.
bronchiseptica)	Bordetella pertussis (ptxP) b,c	The Bordetella pertussis (ptxP) assay can
amplify pertussis toxin pseudogene
sequences in B. bronchiseptica and B.
parapertussis. Cross-reactivity is observed
at high concentration ( $≥1.2E+09$
CFU/mL).
Bordetella bronchisepticaa
(with IS1001 sequences)	Bordetella parapertussis
(IS1001)	Some strains of B. bronchiseptica (rarely
isolated from humans) do carry IS1001
insertion sequences identical to those
carried by most strains of B.
parapertussis. These sequences will be
amplified by the IS1001 assay and
reported by FilmArray RP2plus as
Bordetella parapertussis (IS1001).
Bordetella pertussis and other
Bordetella species	Human Rhinovirus/Enterovirus d,e	The Human Rhinovirus/Enterovirus assay
may amplify off-target sequences found in
strains of B. pertussis, B. bronchiseptica
and B. parapertussis. Cross-reactivity
with B. pertussis was observed at a
concentration of 4.5E+07 CFU/mL or
higher.

Table 24: Predicted and Observed Cross-Reactivity of the FilmArray RP2plus

40

| Influenza A H1N1
(swine origin) | Influenza A H1-2009 f | The Influenza A H1-2009 assay may
react with H1 hemagglutinin gene
sequences from viruses of swine origin.
RP2plus results will be either Influenza A
H1 or Influenza A H1-2009, depending
on the strain and concentration in the
sample. |

------------------------------------	-----------------------	-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

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

b Cross-reactivity was observed only when tested at a high concentration (≥1.2E+09 CFU/mL).

& Cross-reactivity between the Bordetella pertussis (ptxP) assay and B. parapertussis will be reported as a codetection (Bordetella parapertussis (IS1001) Detected and Bordetella pertussis (ptxP) Detected); while crossreactivity with most strains of B. bronchiseptica (that do not carry IS 1001) will be reported only as Bordetella pertussis (ptxP) Detected.

4 Cross-reactivity with B. pertussis was observed when tested at a concentration of 4.5E+07 CFU/mL and higher. Cross-reactivity with B. parapertussis and B. bronchiseptica is predicted based on in silico analysis, but was not observed when tested at a concentration of 1.2E+09 CFU/mL.

e Cross-reactivity between the Human Rhinovirus/Enterovirus assays and B. pertussis or B. parapertussis will be reported as a co-detection (Bordetella pertussis (ntxP) Detected and Human Rhinovirus/Enterovirus Detected or Bordetella parapertussis (IS1001) Detected and Human Rhinovirus Detected); while cross-reactivity with most strains of B. bronchiseptica (that do not carry IS1001) will be reported (falsely) only as Human Rhinovirus/Enterovirus Detected.

f Swine origin Hsw1N1 (A/New Jersey/8/1976 ; ATCC VR-897) was detected as either Influenza A H1 or Influenza A H1-2009 at a concentration of 8.9E+06 CEID50/mL.

g. Assay Cut-off:

The FilmArray RP2plus is part of BioFire Diagnostics' FilmArray system. The FilmArray system is designed to interpret the test data and automatically report the test results to the operator. The FilmArray system uses the results of the Melt Detector to determine each test result. The Melt Detector is part of the FilmArray Analysis Software and assigns a positive or negative result to each reaction on the array through analysis of the melt data collected during the test. These positive and negative results are combined in the FilmArray Analysis Software (using the replicate, assay and interpretation rules) to report the presence or absence of each pathogen in the panel.

To determine the result for each well, the Melt Detector was developed to analyze the melt data. The Melt Detector analyzes the melt data for each well independently, and utilizes curve-specific characteristics (e.g., shape, signal-to-noise, etc.) to assign a positive or negative call to each curve. To maximize the sensitivity and specificity of the Melt Detector, the algorithm was tuned against a large data set comprising typical and atypical melting curves (i.e., training data set) with expert annotation (positive or negative calls) during the development of the original FilmArray RP.

To evaluate the Melt Detector performance for the FilmArray RP2plus, the observed sensitivity and specificity rates for the individual melt curves and assay calls were reported and analyzed. These sensitivity and specificity rates were determined by comparing the FilmArray RP2plus test results obtained from well-characterized samples, collected as part of the clinical evaluation and analytic testing of the FilmArray RP2plus panel, to expert annotation.

41

For individual melt curves, the observed sensitivity and specificity, as compared to expert annotation, of the Melt Detector is 99.65% and 99.94%, respectively. For the Analysis Software, the observed sensitivity and specificity, as compared to expert annotation, of the assay calls are greater than 99.80% for sensitivity and 99.96% specificity. These rates are comparable to the observed rates reported in the previous melt detector validation studies.

h. Interfering Substances

An analytical study was performed to assess the potential inhibitory effects of exogenous and endogenous substances and competitive microorganisms that may be commonly found in nasopharyngeal specimens (NPS). This study also assessed the potential inhibitory effects of disinfecting/cleaning substances and specimen collection materials.

For this study, potentially interfering substances were selected for evaluation based upon whether the substance may normally be found in NPS specimens or may be introduced into specimens during NPS collection or subsequent handling and testing. This included endogenous substances that may be found in specimens at normal or elevated levels, such as blood, mucus/mucin, human genomic DNA, and various commensal or infectious microorganisms (both on-panel and off-panel). Exogenous substances that may be present in specimens, such as medications, treatments, or topical applications for soothing symptoms associated with respiratory infections were also included. Lastly. substances such as disinfectants (e.g., bleach and ethanol) and various swabs and transport media that could contact specimens during collection or testing were evaluated as potential technique- specific interfering substances.

Each test substance was added to a contrived sample at a concentration similar to or greater than the level expected to be found in a clinical NPS specimen. The contrived sample contained a mix of five different FilmArray RP2plus analytes, each present at a concentration near the limit of detection (LoD). Contrived sample with no substance added served as a positive control (no interference) on each day of testing, and a potentially interfering substance in negative sample matrix served as a negative or substance-only control. Samples containing test substances were evaluated for effects of the substance on the internal pouch control assays as well as effects on the ability of the FilmArray RP2plus to provide accurate organism test results compared to the positive control samples.

In total, 39 different endogenous and exogenous substances, potentially competing microorganisms, specimen collection materials (swabs and media), and disinfecting agents were evaluated in this study (see Table 25 below).

|--|

Substance Tested	Concentration Tested	Result
Endogenous Substances
Human Whole Blood	10% v/v	No Interference
Human Mucin (Sputum)	1 swab/mL sample	No Interference
Human Genomic DNA	20 ng/µL	No Interference
Competitive Microorganisms
Coronavirus 229E	1.7E+04 TCID50/mL	No Interference
Adenovirus A12	8.9E+05 TCID50/mL	No Interference
Parainfluenza Virus 3	6.6E+05 TCID50/mL	No Interference
Bordetella pertussis	5.8E+08 CFU/mL	No Interference
Enterovirus D68	1.6E+07 TCID50/mL a	No Interference
Echovirus 6	1.0E+07 TCID50/mL	No Interference
Respiratory Syncytial Virus	4.2E+04 TCID50/mL	No Interference
Staphylococcus aureus	2.5E+07 CFU/mL	No Interference
Streptococcus pneumoniae	1.7E+07 CFU/mL	No Interference
Haemophilus influenzae	6.2E+07 CFU/mL	No Interference
Candida albicans	1.0E+06 CFU/mL	No Interference
Herpes Simplex Virus 1	1.6E+06 TCID50/mL	No Interference
Cytomegalovirus (CMV)	1.2E+06 TCID50/mL	No Interference
Exogenous Substances b
Tobramycin (systemic antibiotic)	0.6 mg/mL	No Interference
Mupirocin
(active ingredient in anti-bacterial ointment)	2% w/v	No Interference
Saline Nasal Spray with Preservatives
(0.65% NaCl, Phenylcarbinol, Benzalkonium chloride)	1% v/v	No Interference
Nasal Decongestant Spray
(Oxymetazoline HCl 0.05%, Benzalkonium chloride, phosphate)	1% v/v	No Interference
Analgesic ointment (VicksVapoRub)	1% w/v	No Interference
Petroleum Jelly (Vaseline)	1% w/v	No Interference
Snuff (Tobacco)	1% w/v	No Interference
Disinfecting/Cleaning Substances
Bleach	1% and 2% v/v
[512, 1024 ppm chlorine]	Interference c
Disinfecting wipes (ammonium chloride d)	½ in²	No Interference
Ethanol	7% v/v	No Interference
DNAZap (Ambion™ AM9891G & AM9892G)	1% v/v	No Interference
RNaseZap (Ambion™ AM9782)	1% v/v	No Interference
Specimen Collection Materials
Rayon Swabs (Copan 168C)	N/A	No Interference
Nylon Flocked Swabs (Copan 553C)	N/A	No Interference
Polyester Swabs (Copan 175KS01)	N/A	No Interference
Calcium Alginate Swabs (Puritan 25-801 A 50)	N/A	No Interference
M4 Transport Medium
(Remel R12500, 3mL/tube)	100%	No Interference
M4-RT Transport Medium
(Remel R12506, 3 mL/tube)	100%	No Interference
M5 Transport Medium
(Remel R12516, 3 mL/tube)	100%	No Interference
M6™ Transport Medium
(Remel R12535, 1.5 mL/tube)
M6™ Transport Medium
(Remel R12535, 1.5 mL/tube)	100%	No Interference
Universal Viral Transport vial
(BD 220220, 3 mL/tube)	100%	No Interference
Sigma-Virocult™ Viral Collection and Transport System – Swabs and Transport Medium (Medical Wire MW951SENT)	100%	No Interference
ESwab™ Sample Collection and Delivery System – Swabs and Liquid Amies Medium (Copan 482C)	100%	No Interference

42

43

a A different lot of this isolate was also tested at a lower concentration of 3.2E+04 TCID50/mL with no interference observed.

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

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

d n-Alkyl (C14, 60%, C16, 30%, C12, 5%, C18, 5%) Dimethyl Benzyl Ammonium Chloride - 0.184% n-Alkyl (C12, 68%, C14, 32%) Dimethyl Ethylbenzyl Ammonium Chloride - 0.184%

Testing showed that none of the substances evaluated had an effect on the FilmArray RP2plus control assays and no interference with pouch function was identified. However, it was demonstrated that exposure of samples to bleach prior to testing could damage the organisms/nucleic acids in the sample and generate inaccurate FilmArrav RP2plus test results (i.e., lack of analyte detection), depending on the concentration and/or length of time the bleach was allowed to interact with the sample.

Interfering Substances Evaluation Conclusion

No interference was observed when testing samples containing potentially inhibitory biological substances (e.g., blood etc.) or high levels of potentially competing microorganisms. Similarly, detection near LoD was robust in samples prepared in a variety of transport media types or when exposed to various swabs that may be used for NPS collection. Saline, decongestants, ointments or other substances that could be introduced into the sample also had no effect on the function of the FilmArray RP2plus.

The only limitation is related to potential damage to the organisms in the sample caused by bleach that could lead to false negative results. A warning describing the potential for sample damage caused by bleach will be included in the product Instructions for Use.

Note: Nasal influenza vaccines (e.g., FluMist) were not evaluated in this study, but are predicted to be reactive with the FilmArray RP2plus Influenza A (including subtype) and Influenza B assays. Therefore, contamination of specimens with vaccine or recent administration of the vaccine prior to NPS specimen collection could lead to accurate detection by the FilmArray RP2plus of the viruses contained in the vaccine, but would not represent infection by those agents.

• i. Carry-Over Contamination:

44

A formal carry-over study in support of this regulatory submission for the FilmArray RP2plus was not performed, since carry-over studies with high positive samples followed by negative samples have been performed for other FDA-cleared FilmArray Panels (i.e., FilmArray RP, BCID, and GI) for both the FilmArray 2.0 and the FilmArray Torch systems, and no carry-over has been observed.

j. Comparator Assay for B. parapertussis Analytical Validation

In the prospective clinical trial for the FilmArray RP2plus, the FilmArray RP2plus results were compared to the results from standard of care testing using the same FDA-cleared multiplexed respiratory pathogens panel performed at the clinical sites for all the FilmArray RP2plus analytes except for Bordetella parapertussis and MERS-CoV.

Two well-validated PCR assays for Bordetella parapertussis (IS1001) followed by bidirectional sequencing (designed to give at least 200 base pairs of sequence information) were performed as the comparator method for Bordetella parapertussis (IS 1001). The two comparator PCR assays target the same gene as the FilmArray RP2plus assay (i.e., the IS1001 genetic element), but the primers identify sequences that do not overlap with the FilmArray RP2plus assays. The two PCR comparator assays used in the clinical studies were designed by BioFire as published assays could not be found that provided adequate amplicon length or quality for sequencing. Additionally, the assays were nested in order to match the sensitivity of the FilmArray RP2plus assay. Replicate assays were run as separate PCR1 reactions so that any cross-contamination events could be detected and resolved via mismatch testing. Each post-PCR1 reaction mixture was then used as template for testing with the appropriate PCR2 assay. PCR plates containing potentially positive amplicons were sent directly to a contract laboratory for ExoSAP clean up and sequencing. A positive result from either assay was considered positive for Bordetella parapertussis (IS1001).

Validation testing demonstrated that the PCR followed by sequencing assays had similar analytical reactivity performance to the FilmArray RP2plus assay, and a LoD that was within 5-fold of the FilmArray RP2plus assay (this was considered "equivalent analytical sensitivity").

2. Comparison Studies:

Due to very limited information available on clinical relevant MERS-CoV viral loads in upper respiratory specimens (including NPS in VTM) from MERS patients and asymptomatic MERS-CoV carriers, and the fact that only three natural clinical specimens that are MERS-CoV positive were available for testing, an additional analytical study was conducted. In order to obtain analytical sensitivity information relative to that of established MERS-CoV nucleic acid-based tests authorized under the FDA Emergency Use Authorization (EUA) program, the CDC Novel Coronavirus 2012 Real-time RT-PCR Assay and the RealStar MERS-CoV RT-PCR Kit U.S. from Altona Diagnostics GmbH a MERS-CoV panel was tested.

45

A MERS-CoV proficiency test panel prepared by the Quality Control for Molecular Diagnostics (QCMD) under a contract with the U.S. government was tested in a blinded fashion utilizing the RealStar MERS-CoV RT-PCR Kit U.S. from Altona Diagnostics GmbH and the FilmArray RP2plus in parallel. This MERS-CoV proficiency test panel contains MERS-CoV samples, as well as samples of common coronaviruses, and was characterized by both the QCMD and the CDC. For the purpose of comparative analysis of analytical sensitivity, panel characterization testing data generated at the CDC using the CDC MERS-CoV N2 rRT-PCR (one of the three assays of the CDC Novel Coronavirus 2012 Real-time RT-PCR test under an EUA) was obtained from QCMD after the parallel testing was completed.

The analytical sensitivity of FilmArray RP2plus detecting MERS-CoV appeared to be at least equivalent to these two established MERS-CoV nucleic acid-based tests that were authorized under the FDA Emergency Use Authorization (EUA) program. See Table 26 below.

46

| PT | Sample
Description | Target
Concentration
(Copies/mL ª) | CDC MERS-CoV
N2 rRT-PCR (EUA) | | Altona RealStar MERS-CoV
RT-PCR (EUA) | | | FilmArray
RP2plus | FilmArray
RP2plus |
|----------------------|-----------------------|------------------------------------------|----------------------------------|------------------------|------------------------------------------|----------|------------------------|----------------------|-----------------------------------------|
| Panel
Sample

| Sample

Description | Target
Concentration
(Copies/mL ª) | N2 Ct b | MERS-
CoV
Result | UpE Ct | orf1a Ct | MERS-
CoV
Result | MERS-
CoV Result | Other
Pathogen
Detected
Result |
| 1 | MERS-CoV | 1.0E+04 | 26.46 | D | 23.66 | 22.85 | D | D | - |
| 2 | MERS-CoV | 1.0E+04 | 26.25 | D | 23.65 | 22.56 | D | D | - |
| 3 | MERS-CoV | 1.0E+03 | 30.20 | D | 26.83 | 26.03 | D | D | - |
| 4 | MERS-CoV | 1.0E+02 | 33.01 | D | 29.65 | 29.03 | D | D | - |
| 5 | MERS-CoV | 1.0E+01 | 35.27 | D | 32.83 | 32.57 | D | D | - |
| 6 | CoV-OC43 | 1.0E+04 | No Ct | ND | No Ct | No Ct | ND | ND | CoV-OC43 |
| 7 | CoV-OC43 | 1.0E+03 | No Ct | ND | No Ct | No Ct | ND | ND | CoV-OC43 |
| 8 | CoV-NL63 | 1.0E+04 | No Ct | ND | No Ct | No Ct | ND | ND | CoV-NL63 |
| 9 | CoV-NL63 | 1.0E+03 | No Ct | ND | No Ct | No Ct | ND | ND | CoV-NL63 |
| 10 | CoV-229E | 1.0E+04 | No Ct | ND | No Ct | No Ct | ND | ND | CoV-229E |
| 11 | CoV-229E | 1.0E+03 | No Ct | ND | No Ct | No Ct | ND | ND | CoV-229E |
| 12 | MERS-CoV | 5.0E+04 | 23.97 | D | 21.74 | 20.90 | D | D | - |

Table 26: QCMD MERS-CoV Proficiency Testing (PT) Panel Results (after un-blinding)

a Generated using QCMD in-house assays.

b Average Ct of two RT-PCR replicates.

D = Detected; ND = Not Detected

3. Clinical Studies:

Prospective Clinical Study

The clinical performance of the FilmArray RP2vlus was established during a multi-center study conducted at three geographically distinct U.S. study sites during portions of the 2015-2016 and 2016-2017 respiratory illness seasons. Each study location was representative of the intended use setting (clinical laboratories) and testing was performed by trained clinical laboratory personnel.

Residual NPS specimens in VTM meeting the following eligibility criteria were prospectively collected and tested using the FilmArray RP2plus during the clinical study:

Inclusion criteria:

• · Specimen is residual NPS in VTM left over from standard of care (SOC) testing under clinician order for respiratory pathogen analysis using the FilmArray RP, an FDAcleared multiplexed respiratory pathogens panel
• · Specimen has been held at room temperature for less than or equal to four hours or 4°C for less than or equal to three days before enrollment (FilmArray RP2plus testing or archiving of specimen aliquots at M. pneumoniae | | | | | 4 | 1 | HRV/EV (1) | |
  | Adenovirus | CoV-HKU1 | | | | | 3 | 2 | Adenovirus (1), CoV-HKU1 (1) | |
  | Adenovirus | PIV3 | | | | | 3 | 3 | Adenovirus (3), PIV3 (1) | |
  | CoV-OC43 | RSV | | | | | 3 | 0 | - | |
  | hMPV | PIV4 | | | | | 3 | 2 | hMPV (1), PIV4 (2) | |
  | FluA H1-2009 | RSV | | | | | 3 | 1 | RSV (1) | |
  | Adenovirus | HRV/EV | PIV3 | RSV | | | 2 | 2 | Adenovirus (1), PIV3 (2) | |
  | Adenovirus | HRV/EV | PIV3 | | | | 2 | 1 | HRV/EV (1) | |
  | Adenovirus | HRV/EV | M. pneumoniae | | | | 2 | 1 | Adenovirus (1), M. pneumoniae (1) | |
  | CoV-HKU1 | CoV-OC43 | RSV | | | | 2 | 2 | CoV-HKU1 (2) | |
  | CoV-HKU1 | HRV/EV | RSV | | | | 2 | 1 | HRV/EV (1) | |
  | hMPV | HRV/EV | PIV4 | | | | 2 | 0 | - | |
  | hMPV | HRV/EV | RSV | | | | 2 | 1 | HRV/EV (1) | |
  | Adenovirus | hMPV | | | | | 2 | 1 | Adenovirus (1) | |
  | Adenovirus | PIV2 | | | | | 2 | 2 | Adenovirus (1), PIV2 (1) | |
  | CoV-229E | HRV/EV | | | | | 2 | 1 | HRV/EV (1) | |
  | CoV-229E | RSV | | | | | 2 | 1 | CoV-229E (1) | |
  | CoV-NL63 | hMPV | | | | | 2 | 1 | hMPV (1) | |
  | CoV-NL63 | FluB | | | | | 2 | 2 | CoV-NL63 (2) | |
  | hMPV | RSV | | | | | 2 | 1 | RSV (1) | |
  | HRV/EV | PIV1 | | | | | 2 | 0 | - | |
  | HRV/EV | B. parapertussis | | | | | 2 | 0 | - | |
  | Adenovirus | CoV-NL63 | hMPV | HRV/EV | PIV4 | RSV | 1 | 1 | HRV/EV (1), PIV4 (1) | |
  | Adenovirus | HRV/EV | PIV3 | PIV4 | RSV | | 1 | 1 | PIV4 (1), RSV (1) | |
  | Adenovirus | HRV/EV | RSV | B. parapertussis | | | 1 | 1 | Adenovirus (1), HRV/EV (1) | |
  | CoV-HKU1 | CoV-OC43 | HRV/EV | RSV | | | 1 | 0 | - | |
  | Adenovirus | CoV-HKU1 | hMPV | | | | 1 | 1 | hMPV (1) | |
  | Adenovirus | CoV-OC43 | HRV/EV | | | | 1 | 0 | - | |
  | Adenovirus | CoV-OC43 | RSV | | | | 1 | 0 | - | |
  | Adenovirus | hMPV | FluA H1-2009 | | | | 1 | 1 | Adenovirus (1) | |
  | Adenovirus | HRV/EV | FluA H3 | | | | 1 | 1 | HRV/EV (1) | |
  | CoV-229E | CoV-HKU1 | hMPV | | | | 1 | 1 | CoV-229E (1) | |
  | CoV-HKU1 | CoV-NL63 | HRV/EV | | | | 1 | 1 | CoV-NL63 (1) | |
  | CoV-HKU1 | CoV-NL63 | RSV | | | | 1 | 1 | CoV-HKU1 (1) | |
  | CoV-HKU1 | hMPV | PIV3 | | | | 1 | 1 | hMPV (1) | |
  | CoV-HKU1 | hMPV | RSV | | | | 1 | 0 | - | |
  | | | | | | | | | | |

54

55

CoV-HKU1	PIV1	RSV	1	0	-
CoV-HKU1	PIV4	RSV	1	1	PIV4 (1)
CoV-NL63	CoV-OC43	PIV1	1	0	-
CoV-NL63	RSV	B.	1	1	RSV (1)
CoV-OC43	HRV/EV	PIV4	1	1	CoV-OC43
hMPV	HRV/EV	FluB	1	1	hMPV (1), HRV/EV (1),
FluB (1)
HRV/EV	FluA H1-2009	RSV	1	0	-
HRV/EV	PIV1	RSV	1	1	PIV1 (1)
HRV/EV	PIV2	RSV	1	1	HRV/EV (1), PIV2 (1)
HRV/EV	RSV	B.	1	0	-
Adenovirus	CoV-NL63		1	0	-
Adenovirus	CoV-OC43		1	1	Adenovirus (1)
Adenovirus	FluA H1-2009		1	1	Adenovirus (1)
Adenovirus	PIV4		1	1	Adenovirus (1)
CoV-229E	FluA H1-2009		1	0	-
CoV-HKU1	hMPV		1	0	-
CoV-HKU1	FluB		1	0	-
CoV-HKU1	PIV4		1	1	PIV4 (1)
CoV-OC43	hMPV		1	1	CoV-OC43 (1)
CoV-OC43	PIV3		1	0	-
CoV-OC43	M. pneumoniae		1	1	CoV-OC43 (1)
hMPV	FluA H1-2009		1	0	-
hMPV	FluB		1	1	FluB (1)
HRV/EV	FluA H1-2009		1	0	-
HRV/EV	FluA H3		1	0	-
HRV/EV	FluB		1	1	HRV/EV (1)
HRV/EV	PIV4		1	1	HRV/EV (1)
HRV/EV	C. pneumoniae		1	0	-
PIV2	RSV		1	1	PIV2
RSV	B. parapertussis		1	0	-
Total Co-infections			245	121	135/554
Total Double Infections			190	86	91/380
Total Triple Infections			49	30	35/147
Total Quadruple Infections			4	3	5/16
Total Quintuple Infections			1	1	2/5
Total Sextuple Infections			1	1	2/6

ª A discrepant co-infection or discrepant analyte was defined as one that was detected by FilmArrayRP2plus but not detected by the comparator method. Of the 135 discrepant analytes), 66 (48.9%) were observed as being present in the specimen during discrepancy investigation; 43/135 (31.9%) were observed using an independent mothod and 27/135 (20.0%) were observed upon comparator method retest.

Additional distinct co-infection combinations detected by the comparator method(s), but

56

not detected by the FilmArray RP2plus in the prospective clinical trial are presented in Table 33 below.

| Distinct Co-infection Combinations
Detected by the FilmArray RP2plus | | | | Total Specimens
with
Co-infections | Number of
Specimens with
Discrepant a | Discrepant
Analyte(s) |
|-------------------------------------------------------------------------|----------------------------|-----------|-----------|------------------------------------------|---------------------------------------------|--------------------------|
| Analyte 1 | Analyte 2 | Analyte 3 | Analyte 4 | | | |
| Adenovirus | FluA H1-2009 | | | 1 | 1 | Adenovirus (1) |
| Adenovirus | PIV3 | | | 2 | 1 | Adenovirus (1) |
| CoV-229E | FluA H1-2009 | | | 2 | 1 | CoV-229E (1) |
| CoV-HKU1 | CoV-OC43 | | | 3 | 3 | CoV-OC43 (3) b |
| CoV-HKU1 | CoV-OC43 | RSV | | 2 | 2 | CoV-OC43 (2) b |
| CoV-HKU1 | CoV-OC43 | HRV/EV | RSV | 2 | 1 | CoV-OC43 (1) b |
| CoV-HKU1 | HRV/EV | | | 5 | 1 | HRV/EV (1) |
| CoV-HKU1 | HRV/EV | RSV | | 1 | 1 | HRV/EV (1) |
| hMPV | HRV/EV | | | 8 | 1 | HRV/EV (1) |
| HRV/EV | PIV2 | | | 5 | 1 | HRV/EV (1) |
| HRV/EV | PIV3 | | | 6 | 1 | HRV/EV (1) |
| HRV/EV | B. parapertussis | | | 3 | 1 | B. parapertussis (1) |
| PIV2 | PIV3 | | | 1 | 1 | PIV2 (1), PIV3 (1) |
| RSV | M. pneumoniae | | | 1 | 1 | M. pneumoniae (1) |
| | Total Co-infections | | | 42 | 17 | 18/91 |
| | Total Double Infections | | | 37 | 13 | 13/74 |
| | Total Triple Infections | | | 3 | 3 | 4/9 |
| | Total Quadruple Infections | | | 2 | 1 | 1/8 |

Table 33: Additional Distinct Co-infection Combinations Detected by the Comparator Method(s), but not detected by the FilmArray RP2plus in the Prospective Clinical Trial

4 This table includes only distinct co-infections that were detected by the comparator method(s) but not by FilmArray RP2plus; the remaining co-infections detected by the comparator method(s) are already represented in Table 32 above.

of the six FilmArray RP2plus FN specimens, all were TP for CoV-HKU1. They were confirmed to be due to a known cross-reactivity with CoV-HKU1 of the comparator method. All six specimens were negative for CoV-OC43 when tested with two independent PCR assays.

Retrospective Clinical Study - Common Respiratory Pathogens

Some of the analytes on the FilmArray RP2plus were of low prevalence and were not encountered in sufficiently large numbers during the prospective study to adequately demonstrate system performance. To supplement the results of the prospective clinical study, an evaluation of preselected archived retrospective specimens was performed at BioFire Diagnostics. These specimens were archived NPS in VTM specimens that were selected because they had previously tested positive for one of the following analytes at the source laboratory: coronavirus 229E, influenza A H1, influenza A H3, influenza B,

57

parainfluenza virus 1, parainfluenza virus 4, Bordetella parapertussis. Bordetella pertussis, Chlamydia pneumoniae, parainfluenza virus 2, parainfluenza virus 3, and Mycoplasma pneumoniae.

A total of 217 clinical specimens were initially received for testing in this retrospective study (Table 34 below). In preparation for testing, a study code number (SCN) was assigned to each specimen and a key was created in order to randomize them such that operators were blinded to the expected test result.

Prior to testing with the FilmArray RP2plus, the composition/integrity of the pre-selected specimens was first confirmed with the same comparator methods employed in the prospective clinical study (i.e., PCR followed by bi-directional sequencing assays for B. parapertussis or an FDA-cleared multiplexed respiratory pathogens panel for all other analytes except for MERS-CoV). Specimens were divided into two different groups for testing based on the method of confirmation testing performed: all specimens containing analytes on the FDA-cleared multiplexed respiratory pathogens panel comparator method were tested in Group 1 and specimens containing B. parapertussis were tested in Group 2. Negative NPS specimens were included in each group. Table 34 below shows the number of specimens for each analyte (and negatives) that were tested with the FilmArray RP2plus in this study.

Analyte	Number of Specimens
Group 1
Coronavirus 229E	19
Influenza A H1	3
Influenza A H3	17
Influenza B	17
Parainfluenza Virus 1 a	16
Parainfluenza Virus 2	17
Parainfluenza Virus 3	17
Parainfluenza Virus 4	17
Bordetella pertussis	32
Chlamydia pneumoniae	18
Mycoplasma pneumoniae a	21
Negative	4
Total	197 a
Group 2
Bordetella parapertussis	16
Negative	4
Total	20
Total Overall	217

Table 34: Archived Specimens Used in the Retrospective Clinical Study - Common Respiratory Pathogens

One specimen contained two analytes of interest.

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The FDA-cleared multiplexed respiratory pathogens panel comparator method was performed on 197 of the 217 achieved specimens only (Group 1). One of the 197 specimens was excluded from performance analysis because of an invalid RP2plus run with insufficient volume to retest. Additionally, two of the 197 specimens were also excluded from performance analysis because a valid FDA-cleared multiplexed respiratory pathogens panel comparator method confirmation result was not obtained and there was insufficient specimen volume for retesting: one comparator run was incomplete and the other comparator run had a pouch control failure. FilmArray RP2plus results for these specimens are shown below in Table 35. Valid comparator method and FilmArray RP2plus results were obtained for 194 of these 197 archived specimens (Group 1).

59

| SCN | Analyte Detected at the Source
Laboratory | Comparator Method
Confirmation
Result | FilmArray RP2plus
Result |
|-----------------|----------------------------------------------|---------------------------------------------|-----------------------------|
| 026979-ARC-0004 | Bordetella pertussis | Bordetella pertussis | Invalid |
| 026979-ARC-0127 | PIV4 | Invalid | PIV4 |
| 026979-ARC-0170 | C. pneumoniae | Invalid | C. pneumoniae |

Table 35: Specimens Excluded Because of Invalid Comparator Test and Insufficient Volume for Retesting

The B. parapertussis PCR followed by bi-directional sequencing comparator assays were performed on 20 of the 217 achieved specimens only (Group 2). The FDA-cleared multiplexed respiratory pathogens panel comparator method was not performed on Group 2 specimens. Valid comparator method and FilmArray RP2plus results were obtained for 20 of these 20 archived specimens.

In addition, all Group 1 and Group 2 positive archived specimens (as determined at the source laboratory) not confirmed by the respective comparator method were also excluded from the performance calculation for each of the respective analyte. FilmArray RP2plus results for these excluded specimens are shown below in Table 36.

| SCN | Analyte Detected at the Source
Laboratory | Comparator Method
Confirmation
Result | FilmArray RP2plus
Result |
|---------------------------------------------------------------------------------------------------------------|----------------------------------------------|---------------------------------------------|-----------------------------|
| Samples Excluded from CoV-229E Performance Analysis Because of Unconfirmed Comparator Method CoV-229E | | | |
| 026979-ARC-0026 | CoV-229E | CoV-229E Not Detected | CoV-229E Not Detected |
| 026979-ARC-0049 | CoV-229E | CoV-229E Not Detected | CoV-229E |
| 026979-ARC-0055 | CoV-229E | CoV-229E Not Detected | CoV-229E Not Detected |
| 026979-ARC-0143 | CoV-229E | CoV-229E Not Detected | CoV-229E |
| Samples Excluded from Flu B Performance Analysis Because of Unconfirmed Comparator Method Flu B Result | | | |
| 026979-ARC-0199 | Influenza B | Influenza B Not Detected | Influenza B |
| Samples Excluded from PIV 2 Performance Analysis Because of Unconfirmed Comparator Method PIV 2 Result | | | |
| 026979-ARC-0014 | PIV 2 | PIV 2 Not Detected | PIV 2 Not Detected |
| Samples Excluded from B. pertussis Performance Analysis Because of Unconfirmed Comparator Method B. pertussis | | | |
| 026979-ARC-0028 | B. pertussis | B. pertussis Not Detected | B. pertussis Not Detected |
| 026979-ARC-0036 | B. pertussis | B. pertussis Not Detected | B. pertussis Not Detected |
| 026979-ARC-0052 | B. pertussis | B. pertussis Not Detected | B. pertussis Not Detected |
| 026979-ARC-0087 | B. pertussis | B. pertussis Not Detected | B. pertussis Not Detected |
| 026979-ARC-0097 | B. pertussis | B. pertussis Not Detected | B. pertussis |
| 026979-ARC-0131 | B. pertussis | B. pertussis Not Detected | B. pertussis Not Detected |
| Samples Excluded from C. pneumoniae Performance Analysis Because of Unconfirmed Comparator Method C. | | | |
| 026979-ARC-0145 | C. pneumoniae | C. pneumoniae Not | C. pneumoniae |
| Samples Excluded from M. pneumoniae Performance Analysis Because of Unconfirmed Comparator Method M. | | | |
| 026979-ARC-0094 | M. pneumoniae | M. pneumoniae Not | M. pneumoniae Not |
| 026979-ARC-0104 | M. pneumoniae | M. pneumoniae Not | M. pneumoniae |
| 026979-ARC-0105 | M. pneumoniae | M. pneumoniae Not | M. pneumoniae |
| 026979-ARC-0138 | M. pneumoniae | M. pneumoniae Not | M. pneumoniae Not |

Table 36: Specimens Excluded Because of Unconfirmed Comparator Method Results

60

026979-ARC-0149	M. pneumoniae	M. pneumoniae Not	M. pneumoniae
-----------------	----------------------	--------------------------	----------------------

| Analyte | Number of Specimens | Number of Specimens Confirmed
by the Comparator Method (%) |
|--------------------------|---------------------|---------------------------------------------------------------|
| Group 1 | | |
| Coronavirus 229E | 19 | 15 (78.9%) |
| Influenza A H1 | 3 | 3 (100%) |
| Influenza A H3 | 17 | 17 (100%) |
| Influenza B | 17 | 16 (94.1%) |
| Parainfluenza Virus 1 a | 16 | 16 (100%) |
| Parainfluenza Virus 2 | 17 | 16 (94.1%) |
| Parainfluenza Virus 3 | 17 | 17 (100%) |
| Parainfluenza Virus 4 | 16 | 16 (100%) |
| Bordetella pertussis | 31 | 25 (80.6%) |
| Chlamydia pneumoniae | 17 | 16 (94.1%) |
| Mycoplasma pneumoniae a | 21 | 16 (76.2%) |
| Negative | 4 | 4 (100%) |
| Total | 194 a | |
| Group 2 | | |
| Bordetella parapertussis | 16 | 16 (100%) |
| Negative | 4 | 4 (100%) |
| Total | 20 | 20 (100%) |
| Total Overall | 214 | |

Table 37: Analyte Confirmation Summary for All Group 1 and Group 2 Specimens

• One specimen contained two analytes of interest.

A summary of the available demographic information of the 214 specimens is provided in Table 38 below.

Table 38: Available Demographic Summary for All Retrospective Specimens (Common Respiratory
Pathogens) Included in the Performance Analysis

Total Specimens	214
Sex	Female (%)	75 (35%)
	Male (%)	81 (38%)
	Unknown	58 (27%)
Age Range