FilmArray Respiratory Panel 2 (RP2)
Device Facts
| Record ID | K170604 |
|---|---|
| Device Name | FilmArray Respiratory Panel 2 (RP2) |
| Applicant | Biofire Diagnostics, LLC |
| Product Code | OCC · Microbiology |
| Decision Date | May 30, 2017 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.3980 |
| Device Class | Class 2 |
Intended Use
The FilmArray® Respiratory Panel 2 (RP2) 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 multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals suspected of respiratory tract infections. The following organism types and subtypes are identified using the FilmArray® RP2: Adenovirus, Coronavirus 229E, Coronavirus HKU1, Coronavirus NL63, Coronavirus OC43, Human Metapneumovirus, Human Rhinovirus/Enterovirus, Influenza A, including subtypes H1, H1-2009, and H3, Influenza B, Parainfluenza Virus 1, Parainfluenza Virus 2, Parainfluenza Virus 3, Parainfluenza Virus 4, Respiratory Syncytial Virus, Bordetella parapertussis (IS1001), Bordetella pertussis (ptxP), Chlamydia pneumoniae, Mycoplasma pneumoniae. The detection and identification of specific viral and bacterial nucleic acids from individuals exhibiting signs and/or symptoms of a respiratory infection aids in the diagnosis of respiratory infection if used in conjunction with other clinical and epidemiological information. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Negative results in the setting of a respiratory illness may be due to infection with pathogens that are not detected by this test, or lower respiratory tract infection that may not be detected by a nasopharyngeal swab specimen. Positive results do not rule out co-infection with other organisms: the agent(s) detected by the FilmArray® RP2 may not be the definite cause of disease. Additional laboratory testing (e.g. bacterial and viral culture, immunofluorescence, and radiography) may be necessary when evaluating a patient with possible respiratory tract infection. Due to the genetic similarity between Human Rhinovirus and Enterovirus, the FilmArray® RP2 cannot reliably differentiate them. A positive FilmArray® RP2 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 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 Story
Device is a multiplexed nucleic acid test for respiratory pathogens; inputs are nasopharyngeal swabs (NPS) in transport media. Pouch contains freeze-dried reagents; user adds hydration solution and sample/buffer mix. Instrument uses inflatable bladders, seal points, and pneumatic pistons to move liquids; Peltier devices control thermal cycling for nested multiplex PCR. First stage is large-volume multiplexed RT-PCR; second stage is singleplex nested PCR in array wells. Detection via melt curve analysis using fluorescent DNA binding dye (LC Green® Plus). Digital camera captures fluorescent images; software automatically interprets results. Used in clinical settings (hospitals, clinics) by laboratory personnel. Provides rapid (~45 min) identification of 21 viral/bacterial targets; aids diagnosis when combined with clinical/epidemiological data. Benefits include faster clinical decision-making and appropriate patient management.
Clinical Evidence
Prospective multi-center study (N=1612) compared RP2 to FDA-cleared multiplexed respiratory panel and sequencing-based comparator for B. parapertussis. Overall PPA ranged from 66.7% to 100% and NPA from 90.2% to 100% across analytes. Retrospective study (N=214) and contrived specimen study (N=48) supplemented performance data for low-prevalence analytes. Reproducibility study (N=480) demonstrated high agreement across sites, operators, and systems.
Technological Characteristics
Multiplex nucleic acid assay; nested PCR; melt curve analysis. Reagents in freeze-dried format. Instrumentation: FilmArray 2.0 or FilmArray Torch. Connectivity: Standalone system. Software: Automated interpretation. Materials: Pouch-based system with internal controls for processing and PCR stages.
Indications for Use
Indicated for individuals suspected of respiratory tract infections exhibiting signs/symptoms of respiratory infection. Used for qualitative detection of 21 viral/bacterial pathogens in nasopharyngeal swabs (NPS).
Regulatory Classification
Identification
A respiratory viral panel multiplex nucleic acid assay is a qualitative in vitro diagnostic device intended to simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or viral culture. The detection and identification of a specific viral nucleic acid from individuals exhibiting signs and symptoms of respiratory infection aids in the diagnosis of respiratory viral infection when used in conjunction with other clinical and laboratory findings. The device is intended for detection and identification of a combination of the following viruses:(1) Influenza A and Influenza B; (2) Influenza A subtype H1 and Influenza A subtype H3; (3) Respiratory Syncytial Virus subtype A and Respiratory Syncytial Virus subtype B; (4) Parainfluenza 1, Parainfluenza 2, and Parainfluenza 3 virus; (5) Human Metapneumovirus; (6) Rhinovirus; and (7) Adenovirus.
Special Controls
*Classification.* Class II (special controls). The special controls are:(1) FDA's guidance document entitled “Class II Special Controls Guidance Document: Respiratory Viral Panel Multiplex Nucleic Acid Assay;” (2) For a device that detects and identifies Human Metapneumovirus, FDA's guidance document entitled “Class II Special Controls Guidance Document: Testing for Human Metapneumovirus (hMPV) Using Nucleic Acid Assays;” and (3) For a device that detects and differentiates Influenza A subtype H1 and subtype H3, FDA's guidance document entitled “Class II Special Controls Guidance Document: Testing for Detection and Differentiation of Influenza A Virus Subtypes Using Multiplex Nucleic Acid Assays.” See § 866.1(e) for the availability of these guidance documents.
Predicate Devices
- FilmArray Respiratory Panel (RP) (K160068)
Related Devices
- K143080 — FilmArray Respiratory Panel (RP) for use with Multi-instrument FilmArray System · Biofire Diagnostics, LLC · Feb 17, 2015
- K120267 — FILMARRAY RP PANEL · Idaho Technology, Inc. · May 15, 2012
- K160068 — FilmArray Respiratory Panel (RP) for use with FilmArray Torch · Biofire Diagnostics, LLC · Feb 8, 2016
- K123620 — FILMARRAY RESPIRATORY PANEL (RP) · Biofire Diagnostics, Inc. · Feb 11, 2013
Submission Summary (Full Text)
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Public Health Service
May 30, 2017
Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-0002
BioFire Diagnostics, LLC Kristen J. Kanack, Ph.D. Vice President, Regulatory and Clinical Affairs 515 Colorow Drive Salt Lake City, UT 84108
Re: K170604
Trade/Device Name: FilmArray® Respiratory Panel 2 (RP2) Regulation Number: 21 CFR 866.3980 Regulation Name: Respiratory Viral Panel Multiplex Nucleic Acid Assay Regulatory Class: II Product Code: OCC, OEM, OEP, OOU, OTG, OZE, OZX, OZY, OZZ, OOI, NSU Dated: February 28, 2017 Received: March 1, 2017
Dear Dr. Kanack:
We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food. Drug. and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.
If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.
Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.
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If you desire specific advice for your device on our labeling regulations (21 CFR Parts 801 and 809), please contact the Division of Industry and Consumer Education at its toll-free number (800) 638 2041 or (301) 796-7100 or at its Internet address
http://www.fda.gov/MedicalDevices/Resourcesfor You/Industry/default.htm. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to
http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.
You may obtain other general information on your responsibilities under the Act from the Division of Industry and Consumer Education at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address
http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.
Sincerely yours,
# Steven R. Gitterman -S
for Uwe Scherf, M.Sc., Ph.D. Director Division of Microbiology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health
Enclosure
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# Indications for Use
#### 510(k) Number (if known) K170604
#### Device Name
The FilmArray® Respiratory Panel 2 (RP2)
#### Indications for Use (Describe)
The FilmArray® Respiratory Panel 2 (RP2) 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 multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals suspected of respiratory tract infections. The following organism types and subtypes are identified using the FilmArray® RP2:
- · 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
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- · Bordetella parapertussis (IS1001)
- Bordetella pertussis (ptxP)
- Chlamydia pneumoniae
- Mycoplasma pneumoniae
The detection and identification of specific viral and bacterial nucleic acids from individuals exhibiting signs and/or symptoms of a respiratory infection aids in the diagnosis of respiratory infection if used in conjunction with other clinical and epidemiological information. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.
Negative results in the setting of a respiratory illness may be due to infection with pathogens that are not detected by this test, or lower respiratory tract infection that may not be detected by a nasopharyngeal swab specimen. Positive results do not rule out co-infection with other organisms: the agent(s) detected by the FilmArray® RP2 may not be the definite cause of disease. Additional laboratory testing (e.g. bacterial and viral culture, immunofluorescence, and radiography) may be necessary when evaluating a patient with possible respiratory tract infection.
Due to the genetic similarity between Human Rhinovirus and Enterovirus, the FilmArray® RP2 cannot reliably differentiate them. A positive FilmArray® RP2 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 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.
| Type of Use (Select one or both, as applicable) | |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| <span style="font-family: Arial, sans-serif;"> <span style="font-size: 11pt;"> <span style="color: black;"> <span style="font-weight: normal;"> <span style="font-style: normal;"> <span style="text-decoration: none;"> <span style="vertical-align: baseline;"> <span style="white-space: pre-wrap;">Prescription Use (Part 21 CFR 801 Subpart D)</span> </span> </span> </span> </span> </span> </span> </span> | <span style="font-family: Arial, sans-serif;"> <span style="font-size: 11pt;"> <span style="color: black;"> <span style="font-weight: normal;"> <span style="font-style: normal;"> <span style="text-decoration: none;"> <span style="vertical-align: baseline;"> <span style="white-space: pre-wrap;">Over-The-Counter Use (21 CFR 801 Subpart C)</span> </span> </span> </span> </span> </span> </span> </span> |
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## 510(k) Summary BioFire Diagnostics, LLC
# FilmArray Respiratory Panel 2 (RP2)
#### Introduction:
According to the requirements of 21 CFR 807.92, the following information provides sufficient detail to understand the basis for a determination of substantial equivalence.
#### Submitted by:
BioFire Diagnostics, LLC 515 Colorow Drive Salt Lake City, UT 84108
Telephone: 801-736-6354 Facsimile: 801-588-0507
Contact: Kristen J. Kanack, ext. 330
Date Submitted: February 28, 2017
#### Device Name and Classification:
Trade Name: FilmArray Respiratory Panel 2 (RP2)
Regulation Number: 21 CFR 866.3980
Classification Name: Respiratory viral panel multiplex nucleic acid assay
#### Predicate Device:
K160068 - FilmArray Respiratory Panel (RP)
#### Intended Use:
The FilmArray® Respiratory Panel 2 (RP2) 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 multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals suspected of respiratory tract infections. The following organism types and subtypes are identified using the FilmArray RP2:
- Adenovirus ●
- Coronavirus 229E ●
- Coronavirus HKU1
- Coronavirus NL63
- Coronavirus OC43
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- 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 individuals exhibiting signs and/or symptoms of a respiratory infection aids in the diagnosis of respiratory infection if used in conjunction with other clinical and epidemiological information. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.
Negative results in the setting of a respiratory illness may be due to infection with pathogens that are not detected by this test, or lower respiratory tract infection that may not be detected by a nasopharyngeal swab specimen. Positive results do not rule out co-infection with other organisms: the agent(s) detected by the FilmArray RP2 may not be the definite cause of disease. Additional laboratory testing (e.g. bacterial and viral culture, immunofluorescence, and radiography) may be necessary when evaluating a patient with possible respiratory tract infection.
Due to the genetic similarity between Human Rhinovirus and Enterovirus, the FilmArray RP2 cannot reliably differentiate them. A positive FilmArray RP2 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 (RP2) is designed to simultaneously identify 21 different potential pathogens (see Table 1) of respiratory tract infection from a single NPS specimen in a
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time frame (~45 minutes) that allows the test results to be used in determining appropriate patient treatment and management. FilmArray RP2 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 RP2 pouch module) is used to perform FilmArray RP2 testing on these systems.
| Viral Targets Detected | Bacterial Targets Detected |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------|
| Adenovirus<br>Coronavirus 229E<br>Coronavirus HKU1<br>Coronavirus NL63<br>Coronavirus OC43<br>Human Metapneumovirus<br>Human Rhinovirus/Enterovirus<br>Influenza A, including subtypes<br>H1, H3 and H1-2009<br>Influenza B<br>Parainfluenza Virus 1<br>Parainfluenza Virus 2<br>Parainfluenza Virus 3<br>Parainfluenza Virus 4<br>Respiratory Syncytial Virus | Bordetella parapertussis (IS1001)<br>Bordetella pertussis (ptxP)<br>Chlamydia pneumoniae<br>Mycoplasma pneumoniae |
Table 1. Pathogens Detected by the FilmArray RP2
A test is initiated by loading Hydration Solution into one port of the FilmArray pouch and an NPS sample (in transport media) mixed with the provided Sample Buffer into the other port of the FilmArray RP2 pouch and placing it in a FilmArray instrument. The pouch contains all of 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. The products from first
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stage PCR are then diluted and combined with a fresh, primer-free master mix and a fluorescent double stranded DNA binding dye (LC Green® Plus, BioFire Diagnostics). 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, 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.
## Substantial Equivalence:
The FilmArray Respiratory Panel 2 is substantially equivalent to the FilmArray Respiratory Panel (K160068), which was most recently cleared on February 8, 2016 and determined to be a Class II device.
Table 2 compares the FilmArray Respiratory Panel 2 (RP2) to the FilmArray Respiratory Panel (RP) and outlines the similarities and differences between the two tests.
| Element | New Device:<br>FilmArray Respiratory Panel 2 (RP2) | Predicate:<br>FilmArray Respiratory Panel (RP)<br>(K160068) |
|-----------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------|
| Specimen Types | NPS in VTM | Same |
| Organisms<br>Detected | Adenovirus, Coronavirus 229E, Coronavirus<br>HKU1, Coronavirus NL63, Coronavirus OC43,<br>Human Metapneumovirus, Influenza A,<br>Influenza A subtype H1, Influenza A subtype<br>H3, Influenza A subtype H1-2009, Influenza B,<br>Parainfluenza Virus 1, Parainfluenza Virus 2,<br>Parainfluenza Virus 3, Parainfluenza Virus 4,<br>Human Rhinovirus/Enterovirus, Respiratory<br>Syncytial Virus, Bordetella parapertussis,<br>Bordetella pertussis, Chlamydia pneumoniae,<br>and Mycoplasma pneumoniae | Does not detect Bordetella parapertussis<br>Reports Chlamydia pneumoniae by alternate<br>name: Chlamydophila pneumoniae |
| Analyte | DNA/RNA | Same |
| Technological<br>Principles | Multiplex nucleic acid | Same |
| Instrumentation | FilmArray 2.0 or FilmArray Torch | FilmArray, FilmArray 2.0, or FilmArray Torch |
| Time to result | About 45 minutes | About 1 hour |
| Reagent Storage | Room temperature | Same |
| Test<br>Interpretation | Automated test interpretation and report<br>generation. User cannot access raw data. | Same |
## Table 2. Comparison of the FilmArray Respiratory Panel 2 (RP2) and the FilmArray Respiratory Panel (RP)
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| Controls | Two controls are included in each reagent pouch<br>to control for sample processing and both stages<br>of PCR and melt analysis. | Same |
|-----------------|----------------------------------------------------------------------------------------------------------------------------------|------|
| User Complexity | Moderate/Low | Same |
## Summary of Performance Data
## Clinical Performance
The clinical performance of the FilmArray RP2 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. A total of 1635 residual NPS specimens in viral transport media (VTM) were acquired for the prospective clinical study. Between January and March 2016. specimens were prospectively collected from all comers meeting the study eligibility criteria and immediately frozen (N=695 specimens) for later testing as prospective archived/frozen (Category II) specimens. Between September 2016, specimens were prospectively collected from all comers meeting the study eligibility criteria and tested fresh (N=940 specimens) as prospective fresh (Category I) specimens. Category II specimens were distributed to study sites beginning in September 2016. Study sites also began testing Category I specimens at this time. At each site, Category II specimens were thawed and tested according to the study procedures as time permitted over the remaining duration of the clinical study. A total of 23 prospective specimens (Category I and II specimens) were excluded from the final performance data analysis due to incompliance with the study protocol. The most common reasons for specimen exclusion were that a valid external control was not completed on the day of testing, that specimens were tested outside the 3-day refrigerated storage window, or that the specimen was found to not meet the inclusion criteria after the specimen had been enrolled. The final data set consisted of 1612 prospective specimens. Table 3 provides a summary of demographic information for the 1612 specimens included in the prospective study.
| | | Overall | Site 1 | Site 2 | Site 3 |
|--------|---------------|--------------|--------------------------|--------------|--------------|
| | Male | 867<br>(54%) | 331<br>(57%) | 271<br>(51%) | 265<br>(53%) |
| Sex | Female | 745<br>(46%) | 250<br>(43%) | 256<br>(49%) | 239<br>(47%) |
| Age | < 5 years | 885<br>(55%) | 379<br>(65%) | 170<br>(32%) | 336<br>(67%) |
| | 6 - 21 years | 331<br>(21%) | 132<br>89 (17%)<br>(23%) | | 110<br>(22%) |
| | 22 - 49 years | 128 (8%) | 27 (5%) | 79 (15%) | 22 (4%) |
| | 50+ years | 268<br>(17%) | 43 (7%) | 189<br>(36%) | 36 (7%) |
| | Outpatient | 329<br>(20%) | 77 (13%) | 66 (13%) | 186<br>(37%) |
| Status | Hospitalized | 640<br>(40%) | 229<br>(39%) | 197<br>(37%) | 214<br>(42%) |
| | Emergency | 643<br>(40%) | 275<br>(47%) | 264<br>(50%) | 104<br>(21%) |
| | Total | 1612 | 581 | 527 | 504 |
Table 3. Demographic Summary for Prospective FilmArray RP2 Clinical Evaluation
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The performance of the FilmArray RP2 was evaluated by comparing the FilmArray RP2 test results with those from an FDA-cleared multiplexed respiratory pathogen panel (the main comparator method) as well as with results from two analytically-validated PCR assays followed by bi-directional sequencing for B. parapertussis (this analyte is not detected by the FDA-cleared multiplexed respiratory pathogen panel). The B. parapertussis comparator assays were designed to amplify a different sequence than that amplified by the FilmArray RP2. Any specimen that had bi-directional sequencing data meeting pre-defined quality acceptance criteria that matched organism-specific sequences deposited in the NCBI GenBank database (www.ncbi.nlm.nih.gov) with acceptable E-values was considered Positive. Any specimen that tested negative by both of the comparator assays was considered Negative.
Positive Percent Agreement (PPA) for each analyte was calculated as 100% x (TP / (TP + FN)). True positive (TP) indicates that both the FilmArray RP2 and the comparator method had a positive result for this specific analyte, and false negative (FN) indicates that the FilmArray RP2 result was negative while the comparator result was positive. Negative Percent Agreement (NPA) was calculated as 100% x (TN / (TN + FP)). True negative (TN) indicates that both the FilmArray RP2 and the comparator method had negative results, and a false positive (FP) indicates that the FilmArray RP2 result was positive but the comparator result was negative. The exact binomial two-sided 95% confidence interval was calculated. Samples for which false positive and/or false negative results (i.e., discrepant results) were obtained when comparing the FilmArray RP2 results to the comparator method results were further investigated. The discrepancy investigation was mainly conducted by performing independent molecular methods with primers that are different from that of the FilmArray RP2 and/or comparator method retesting. The prospective clinical study results are summarized in Table 4.
| Analyte | | Positive Percent Agreement | | | Negative Percent Agreement | | |
|-------------------------------|---------|----------------------------|------|-----------|----------------------------|------|-----------|
| | | TP/(TP + FN) | % | 95%CI | TN/(TN + FP) | % | 95%CI |
| Viruses | | | | | | | |
| Adenovirusa | Fresh | 36/38 | 94.7 | 82.7-98.5 | 850/880 | 96.6 | 95.2-97.6 |
| | Frozen | 34/36 | 94.4 | 81.9-98.5 | 640/658 | 97.3 | 95.7-98.3 |
| | Overall | 70/74 | 94.6 | 86.9-97.9 | 1490/1538 | 96.9 | 95.9-97.6 |
| CoV-229Eb | Fresh | 5/5 | 100 | 56.6-100 | 909/913 | 99.6 | 98.9-99.8 |
| | Frozen | 6/7 | 85.7 | 48.7-97.4 | 686/687 | 99.9 | 99.2-100 |
| | Overall | 11/12 | 91.7 | 64.6-98.5 | 1595/1600 | 99.7 | 99.3-99.9 |
| CoV-HKU1c | Fresh | 1/1 | 100 | - | 917/917 | 100 | 99.6-100 |
| | Frozen | 42/42 | 100 | 91.6-100 | 640/652 | 98.2 | 96.8-98.9 |
| | Overall | 43/43 | 100 | 91.8-100 | 1557/1569 | 99.2 | 98.7-99.6 |
| CoV-NL63d | Fresh | 0/0 | - | - | 917/918 | 99.9 | 99.4-100 |
| | Frozen | 40/40 | 100 | 91.2-100 | 645/654 | 98.6 | 97.4-99.3 |
| | Overall | 40/40 | 100 | 91.2-100 | 1562/1572 | 99.4 | 98.8-99.7 |
| CoV-OC43e | Fresh | 11/13 | 84.6 | 57.8-95.7 | 904/905 | 99.9 | 99.4-100 |
| | Frozen | 22/28 | 78.6 | 60.5-89.8 | 662/666 | 99.4 | 98.5-99.8 |
| | Overall | 33/41 | 80.5 | 66.0-89.8 | 1566/1571 | 99.7 | 99.3-99.9 |
| hMPVf | Fresh | 5/5 | 100 | 56.6-100 | 913/913 | 100 | 99.6-100 |
| Analyte | | Positive Percent Agreement | | | Negative Percent Agreement | | |
| | | TP/(TP + FN) | % | 95%CI | TN/(TN + FP) | % | 95%CI |
| | Frozen | 68/70 | 97.1 | 90.2-99.2 | 616/624 | 98.7 | 97.5-99.3 |
| | Overall | 73/75 | 97.3 | 90.8-99.3 | 1529/1537 | 99.5 | 99.0-99.7 |
| | Fresh | 320/328 | 97.6 | 95.3-98.8 | 532/590 | 90.2 | 87.5-92.3 |
| HRV/EVg | Frozen | 105/108 | 97.2 | 92.1-99.1 | 567/586 | 96.8 | 95.0-97.9 |
| | Overall | 425/436 | 97.5 | 95.5-98.6 | 1099/1176 | 93.5 | 91.9-94.7 |
| | Fresh | 3/3 | 100 | 43.9-100 | 915/915 | 100 | 99.6-100 |
| FluAh | Frozen | 75/75 | 100 | 95.1-100 | 616/616 | 100 | 99.4-100 |
| | Overall | 78/78 | 100 | 95.3-100 | 1531/1531 | 100 | 99.7-100 |
| | Fresh | 0/0 | - | - | 918/918 | 100 | 99.6-100 |
| FluA H1 | Frozen | 0/0 | - | - | 691/691 | 100 | 99.4-100 |
| | Overall | 0/0 | - | - | 1609/1609 | 100 | 99.8-100 |
| | Fresh | 0/0 | - | - | 918/918 | 100 | 99.6-100 |
| FluA H1-2009 | Frozen | 74/74 | 100 | 95.1-100 | 617/617 | 100 | 99.4-100 |
| | Overall | 74/74 | 100 | 95.1-100 | 1535/1535 | 100 | 99.8-100 |
| | Fresh | 3/3 | 100 | 43.9-100 | 915/915 | 100 | 99.6-100 |
| FluA H3 | Frozen | 1/1 | 100 | - | 690/690 | 100 | 99.4-100 |
| | Overall | 4/4 | 100 | 51.0-100 | 1605/1605 | 100 | 99.8-100 |
| | Fresh | 0/0 | - | - | 918/918 | 100 | 99.6-100 |
| FluBi | Frozen | 14/14 | 100 | 78.5-100 | 678/680 | 99.7 | 98.9-99.9 |
| | Overall | 14/14 | 100 | 78.5-100 | 1596/1598 | 99.9 | 99.5-100 |
| | Fresh | 0/0 | - | - | 918/918 | 100 | 99.6-100 |
| MERS-CoV | Frozen | 0/0 | - | - | 694/694 | 100 | 99.4-100 |
| | Overall | 0/0 | - | - | 1612/1612 | 100 | 99.8-100 |
| | Fresh | 5/5 | 100 | 56.6-100 | 913/913 | 100 | 99.6-100 |
| PIV1j | Frozen | 4/4 | 100 | 51.0-100 | 689/690 | 99.9 | 99.2-100 |
| | Overall | 9/9 | 100 | 70.1-100 | 1602/1603 | 99.9 | 99.6-100 |
| | Fresh | 46/47 | 97.9 | 88.9-99.6 | 863/871 | 99.1 | 98.2-99.5 |
| PIV2k | Frozen | 0/0 | - | - | 694/694 | 100 | 99.4-100 |
| | Overall | 46/47 | 97.9 | 88.9-99.6 | 1557/1565 | 99.5 | 99.0-99.7 |
| | Fresh | 40/42 | 95.2 | 84.2-98.7 | 867/876 | 99.0 | 98.1-99.5 |
| PIV3l | Frozen | 3/3 | 100 | 43.9-100 | 690/691 | 99.9 | 99.2-100 |
| | Overall | 43/45 | 95.6 | 85.2-98.8 | 1557/1567 | 99.4 | 98.8-99.7 |
| | Fresh | 6/6 | 100 | 61.0-100 | 910/912 | 99.8 | 99.2-99.9 |
| PIV4m | Frozen | 3/3 | 100 | 43.9-100 | 686/691 | 99.3 | 98.3-99.7 |
| | Overall | 9/9 | 100 | 70.1-100 | 1596/1603 | 99.6 | 99.1-99.8 |
| | Fresh | 44/45 | 97.8 | 88.4-99.6 | 867/873 | 99.3 | 98.5-99.7 |
| RSVn | Frozen | 131/131 | 100 | 97.2-100 | 545/563 | 96.8 | 95.0-98.0 |
| | Overall | 175/176 | 99.4 | 96.9-99.9 | 1412/1436 | 98.3 | 97.5-98.9 |
| Bacteria | | | | | | | |
| B. parapertussis<br>(IS1001)o | Fresh | 4/5 | 80.0 | 37.6-96.4 | 913/913 | 100 | 99.6-100 |
| | Frozen | 2/2 | 100 | 34.2-100 | 692/692 | 100 | 99.4-100 |
| | Overall | 6/7 | 85.7 | 48.7-97.4 | 1605/1605 | 100 | 99.8-100 |
| B. pertussis<br>(ptxP)p | Fresh | 2/2 | 100 | 34.2-100 | 915/916 | 99.9 | 99.4-100 |
| | Frozen | 0/1 | 0.0 | - | 693/693 | 100 | 99.4-100 |
| Analyte | | Positive Percent Agreement | | | Negative Percent Agreement | | |
| | | TP/(TP + FN) | % | 95%CI | TN/(TN + FP) | % | 95%CI |
| C. pneumoniaeq | Overall | 2/3 | 66.7 | 20.8-93.9 | 1608/1609 | 99.9 | 99.6-100 |
| | Fresh | 2/2 | 100 | 34.2-100 | 915/916 | 99.9 | 99.4-100 |
| | Frozen | 3/3 | 100 | 43.9-100 | 691/691 | 100 | 99.4-100 |
| | Overall | 5/5 | 100 | 56.6-100 | 1606/1607 | 99.9 | 99.6-100 |
| M. pneumoniaer | Fresh | 17/17 | 100 | 81.6-100 | 897/901 | 99.6 | 98.9-99.8 |
| | Frozen | 6/7 | 85.7 | 48.7-97.4 | 686/687 | 99.9 | 99.2-100 |
| | Overall | 23/24 | 95.8 | 79.8-99.3 | 1583/1588 | 99.7 | 99.3-99.9 |
Table 4. FilmArray RP2 Prospective Clinical Performance Summary
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BioFire Diagnostics 510(k)
FilmArray Respiratory Panel 2 (RP2)
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Adenovirus was detected in 34 FN speciment molecular method. Adenovirus was detected in 38/48 FP specimens using an independent molecular method; an additional two FP specimens were collected from subjects with an acute history of adenovirus infection.
9 The single FN specimen was negative for CoV-229E when tested using an independent molecular method. All five FP specimens were negative for CoV-229E when tested using an independent molecular method.
CoV-HKU1 was detected in 3/12 FP specimens upon comparator method retest.
9 CoV-NL63 was detected in 3/10 FP specimens during discription; two were detected using an independent molecular method and one was detected upon comparator method retest.
° Of the eight FN specimens, six were TP for Col-HKU1. They were confirmed to be due to a known cross-neactivity with CoV-HKU1 by the comparator method; All six specimens were negative for Col-OC43 when tested with two independent PCR assays; the remaining two FN specimens were negative for CN-0043 when tested using an independent molecular method. CoV-0043 was detected in 2/5 FP specimens upon comparator method retest.
Both FN specimens were negative for hMPV when tested using an independent molecular method. MMPV was detected in 6/8 FP specimens during discrepancy investigation; one was detected using an ine were detected upon comparator method retest.
9 HRV/EV was detected in 5/11 FN specimens during discrepation; one was detected using an independent molecular method and four were detected upon FilmArray RP2 retest. HRV/EV was detected in 3377 FP specimens during discrepancy investigation; four were detected using an independent molecular method and 29 were detected upon comparator method retest.
" Three specimens were excluded from influenza A analysis: one with a comparator method result of Influenza A (No Subtype Detected) and two FilmArray RP2 Influenza A (Equivocal) detections.
' FluB was detected in both FP specimens during discripation; one was detected using an independent molecular method and one was detected upon comparator method retest.
1 The single FP specimen was negative for PIV1 when tested using an independent molecular method.
^ The single FN specimen was negative for PV2 when tested using an independent molecular method. PV2 FP specimens during discrepancy investigation; one was detected using an method and four were detected upon comparator method retest.
PV3 was detected in both FN specimens during discrepancy investigation; one was detected using an independent molecular method and one was detected upon FilmArray RP2 retest. PIV3 was detected in 4/10 FP specimens during discrepancy investigation; two were detected using an independent molecular method and two were detected upon comparator method retest.
"PIV4 was detected in 1/7 FP specimens using an independent molecular method.
" The single FN specimen was negative for RSV when tested using an independent molecular method. RSV was detected in 8/24 FP specimens during discrepancy investigation; three were detected using an independent mothod and five were detected upon comparator method retest.
· B. parapertussis was detected in the single FN specimen upon FilmArray RP2 retest.
° B. pertussis was detected in the FN and FP specimens using an independent molecular method.
9 C. pneumoniae was detected in the single FP specimen using an independent molecular method.
[ M. pneumoniae was detected in the single FN specimen upon FilmArray RP2 retest. M. pneumoniae was detected in all five FP specimens during discrepancy investigation: three were detected using an independent mothod and two were detected upon comparator method retest.
FilmArray RP2 reported a total of 245 specimens with discernible multiple organism detections (15.2% of all specimens, 245/1612; and 24.0% of positive specimens, 245/1020; Table 5). The majority of multiple detections (190/245; 77.6%) contained two organisms, while 20.0% (49/245) contained three organisms, 1.6% (4/245) contained four organisms, 0.4% (1/245) contained five organisms, and 0.4% (1/245) contained six organisms. Out of the 245 specimens with multiple detections, 124 specimens (50.6%; 124/245) were concordant with the comparator methods. One hundred twenty-one (121) specimens (49.4%; 121/245) contained one or more organisms that had not been detected by the comparator methods (i.e. false positive results).
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The three organisms that were most prevalent in multiple detections were also the three most prevalent organisms in the study as a whole (i.e. HRV/EV, RSV, and adenovirus). The most prevalent multiple detections (≥5 instances) are shown in Table 6.
| Analyte | Prevalence in Multiple<br>Detections (N=245) | |
|---------------------------|----------------------------------------------|-------|
| Viruses | | |
| Adenovirus | 85 | 34.7% |
| CoV-229E | 6 | 2.4% |
| CoV-HKU1 | 41 | 16.7% |
| CoV-NL63 | 31 | 12.7% |
| CoV-OC43 | 19 | 7.8% |
| hMPV | 33 | 13.5% |
| HRV/EV | 150 | 61.2% |
| FluA H1 | 0 | 0% |
| FluA H1-2009 | 9 | 3.7% |
| FluA H3 | 2 | 0.8% |
| FluB | 6 | 2.4% |
| PIV1 | 5 | 2.0% |
| PIV2 | 15 | 6.1% |
| PIV3 | 21 | 8.6% |
| PIV4 | 12 | 4.9% |
| RSV | 105 | 42.9% |
| Bacteria | | |
| B. parapertussis (IS1001) | 6 | 2.4% |
| B. pertussis (ptxP) | 0 | 0% |
| C. pneumoniae | 1 | 0.4% |
| M. pneumoniae | 7 | 2.9% |
Table 5. Prevalence of Analytes in Multiple Detections as determined by the FilmArray RP2
The most prevalent multiple detection was adenovirus with HRV/EV (1.9% of all specimens; 30/1612) followed by HRV/EV with RSV (1.4% of all specimens; 22/1612); as previously stated these were also the most prevalent organisms detected in the study.
Table 6. Multiple Detection Combinations (≥5 instances) as Determined by the FilmArray RP2
| Distinct Co-Detection Combinations | | | Total<br>Multiple<br>Detections | Number of<br>Specimens with<br>False Positive<br>Results | False Positive Analyte(s)a |
|------------------------------------|-----------|-----------|---------------------------------|----------------------------------------------------------|-----------------------------|
| Analyte 1 | Analyte 2 | Analyte 3 | | | |
| Adenovirus | HRV/EV | | 30 | 15 | Adenovirus (15), HRV/EV (1) |
| HRV/EV | RSV | | 22 | 7 | HRV/EV (3), RSV (4) |
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| CoV-HKU1 | RSV | 13 | 7 | CoV-HKU1 (4), RSV (3) | |
|------------|--------|-----|---|--------------------------|-------------------------------------|
| CoV-NL63 | RSV | 13 | 3 | CoV-NL63 (2), RSV (1) | |
| HRV/EV | PIV2 | 11 | 7 | HRV/EV (6), PIV2 (2) | |
| HRV/EV | PIV3 | 11 | 6 | HRV/EV (3), PIV3 (4) | |
| Adenovirus | RSV | 10 | 5 | Adenovirus (4), RSV (1) | |
| Adenovirus | HRV/EV | RSV | 9 | 5 | Adenovirus (2), HRV/EV (3), RSV (1) |
| CoV-NL63 | HRV/EV | 8 | 2 | CoV-NL63 (2) | |
| CoV-HKU1 | HRV/EV | 5 | 2 | CoV-HKU1 (1), HRV/EV (1) | |
| CoV-OC43 | HRV/EV | 5 | 3 | HRV/EV (3) | |
| hMPV | HRV/EV | 5 | 1 | HRV/EV | |
ª Of the 67 discrepant analytes (out of 293 total analytes), 32 (47.8%) were observed as being present in the specimen during discrepancy investigation; 22/67 (32.8%) were observed using an method and 13/67 (19.4%) were observed upon comparator method retest.
The overall success rate for initial specimen tests in the prospective study was 99.3% (1611/1623) (95% CI: 98.7% - 99.6%); 12 tests were unsuccessful (one due to an incomplete test, one due to an instrument error, and ten due to control failures). Two tests (2/1623; 0.1%) did not complete on the initial run, resulting in an instrument success rate of 99.9% (1621/1623) (95% CI: 99.6% - 100%) for initial specimen tests. Both specimens were able to be retested and valid results were produced after a single retest. Ten tests (10/1621; 0.6%) did not produce valid pouch controls, resulting in a pouch control success rate of 99.4% (1611/1621) (95% CI: 98.9% -99.7%) for completed runs in the initial specimen tests. Nine of the 10 invalid specimens were able to be retested and produced valid control results after a single retest; one was not able to be retested due to insufficient specimen volume.
# Testing of Preselected Archived Specimens
Some of the analytes on the FilmArray RP2 were of low prevalence and were not encountered in large enough 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. These specimens were archived NPS in VTM specimens that were selected because they had previously tested positive for one of the following analytes: coronavirus 229E, influenza A H1, influenza A H3, influenza B, parainfluenza virus 1, parainfluenza virus 4, Bordetella parapertussis, B. pertussis, and Chlamydia pneumoniae. Parainfluenza virus 2, parainfluenza virus 3, and Mycoplasma pneumoniae were also expected to be low prevalence based on BioFire data collected during the 2015-2016 respiratory season, therefore archived testing was performed for these analytes as well and included in the study data (although ultimately they were observed in larger numbers during the prospective clinical study).
A total of 217 preselected archived retrospective clinical specimens were initially received for testing in this retrospective study. Prior to testing with the FilmArray RP2, the composition/integrity of the specimens was first confirmed with confirmatory molecular methods (PCR followed by bi-directional sequencing for B. parapertussis or an FDA-cleared multiplexed respiratory pathogen panel.
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The 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 also included in each group for testing.
The FDA-cleared multiplexed respiratory pathogen panel comparator method was performed on 197 of the 217 preselected archived retrospective clinical specimens only (Group 1). One of the 197 specimens was excluded from performance analysis because of an invalid FilmArray RP2 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 control failure. Valid comparator method and FilmArray RP2 results were obtained for 194 of these 197 archived specimens (Group 1).
The B. parapertussis PCR followed by bi-directional sequencing comparator assays were performed on 20 of the 217 preselected archived retrospective clinical 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 RP2 results were obtained for 20 of these 20 archived specimens.
A summary of the available demographic information of these 214 valid archived specimens is provided in Table 7.
| Total Specimens | 214 | |
|-----------------|---------------|----------|
| Sex | Female (%) | 75 (35%) |
| | Male (%) | 81 (38%) |
| | Unknown | 58 (27%) |
| Age Range | ≤ 5 years | 78 (36%) |
| | 6 - 21 years | 46 (21%) |
| | 22 - 49 years | 13 (6%) |
| | 50+ years | 19 (9%) |
| | Unknown | 58 (27%) |
Table 7. Available Demographic Summary for All Valid Archived Specimens
All Group 1 and Group 2 positive archived specimens (as determined at the source laboratory) that were not confirmed by the respective comparator method were further excluded from the performance calculation for each of the respective analytes.
The FilmArray RP2 retrospective specimens testing performance data against the comparator methods are provided in Table 8 by analyte.
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| | Positive Percent Agreement | | | Negative Percent Agreement | | |
|------------------------------------|----------------------------|------|-----------|----------------------------|------|-----------|
| Analyte | TP/(TP + FN) | % | 95% CI | TN/(TN + FP) | % | 95% CI |
| Viruses | | | | | | |
| Adenovirus | 0/0 | 0 | N/A | 189/194 | 97.4 | 94.1-98.9 |
| CoV- 229Ea | 15/15 | 100 | 79.6-100 | 175/175 | 100 | 97.9-100 |
| CoV-HKU1 | 0/0 | 0 | N/A | 194/194 | 100 | 98.1-100 |
| CoV-NL63 | 2/2 | 100 | 34.2-100 | 192/192 | 100 | 98.0-100 |
| CoV-OC43 | 0/0 | 0 | N/A | 194/194 | 100 | 98.1-100 |
| hMPV | 1/1 | 100 | 20.7-100 | 192/193 | 99.5 | 97.1-99.9 |
| HRV/EV | 18/19 | 94.7 | 75.4-99.1 | 168/175 | 96.0 | 92.0-98.0 |
| Influenza A | 22/22 | 100 | 85.1-100 | 172/172 | 100 | 97.8-100 |
| Influenza A H1 | 3/3 | 100 | 43.9-100 | 191/191 | 100 | 98.0-100 |
| Influenza A 2009-H1 | 1/1 | 100 | 20.7-100 | 193/193 | 100 | 98.0-100 |
| Influenza A H3 | 18/18 | 100 | 82.4-100 | 176/176 | 100 | 97.9-100 |
| Influenza Bb | 16/16 | 100 | 80.6-100 | 177/177 | 100 | 97.9-100 |
| Parainfluenza Virus 1 | 16/16 | 100 | 80.6-100 | 178/178 | 100 | 97.9-100 |
| Parainfluenza Virus 2c | 16/16 | 100 | 80.6-100 | 177/177 | 100 | 97.9-100 |
| Parainfluenza Virus 3 | 17/17 | 100 | 81.6-100 | 175/177 | 98.9 | 96.0-99.7 |
| Parainfluenza Virus 4 | 17/17 | 100 | 81.6-100 | 174/177 | 98.3 | 95.1-99.4 |
| RSV | 2/2 | 100 | 34.2-100 | 191/192 | 99.5 | 97.1-99.9 |
| Bacteria | | | | | | |
| Bordetella parapertussis (IS1001)d | 16/16 | 100 | 80.6-100 | 4/4 | 100 | 51.0-100 |
| Bordetella pertussis (ptxP)e | 25/26 | 96.2 | 81.1-99.3 | 160/162 | 98.8 | 95.6-99.7 |
| Chlamydia pneumoniaef | 17/17 | 100 | 81.6-100 | 176/176 | 100 | 97.9-100 |
| Mycoplasma pneumoniaeg | 16/16 | 100 | 80.6-100 | 171/173 | 98.8 | 95.9-99.7 |
#### Table 8, FilmArray RP2 Archived Specimen Performance Data Summary
ª Four of 19 CoV-229E positive archived specimens by the source laboratory were not confirmed by the comparator method and therefore were excluded from the performance calculation for CoV-229E
* One of the 17 Influenza B positive archived specimens by the source laboratory was not confirmed by the comparator method and therefore was excluded from the performance calculation for Influenza B.
^ One of the 17 Parainfluenza Virus 2 positived specimens the source laboratory was not confirmed by the comparator method and therefore was excluded from the performance calculation for Parainfluenza Virus 2 .
4 The comparator B. parapertussis PCR followed by sequencing assays were performed on 20 achieved specimens only (Group 2). The comparator method for the other analytes was not performed on these 20 specimens.
Six of the 31 B. pertussis positive archived specifical by the source aboratory were not confirmed by the comparator were excluded from the performance calculation for B. pertussis.
f One of the 17 C. pneumoniae positived specimens by the souce laboratory was not confirmed by the comparator method and therefore was excluded from the performance calculation for C. pneumoniae.
s Five of the 21 M. pneumoniae positived specimens by the source laboratory were not confirmed by the comparator method and therefore were excluded from the performance calculation for M. pneumoniae.
#### Testing of Contrived Specimens
Influenza A H1 is of such rarity that both prospective and retrospective archived testing efforts were insufficient to demonstrate system performance. To supplement the prospective and retrospective data, an evaluation of contrived specimens was performed at one of the three clinical testing sites participating in the prospective evaluation. Contrived clinical specimens were prepared using individual unique residual NPS specimens that had previously tested
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negative by the FDA-cleared multiplexed respiratory pathogen panel (i.e., the same test as the comparator method employed in the prospective and retrospective clinical evaluations) at the source laboratory. Spiking was performed using multiple quantified isolates of Influenza A H1. The spiking scheme was such that at least 25 of the contrived positive specimens had analyte concentrations at 2 × the limit of detection (LoD), while the remaining 25 contrived positive specimens were at additional concentrations that spanned the clinically relevant range which was based on FilmArray® RP2 Cp observations of influenza A (A H1, A H-2009, and H3) from the prospective and archived specimen studies. Contrived positive specimens were prepared and randomized along with 50 un-spiked influenza A H1 negative specimens such that the analyte status of each contrived specimen was unknown to the users performing the testing. The results of the FilmArray RP2 testing contrived specimens are presented in Table 9.
| | Positive Percent Agreement | | | | | Negative Percent Agreement | |
|----------------|----------------------------|--------------|-------|-----------|--------------|----------------------------|----------|
| Analyte | × LoD | TP/(TP + FN) | % | 95% CI | TN/(TN + FP) | % | 95% CI |
| Influenza A H1 | 2 | 22/23 a | 95.7% | 79.0-99.2 | 50/50 | 100 | 92.9-100 |
| | 10 | 10/10 | 100% | 72.3-100 | | | |
| | 50 | 5/5 | 100% | 56.6-100 | | | |
| | 200 | 5/5 | 100% | 56.6-100 | | | |
| | 1000 | 5/5 | 100% | 56.6-100 | | | |
| | Combined | 47/48 a | 97.9% | 89.1-99.6 | | | |
#### Table 9. FilmArray RP2 Performance Using Contrived Specimens
4 The FN specimen was spiked with influenza A/Weiss/43; this strain was detected at all other concentrations. Two specimens (also spiked with strain A/Weiss/43) had a result of Influenza A Equivocal and were excluded from Influenza A H1 performance calculation.
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# Selected Analytical Studies
# Limit of Detection
The limit of detection (LoD) for FilmArray RP2 analytes was estimated by testing dilutions of contrived samples containing known concentrations of organisms. Confirmation of LoD was achieved by testing 20 replicates on FilmArray 2.0 and 20 replicates on FilmArray Torch. LoD was confirmed when the organism was detected in at least 19 of the 20 replicates tested (19/20 = 95%). The confirmed LoD for each FilmArray RP2 analyte is listed in Table 10 in viable units. An equivalent DNA or RNA copies/mL was calculated based on an alternate quantitative PCR assay for each analyte. LoD is equivalent when testing on FilmArray 2.0 and FilmArray Torch systems.
| RP2 Analyte | Isolate | LoD<br>Concentration | FilmArray<br>2.0 | FilmArray Torch |
|-----------------------------------|---------------------------------------------------------------------|----------------------------------------|------------------|-----------------|
| | | Viruses | | |
| | Species A, Serotype 18<br>ATCC VR-19 | 5.0E+00 TCID50/mL<br>7.6E+03 copies/mL | 20/20<br>100% | 19/20<br>95% |
| | Species B, Serotype 7A<br>Zeptometrix 0810021CF | 5.0E-02 TCID50/mL<br>3.9E+01 copies/mL | 20/20<br>100% | 20/20<br>100% |
| Adenovirusa | Species C, Serotype 2<br>ATCC VR-846 | 2.0E+00 TCID50/mL<br>3.7E+01 copies/mL | 19/20<br>95% | 20/20<br>100% |
| | Species D, Serotype 37<br>Zeptometrix 0810119CF | 5.0E-02 TCID50/mL<br>9.0E+00 copies/mL | 20/20<br>100% | 20/20<br>100% |
| | Species E, Serotype 4a<br>S. Carolina/2004, UIRF | 1.0E+01 TCID50/mL<br>3.0E+03 copies/mL | 19/20<br>95% | 19/20<br>95% |
| | Species F, Serotype 41<br>Tak, ATCC VR-930 | 1.0E+00 TCID50/mL<br>1.2E+02 copies/mL | 20/20<br>100% | 20/20<br>100% |
| Coronavirus 229E | ATCC VR-740 | 4.0E-01 TCID50/mL<br>6.5E+01 copies/mL | 20/20<br>100% | 20/20<br>100% |
| Coronavirus HKU1 | Clinical specimen | 2.0E+03 RNA copies/mLb | 20/20<br>100% | 20/20<br>100% |
| Coronavirus NL63 | BEI NR-470 | 2.5E-01 TCID50/mL<br>5.4E+01 copies/mL | 20/20<br>100% | 20/20<br>100% |
| Coronavirus OC43 | ATCC VR-759 | 3.0E+01 TCID50/mL<br>5.6E+02 copies/mL | 19/20<br>95% | 20/20<br>100% |
| Human Metapneumovirus | 16, Type A1 IA10-2003<br>Zeptometrix 0810161CF | 1.0E+01 TCID50/mL<br>1.2E+03 copies/mL | 20/20<br>100% | 20/20<br>100% |
| Human Rhinovirus/<br>Enterovirusa | Human Rhinovirus<br>Type 1A<br>Zeptometrix 0810012CFN | 1.0E-01 TCID50/mL<br>8E+01 copies/mL | 20/20<br>100% | 20/20<br>100% |
| | Enterovirus D68<br>ATCC VR-1823 | 3.0E+02 TCID50/mL<br>2.6E+01 copies/mL | 20/20<br>100% | 20/20<br>100% |
| Influenza A H1 | Influenza A H1N1<br>A/New Caledonia/20/99<br>Zeptometrix 0810036CF | 1.0E+03 TCID50/mL<br>1.4E+02 copies/mL | 20/20<br>100% | 20/20<br>100% |
| Influenza A H1-2009 | Influenza A H1N1 pdm<br>A/Swine/NY/03/2009<br>Zeptometrix 0810249CF | 5.0E-01 TCID50/mL<br>3.3E+02 copies/mL | 20/20<br>100% | 20/20<br>100% |
| Influenza A H3 | Influenza H3N2<br>A/Port Chalmers/1/73<br>ATCC VR-810 | 1.0E-01 TCID50/mL<br>2.1E+01 copies/mL | 20/20<br>100% | 20/20<br>100% |
| Influenza B | B/FL/04/06<br>Zeptometrix 0810255CF | 5.0E+00 TCID50/mL<br>3.4E+01 copies/mL | 20/20<br>100% | 20/20<br>100% |
#### Table 10. Summary of Limit of Detection (LoD) for FilmArray RP2 Analytes
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| RP2 Analyte | Isolate | LoD<br>Concentration | FilmArray<br>2.0 | FilmArray Torch |
|-----------------------------------------------|----------------------------------|--------------------------------------------------------|------------------|-----------------|
| Parainfluenza Virus 1 | Type 1<br>Zeptometrix 0810014CF | $5.0E+00$ TCID50/mL<br>$1.0E+03$ copies/mL | 20/20<br>100% | 20/20<br>100% |
| Parainfluenza Virus 2 | Type 2<br>Zeptometrix 0810015CF | $5.0E-01$ TCID50/mL<br>$3.0E+01$ copies/mL | 20/20<br>100% | 20/20<br>100% |
| Parainfluenza Virus 3 | Type 3<br>Zeptometrix 0810016CF | $2.5E+00$ TCID50/mL<br>$3.8E+01$ copies/mL | 19/20<br>95% | 20/20<br>100% |
| Parainfluenza Virus 4 | Type 4a<br>Zeptometrix 0810060CF | $5.0E+01$ TCID50/mL<br>$1.6E+03$ copies/mL | 20/20<br>100% | 20/20<br>100% |
| Respiratory Syncytial Virus | Type A<br>Zeptometrix 0810040ACF | $2.0E-02$ TCID50/mL<br>$9.0E+00$ copies/mL | 19/20<br>95% | 20/20<br>100% |
| Bacteria | | | | |
| Bordetella parapertussis<br>( <i>IS1001</i> ) | A747<br>Zeptometrix 0801461 | $4.1E+01$ CFU/mL<br>$6.0E+01$ <i>IS1001</i> copies/mLc | 20/20<br>100% | 19/20<br>95% |
| Bordetella pertussis (ptxP) | A639<br>Zeptometrix 0801459 | $1.0E+03$ CFU/mL | 20/20<br>100% | 20/20<br>100% |
| Chlamydia pneumoniae | TW183<br>ATCC VR-2282 | $1.0E-01$ TCID50/mL<br>$6.6E+01$ copies/mL | 20/20<br>100% | 19/20<br>95% |
| Mycoplasma pneumoniae | M129<br>Zeptometrix 0801579 | $1.0E+01$ TCID50/mL<br>$4.6E+02$ copies/mLd | 20/20<br>100% | 20/20<br>100% |
"The differences in LoD concentration between Adenovines species (5.0E-02 - 1.0E-01 TCD-J/mL) or between Human Rhinovirus (1.0E-01 - 3.0E+02TCID=ymL) may not indicate actual different species or serotypes, but ather the variability of the TCD-measurement method.
" A cultured isolate of Coronavirus HKU1 was not available for testing. LoD for Coronavirus HKU1 was theremined by testing dilutions of a clinical NPS specimen known to contain the virus. The anount of viral RNA copies/mL) was determined by real-ime RT-PCR against a standard curve.
* IS/001 sequences can be present in more than one copy per cell, so the relationship between CFU/mL and copies/mL may vary from strain and culture to culture. LoD was determined based on the copy number of IS 001 measured by an independent quantitative real-ime PCR assay and for this culture.
The copy number of Mycoplasma pneumoniae was determined by a multicopy assay (16S rRNA) and may not accurately reflect the number of copies detected by the single-copy FilmArray RP2 gene target.
Note: LoD concentrations of the cultured viruses and obligate intracelleular bacteria (C. pneumoniae and M. pneumoniae) are provided in units of TCID-6 (50% Tissue Culture Infectious Dose). TCID50 is an indirect measure of viral or bacterial concentration based on infectivity and cytotoxicity and will therefore vary considerably depending on technique and methodology (including cell type, culture media and conditions, cytotoxicity of the virus, etc.). It is not appropriate to make determinations on relative sensitivity of different molecular assays for detection of viruses and bacteria based on LoD values measured in TCID50/mL
#### Inclusivity
Analytical reactivity (inclusivity) was evaluated with a collection of 177 isolates that represent temporal and geographic diversity of FilmArray RP2 analytes, including relevant species, strains, serotypes, or genotypes. All isolates tested were detected by the FilmArray RP2 at concentrations within 10× LoD. When possible, in silico analysis of sequence data was used to make predictions of assay reactivity for less common strains or serotypes that were not tested but that may be detected by the FilmArray RP2 (Table 11 - Table 22).
{20}------------------------------------------------
RP2 influenza A assays will react variably with non-human influenza A viruses and rarely encountered human influenza A viruses that are not H1, H1-2009 or H3; generally producing Influenza A Equivocal or Influenza A (no subtype detected) results.
Note: FilmArray RP2 Influenza A (subtype) and Influenza B assays are predicted to react with attenuated viruses used in vaccines.
| Speciesa | Serotype | Isolate ID/Source | [Strain/Location/Year] | xLoD<br>Dectected | Result |
|----------|----------|----------------------------------------|------------------------------------|-------------------|------------------------|
| A | 12 | ATCC VR-863 | [Huie/Massachusetts] | 3x | |
| A | 18 | ATCC VR-19 | [Washington DC/1954] | 1x | |
| A | 31 | Zeptometrix 0810073CF | - | 3x | |
| A | 3 | Zeptometrix 0810062CF | - | 3x | |
| A | 7A | Zeptometrix 0810021CF | - | 1x | |
| B | 7d/d2 | Univ of Iowa Research Foundation | [Iowa/2001] | 3x | |
| B | 7h | Univ of Iowa Research Foundation | [Iowa/1999] | 3x | |
| B | 11 | Univ of Iowa Research Foundation | [Wisconsin/2005] | 3x | |
| B | 14 | Univ of Iowa Research Foundation | [Missouri/2005] | 3x | |
| B | 16 | ATCC VR-17 | [CH.79/Saudia Arabia/1955] | 3x | |
| B | 21 | Univ of Iowa Research Foundation | [Missouri/2005] | 3x | |
| B | 34 | ATCC VR-716 | [Compton/1972] | 3x | |
| B | 35 | ATCC VR-718 | [Holden] | 3x | |
| B | 50 | ATCC VR-1602 | [Wan/Amsterdam/1988] | 3x | Adenovirus<br>Detected |
| C | 1 | Zeptometrix 0810050CF | - | 3x | |
| C | 2 | ATCC VR-846 | [Adenoid 6] | 1x | |
| C | 5 | Zeptometrix 0810020CF | - | 3x | |
| C | 6 | ATCC VR-6 | [Tonsil 99/Washington DC] | 3x | |
| D | 8 | Zeptometrix 0810069CF | - | 3x | |
| D | 20 | Zeptometrix 0810115CF | - | 3x | |
| D | 37 | Zeptometrix 0810119CF | - | 1x | |
| E | 4a | Univ of Iowa Research Foundation | [S Carolina/2004] | 1x | |
| E | 4 | Zeptometrix 0810070CF | - | 3x | |
| E | 40 | Zeptometrix 0810084CF<br>NCPV 0101141v | - | 3x | |
| F | 41 | ATCC VR-930 | [Tak/73-<br>3544/Netherlands/1973] | 1x | |
| F | 41 | Zeptometrix 0810085CF | - | 3x | |
Table 11. Adenovirus Isolates Tested and Detected by FilmArray RP2
ª In silco analysis of available sequences predicts that the FilmArray RP2 will also react with Adenovirus B55, C57, species D serotypes, and G52.
| Table 12. Coronavirus Isolates/Specimens Tested and Detected by FilmArray RP2 |
|------------…
