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 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 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.

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 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.

AI/ML Overview

Here's a breakdown of the requested information about the FilmArray® Respiratory Panel 2 (RP2) based on the provided text:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria for each analyte are implicitly demonstrated by the Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) values meeting a high standard, generally interpreted as near 100% agreement. While explicit numerical acceptance targets are not stated (e.g., "PPA must be >95%"), the reported performance in both prospective and archived studies consistently shows high agreement. For brevity, I've listed a representative sample of analytes.

Analyte (Category)	Acceptance Criteria (Implied)	Reported Performance (Overall Prospective Clinical Study)	Reported Performance (Archived Specimen Study)
Viruses
Adenovirus	High PPA and NPA	PPA: 94.6% (70/74), NPA: 96.9% (1490/1538)	NPA: 97.4% (189/194) (No PPA data for archived)
Coronavirus 229E	High PPA and NPA	PPA: 91.7% (11/12), NPA: 99.7% (1595/1600)	PPA: 100% (15/15), NPA: 100% (175/175)
Coronavirus HKU1	High PPA and NPA	PPA: 100% (43/43), NPA: 99.2% (1557/1569)	NPA: 100% (194/194) (No PPA data for archived)
Human Metapneumovirus (hMPV)	High PPA and NPA	PPA: 97.3% (73/75), NPA: 99.5% (1529/1537)	PPA: 100% (1/1), NPA: 99.5% (192/193)
Human Rhinovirus/Enterovirus	High PPA and NPA	PPA: 97.5% (425/436), NPA: 93.5% (1099/1176)	PPA: 94.7% (18/19), NPA: 96.0% (168/175)
Influenza A	High PPA and NPA	PPA: 100% (78/78), NPA: 100% (1531/1531)	PPA: 100% (22/22), NPA: 100% (172/172)
RSV	High PPA and NPA	PPA: 99.4% (175/176), NPA: 98.3% (1412/1436)	PPA: 100% (2/2), NPA: 99.5% (191/192)
Bacteria
Bordetella parapertussis	High PPA and NPA	PPA: 85.7% (6/7), NPA: 100% (1605/1605)	PPA: 100% (16/16), NPA: 100% (4/4)
Bordetella pertussis	High PPA and NPA	PPA: 66.7% (2/3), NPA: 99.9% (1608/1609)	PPA: 96.2% (25/26), NPA: 98.8% (160/162)
Chlamydia pneumoniae	High PPA and NPA	PPA: 100% (5/5), NPA: 99.9% (1606/1607)	PPA: 100% (17/17), NPA: 100% (176/176)
Mycoplasma pneumoniae	High PPA and NPA	PPA: 95.8% (23/24), NPA: 99.7% (1583/1588)	PPA: 100% (16/16), NPA: 98.8% (171/173)

2. Sample Size Used for the Test Set and Data Provenance

Prospective Clinical Study:
- Sample Size: 1612 valid nasopharyngeal swab (NPS) specimens.
- Data Provenance: Obtained from individuals suspected of respiratory tract infections at three geographically distinct U.S. study sites during portions of the 2015-2016 and 2016-2017 respiratory illness seasons. Specimens were both prospectively collected and immediately tested ("fresh," N=940) and prospectively collected and frozen for later testing ("archived/frozen," N=695, with a total of 1635 acquired, 23 excluded, leaving 1612 in the final dataset).
Archived Specimens Study:
- Sample Size: 214 valid preselected archived retrospective clinical specimens. These were chosen to supplement low-prevalence analytes from the prospective study.
- Data Provenance: Archived NPS in VTM specimens that had previously tested positive for specific analytes (Coronavirus 229E, Influenza A H1, H3, Influenza B, Parainfluenza Virus 1, 4, Bordetella parapertussis, B. pertussis, Chlamydia pneumoniae, Parainfluenza Virus 2, 3, Mycoplasma pneumoniae). These were sourced from a BioFire internal collection.
Contrived Specimens Study:
- Sample Size: 50 contrived positive specimens (spiked with Influenza A H1 at various concentrations) and 50 un-spiked negative specimens, totaling 100 specimens.
- Data Provenance: Prepared using individual unique residual NPS specimens that had previously tested negative by the FDA-cleared multiplexed respiratory pathogen panel at the source laboratory. Tested at one of the clinical testing sites participating in the prospective evaluation.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Their Qualifications

The document does not explicitly state the number of experts or their specific qualifications for establishing the initial ground truth. However, the ground truth was established by:

Primary Comparator: An FDA-cleared multiplexed respiratory pathogen panel. This implies that the results from this predicate device served as the primary reference, which itself would have undergone rigorous validation.
Secondary Comparator (for B. parapertussis): Two analytically-validated PCR assays followed by bi-directional sequencing. This is a highly specific and expert-level molecular technique.
Discrepancy Resolution: Discrepancies were investigated by performing independent molecular methods with primers different from the FilmArray RP2 and/or comparator method retesting. This suggests review and confirmation by skilled laboratory personnel, capable of performing and interpreting advanced molecular diagnostics.

4. Adjudication Method

The adjudication method described for the prospective clinical study's discrepant results was:

"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."
For B. parapertussis, the bi-directional sequencing data "meeting pre-defined quality acceptance criteria that matched organism-specific sequences deposited in the NCBI GenBank database" was considered Positive.

This indicates a multi-methodological approach to resolve conflicting results between the investigational device and the primary comparator, rather than a purely expert consensus reading without additional testing.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

No MRMC comparative effectiveness study was mentioned or performed. This device is an in vitro diagnostic (IVD) assay designed for qualitative detection of nucleic acids, not an imaging device requiring human interpretation, therefore a human-in-the-loop study with AI assistance is not applicable. The device provides automated results.

6. Standalone Performance (Algorithm Only without Human-in-the-Loop Performance)

Yes, standalone performance was done. The entire clinical and analytical performance evaluation describes the algorithm's (FilmArray RP2 test's) direct performance against comparator methods and ground truth. The device is intended for "simultaneous qualitative detection and identification" which is an automated process without human interpretation of raw 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."

7. Type of Ground Truth Used

The ground truth for the test sets was primarily established using a combination of:

FDA-cleared multiplexed respiratory pathogen panel: For the majority of analytes in both prospective and archived studies.
Analytically-validated PCR assays followed by bi-directional sequencing: Specifically for Bordetella parapertussis.
Independent molecular methods and/or retesting: For discrepancy resolution.
Known concentrations of organisms: For analytical studies like Limit of Detection and Inclusivity.

This represents a high standard of ground truth, relying on established and highly sensitive molecular diagnostic techniques.

8. Sample Size for the Training Set

The document describes a clinical study evaluating the device's performance, but it does not mention a separate "training set" for the algorithm's development. This is typical for IVD submissions where the device's analytical and clinical performance is validated rather than its algorithm being developed and trained on a dataset. The development of the assays and their internal thresholds would have occurred during the R&D phase prior to these validation studies.

9. How the Ground Truth for the Training Set Was Established

As no specific "training set" for the algorithm was mentioned in the context of this submission, the method for establishing its ground truth is not provided. The performance data presented are for the validation of the already-developed device.

Summary

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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)

· 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

Type of Use (Select one or both, as applicable)
Prescription Use (Part 21 CFR 801 Subpart D)	Over-The-Counter Use (21 CFR 801 Subpart C)

CONTINUE ON A SEPARATE PAGE IF NEEDED.

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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:

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 ●

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
AdenovirusCoronavirus 229ECoronavirus HKU1Coronavirus NL63Coronavirus OC43Human MetapneumovirusHuman Rhinovirus/EnterovirusInfluenza A, including subtypesH1, H3 and H1-2009Influenza BParainfluenza Virus 1Parainfluenza Virus 2Parainfluenza Virus 3Parainfluenza Virus 4Respiratory Syncytial Virus	Bordetella parapertussis (IS1001)Bordetella pertussis (ptxP)Chlamydia pneumoniaeMycoplasma pneumoniae

Table 1. Pathogens Detected by the FilmArray RP2

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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.

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:FilmArray Respiratory Panel 2 (RP2)	Predicate:FilmArray Respiratory Panel (RP)(K160068)
Specimen Types	NPS in VTM	Same
OrganismsDetected	Adenovirus, Coronavirus 229E, CoronavirusHKU1, Coronavirus NL63, Coronavirus OC43,Human Metapneumovirus, Influenza A,Influenza A subtype H1, Influenza A subtypeH3, Influenza A subtype H1-2009, Influenza B,Parainfluenza Virus 1, Parainfluenza Virus 2,Parainfluenza Virus 3, Parainfluenza Virus 4,Human Rhinovirus/Enterovirus, RespiratorySyncytial Virus, Bordetella parapertussis,Bordetella pertussis, Chlamydia pneumoniae,and Mycoplasma pneumoniae	Does not detect Bordetella parapertussisReports Chlamydia pneumoniae by alternatename: Chlamydophila pneumoniae
Analyte	DNA/RNA	Same
TechnologicalPrinciples	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
TestInterpretation	Automated test interpretation and reportgeneration. 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 pouchto control for sample processing and both stagesof 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(54%)	331(57%)	271(51%)	265(53%)
Sex	Female	745(46%)	250(43%)	256(49%)	239(47%)
Age	< 5 years	885(55%)	379(65%)	170(32%)	336(67%)
	6 - 21 years	331(21%)	13289 (17%)(23%)		110(22%)
	22 - 49 years	128 (8%)	27 (5%)	79 (15%)	22 (4%)
	50+ years	268(17%)	43 (7%)	189(36%)	36 (7%)
	Outpatient	329(20%)	77 (13%)	66 (13%)	186(37%)
Status	Hospitalized	640(40%)	229(39%)	197(37%)	214(42%)
	Emergency	643(40%)	275(47%)	264(50%)	104(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(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(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 MultipleDetections (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			TotalMultipleDetections	Number ofSpecimens withFalse PositiveResults	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	LoDConcentration	FilmArray2.0	FilmArray Torch
		Viruses
	Species A, Serotype 18ATCC VR-19	5.0E+00 TCID50/mL7.6E+03 copies/mL	20/20100%	19/2095%
	Species B, Serotype 7AZeptometrix 0810021CF	5.0E-02 TCID50/mL3.9E+01 copies/mL	20/20100%	20/20100%
Adenovirusa	Species C, Serotype 2ATCC VR-846	2.0E+00 TCID50/mL3.7E+01 copies/mL	19/2095%	20/20100%
	Species D, Serotype 37Zeptometrix 0810119CF	5.0E-02 TCID50/mL9.0E+00 copies/mL	20/20100%	20/20100%
	Species E, Serotype 4aS. Carolina/2004, UIRF	1.0E+01 TCID50/mL3.0E+03 copies/mL	19/2095%	19/2095%
	Species F, Serotype 41Tak, ATCC VR-930	1.0E+00 TCID50/mL1.2E+02 copies/mL	20/20100%	20/20100%
Coronavirus 229E	ATCC VR-740	4.0E-01 TCID50/mL6.5E+01 copies/mL	20/20100%	20/20100%
Coronavirus HKU1	Clinical specimen	2.0E+03 RNA copies/mLb	20/20100%	20/20100%
Coronavirus NL63	BEI NR-470	2.5E-01 TCID50/mL5.4E+01 copies/mL	20/20100%	20/20100%
Coronavirus OC43	ATCC VR-759	3.0E+01 TCID50/mL5.6E+02 copies/mL	19/2095%	20/20100%
Human Metapneumovirus	16, Type A1 IA10-2003Zeptometrix 0810161CF	1.0E+01 TCID50/mL1.2E+03 copies/mL	20/20100%	20/20100%
Human Rhinovirus/Enterovirusa	Human RhinovirusType 1AZeptometrix 0810012CFN	1.0E-01 TCID50/mL8E+01 copies/mL	20/20100%	20/20100%
	Enterovirus D68ATCC VR-1823	3.0E+02 TCID50/mL2.6E+01 copies/mL	20/20100%	20/20100%
Influenza A H1	Influenza A H1N1A/New Caledonia/20/99Zeptometrix 0810036CF	1.0E+03 TCID50/mL1.4E+02 copies/mL	20/20100%	20/20100%
Influenza A H1-2009	Influenza A H1N1 pdmA/Swine/NY/03/2009Zeptometrix 0810249CF	5.0E-01 TCID50/mL3.3E+02 copies/mL	20/20100%	20/20100%
Influenza A H3	Influenza H3N2A/Port Chalmers/1/73ATCC VR-810	1.0E-01 TCID50/mL2.1E+01 copies/mL	20/20100%	20/20100%
Influenza B	B/FL/04/06Zeptometrix 0810255CF	5.0E+00 TCID50/mL3.4E+01 copies/mL	20/20100%	20/20100%

Table 10. Summary of Limit of Detection (LoD) for FilmArray RP2 Analytes

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RP2 Analyte	Isolate	LoDConcentration	FilmArray2.0	FilmArray Torch
Parainfluenza Virus 1	Type 1Zeptometrix 0810014CF	$5.0E+00$ TCID50/mL$1.0E+03$ copies/mL	20/20100%	20/20100%
Parainfluenza Virus 2	Type 2Zeptometrix 0810015CF	$5.0E-01$ TCID50/mL$3.0E+01$ copies/mL	20/20100%	20/20100%
Parainfluenza Virus 3	Type 3Zeptometrix 0810016CF	$2.5E+00$ TCID50/mL$3.8E+01$ copies/mL	19/2095%	20/20100%
Parainfluenza Virus 4	Type 4aZeptometrix 0810060CF	$5.0E+01$ TCID50/mL$1.6E+03$ copies/mL	20/20100%	20/20100%
Respiratory Syncytial Virus	Type AZeptometrix 0810040ACF	$2.0E-02$ TCID50/mL$9.0E+00$ copies/mL	19/2095%	20/20100%
Bacteria
Bordetella parapertussis( IS1001 )	A747Zeptometrix 0801461	$4.1E+01$ CFU/mL$6.0E+01$ IS1001 copies/mLc	20/20100%	19/2095%
Bordetella pertussis (ptxP)	A639Zeptometrix 0801459	$1.0E+03$ CFU/mL	20/20100%	20/20100%
Chlamydia pneumoniae	TW183ATCC VR-2282	$1.0E-01$ TCID50/mL$6.6E+01$ copies/mL	20/20100%	19/2095%
Mycoplasma pneumoniae	M129Zeptometrix 0801579	$1.0E+01$ TCID50/mL$4.6E+02$ copies/mLd	20/20100%	20/20100%

"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).

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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]	xLoDDectected	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	AdenovirusDetected
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 0810084CFNCPV 0101141v	-	3x
F	41	ATCC VR-930	[Tak/73-3544/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
-------------------------------------------------------------------------------

CoronavirusType	Isolate ID/Source	[Location/Year]	xLoDDectected	Result
229E	ATCC VR-740	-	1x	Coronavirus 229E
229E	Zeptometrix 0810229CF	-	3x	Coronavirus 229E
HKU1	Clinical Specimen	[Utah/2015]	1x
HKU1	Clinical Specimen	[Utah/2015]	3x
HKU1	Clinical Specimen	[Utah/2015]	3x	Coronavirus HKU1
HKU1	Clinical Specimen	[S. Carolina/2010]	3x	Coronavirus HKU1
HKU1	Clinical Specimen	[Detroit/2010]	3x	Coronavirus HKU1
NL63	BEI NR-470a	[Amsterdam/2003]	1x	Coronavirus NL63
NL63	Zeptometrix 0810228CF	-	3x	Coronavirus NL63
OC43	ATCC VR-759b	-	1x	Coronavirus OC43
OC43	Zeptometrix 0810024CF	-	3x	Coronavirus OC43

ª Organism obtained through the NIH Biodefense and Emerging Infections Research Resources Repository, NIAID, NIH: Human Coronavirus NL63, NR-470.

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b Discontinued part number; see ATCC VR-1558.

Genotype	Serotype	Isolate ID/Source	[Location/Year]	xLoDDetected	Result
A1	16	Zeptometrix 0810161CF	[Iowa10/2003]	1x	HumanMetapneumovirus
A1	9	Zeptometrix 0810160CF	[Iowa3/2002]	3x
A2	20	Zeptometrix 0810163CF	[Iowa14/2003]	3x
A2	27	Zeptometrix 0810164CF	[Iowa27/2004]	3x
B1	3	Zeptometrix 0810156CF	[Peru2/2002]	3x
B1	5	Zeptometrix 0810158CF	[Peru3/2003]	3x
B1	13	Univ of Iowa Research Foundation	[Iowa7/2003]	3x
B2	4	Zeptometrix 0810157CF	[Peru1/2002]	3x
	8	Zeptometrix 0810159CF	[Peru6/2003]	3x
	18	Zeptometrix 0810162CF	[Iowa18/2003]	3x
	22	Univ of Iowa Research Foundation	[Iowa16/2003]	3x

Table 13. Human Metapneumovirus Isolates Tested and Detected by FilmArray RP2

Table 14. Human Rhinovirus and Enterovirus Isolates Tested and Detected by FilmArray RP2

Species	Serotype	Isolate ID/Source	[Strain/Location/Year]	xLODDetected	Result
Human Rhinovirus
	1	Zeptometrix 0810012CFN	[1A]	1x
	2	ATCC VR-482	[HGP]	3x
	7	ATCC VR-1601	[68-CV11]	3x
A	16	ATCC VR-283	[11757/WashingtonDC/1960]	3x
	34	ATCC VR-507ª	[137-3]	3x
	57	ATCC VR-1600	[Ch47]	3x	HumanRhinovirus/Enterovirus
	77	ATCC VR-1187	[130-63]	3x
	85	ATCC VR-1195	[50-525-CV54]	3x
	3	ATCC VR-483	[FEB]	3x
	14	ATCC VR-284	[1059/S Carolina/1959]	3x
B	17	ATCC VR-1663	[33342/N Carolina/1959]	3x
	27	ATCC VR-1137	[5870]	3x
	42	ATCC VR-338	[56822]	3x
	83	ATCC VR-1193	[Baylor 7]	3x
Enterovirus
A	Coxsackievirus 10	ATCC VR-168	[NY/1950]	3x
	Enterovirus 71	ATCC VR-1432	[H]	3x
	Coxsackievirus A9	Zeptometrix 0810017CF	-	3x	HumanRhinovirus/Enterovirus
	Coxsackievirus B3	Zeptometrix 0810074CF	-	3x
	Coxsackievirus B4	Zeptometrix 0810075CF	-	3x
B	Echovirus 6	Zeptometrix 0810076CF	-	3x
	Echovirus 9	Zeptometrix 0810077CF	-	3x
	Echovirus 11	Zeptometrix 0810023CF	-	3x
C	Coxsackievirus A21	ATCC VR-850	[Kuykendall/California/1952]	3x
C	Coxsackievirus A24	ATCC VR-583	[DN-19/Texas/1963]	3x
D	68	ATCC VR-1823	[US/MO/2014-18947]	1x
Type	Isolate ID/Source	[Strain/Location/Year]	xLoDDetected	Result
	Zeptometrix 0810036CF	[New Caledonia/20/1999]	1x
H1N1	Human	ATCC VR-219	[NWS/1933]	3x	Influenza AH1
		ATCC VR-95	[PR/8/1934]	10xa
		ATCC VR-96	[Weiss/1943]	3x
		ATCC VR-97	[FM/1/1947]	3x
		ATCC VR-98	[Mal/302/1954]	3x
		ATCC VR-546	[Denver/1/1957]	3x
	Swine	Zeptometrix 0810036CFN	[Solomon Isl/03/2006]	3x
		Zeptometrix 0810244CF	[Brisbane/59/2007]	3x
		ATCC VR-333	[A/Swine/Iowa/15/1930]	3x
	Swine	ATCC VR-99	[A/Swine/1976/1931]	3x
		ATCC VR-897	[A/New Jersey/8/76 (Hsw1N1)]	10xa
H1N2	Recombinant	BEI NR-9677b	[Kilbourne F63, A/NWS/1934 (HA) xA/Rockefeller Institute/5/1957 (NA)]	3x
H1N1pdm09	Human	Zeptometrix 0810249CFN	[SwineNY/03/2009]	1x	Influenza AH1-2009
		Zeptometrix 0810248CFN	[SwineNY/01/2009]	3x
		Zeptometrix 0810109CFN	[SwineNY/02/2009]	3x
		Zeptometrix 0810109CFJ	[Canada/6294/2009]	3x
		Zeptometrix 0810165CF	[California/07/2009]	3x
		Zeptometrix 0810166CF	[Mexico/4108/2009]	3x
		BEI NR-19823c	[Netherlands/2629/2009]	3x
		BEI NR-44345d	[Hong Kong/H090-761-V1(0)/2009]	10xe
		BEI NR-42938f	[Georgia/F32551/2012]	3x
	Human	ATCC VR-810	[Port Chalmers/1/1973]	1x
		ATCC VR-776	[Alice (live attenuated vaccine)]	3x
		Zeptometrix 0810238CF	[Texas/50/2012]	3x
		ATCC VR-547	[Aichi/2/1968]	3x	Influenza AH3
H3N2g		ATCC VR-544	[Hong Kong/8/1968]	3x
		ATCC VR-822	[Victoria/3/1975]	3x
		Zeptometrix 0810252CF	[Wisconsin/67/2005]	3x
		Zeptometrix 0810138CF	[Brisbane/10/2007]	3x
	Recombinant	ATCC VR-777	[MCR2(A/England/42/72xA/PR8/34)]	3x
H3N2v	Human	Clinical Specimen	[Ohio/2012]	3x
H2N2	Human	BEI NR-2775h	[Japan/305/1957]	10xe	Influenza A(no subtypedetected)
	Recombinant	BEI NR-9679i	[Korea/426/1968xPuerto Rico/8/1934 ]	10xe	Influenza A(no subtypedetected)
H2N3		MRI Globalj	[Mallard/Alberta/79/2003]	3x	Influenza AEquivocal
H5N1	Avian	MRI Globalj	[A/Chicken/Yunnan/1251/2003]	3x
H5N2		MRI Globalj	[A/Northernpintail/Washington/40964/2014]	3x	Influenza A
H5N3		BEI NR-9682k	[A/Duck/Singapore/645/1997]	3x	(no subtypedetected)
H5N8		MRI Globalj	[A/Gryfalcon/Washington/41088-6/2014]	3x
H7N7		MRI Globalj	[A/Netherlands/219/2003]	3x
H7N9		MRI Globalj	[A/Anhui/01/2013]	3x
H10N7		BEI NR-2765l	[Chicken/Germany/N/49]	3x	Influenza AEquivocal
Lineage	Isolate ID/Source	[Strain/Location/Year]	xLoDDetected	Result
N/A	ATCC VR-101	[Lee/1940]	3x
	ATCC VR-102	[Allen/1945]	3x
	ATCC VR-103	[GL/1739/1954]	3x
	ATCC VR-296	[1/Maryland/1959]	3x
	ATCC VR-295	[2/Taiwan/1962]	3x
	ATCC VR-786	[Brigit/Russia/1969]	3x
Victoria	ATCC VR-823	[5/Hong Kong/1972]	3x	Influenza B
	Zeptometrix 0810258CF	[2506/Malaysia/2004]	3x
	CDC 2005743348	[1/Ohio/2005]	3x
Yamagata	Zeptometrix 0810256CF	[07/Florida/2004]	3x
	Zeptometrix 0810255CF	[04/Florida/2006]	1x
	Zeptometrix 0810241CF	[1/Wisconsin/2010]	3x
	Zeptometrix 0810239CF	[2/Massachusetts/2012]	3x

ª Discontinued part number; see ATCC VR-1365.

Table 15. Influenza A Isolates Tested and Detected by FilmArray RP2

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4 Reported as Influenza A (no subtype detected) at 3× LoD.

b Genomic RNA obtained through the NIH Biodefense and Emerging Infections Resources Respiratory NAID, NIH Kilbourne F63: A/NWS/1934 (HA) x A/Rockefeller Institute/5/1957 (NA) (H1N2), Reassortant NWS-F, NR-9677.

& Organism obtained through BEI Resources, NIAID, NIH: Influenza A Virus, A/Netherlands/2629/2009 (H1N1)pdm09, NR-19823.

4 Organism obtained through BEI Resources, NIAID, NIH: Influenza A Virus, A/Hong Kong/H090-761-V (0)/2009 (H1N1)pdm09, NR-44345. e Reported as Influenza A Equivocal or Influenza A (no subtype detected) at 3× LoD.

f Organism obtained through BEI Resources, NIAID, NIH: Influenza A Virus, A/Georgia/F32551/2012 (HIN1)pdm09, NR-42938.

8 Human isolates of recent swine variant H3N2v) were not available for testing, but reactivity near LoD is predicted by sequence analysis.

4 Genomic RNA obtained through the NIH Biodefense and Emerging Infections Resources Repository, NIAID, NIH: Genomic RNA from Influenza A Virus, A/Japan/305/1957 (H2N2), NR-2775.

{23}------------------------------------------------

1 Genomic RNA obtained through the NIH Biodefense and Emerging Infections Resources Repository, NIAID, NIH: Genomic RNA from Kilbourne F38: A/Korea/426/1968 (HA, NA) x A/Puerto Rico/8/1934 (H2N2), NR-9679.

J Isolate provided and tested by MRI Global, Kansas City, MO.

Genomic RNA obtained through the NIH Biodefense and Emerging Infections Research Resources Repository NIAID, NIH: Genomic RNA from Kilbourne F181: A/duck/Singapore/645/1997 (H5N3), Wild Type, NR-9682.

1 Genomic RNA obtained through the NIH Biodefense and Emerging Infections Resources Repository NIAID, NIH: Genomic RNA from Influenza A Virus, A/chicken/Germany/N/1949 (H10N7), NR-2765

Table 16. Influenza B Isolates Tested and Detected by FilmArray RP2

Table 17. Parainfluenza Virus Isolates Tested and Detected by FilmArray RP2

Type	Subtype	Isolate ID/Source	[Strain/Location/Year]	xLoDDetected	Result
1		Zeptometrix 0810014CF	-	1x	Parainfluenza Virus1
		ATCC VR-94	[C-35/Washington DC/1957]	3x
		BEI NR-3226a	[C39]	3x
		BEI NR-48680b	[FRA/29221106/2009]	3x
2		Zeptometrix 0810015CF	-	1x	Parainfluenza Virus2
		ATCC VR-92	[Greer/Ohio/1955]	3x
3		Zeptometrix 0810016CF	-	1x	Parainfluenza Virus3
		ATCC VR-93	[C-243/Washington DC/1957]	3x
		BEI NR-3233c	[NIH 47885, Wash/47885/57]	3x
4	A	Zeptometrix 0810060CF	-	1x	Parainfluenza Virus4
		ATCC VR-1378	[M-25/1958]	3x
	B	Zeptometrix 0810060BCF	-	3x
		ATCC VR-1377	[CH-19503/WashingtonDC/1962]	3x

ª Discontinued part number.

Obtained through BEI Resources, NIAID, NIH: Human Parainfluenza Virus 1, HPV 1/FRA/29221106/2009, NR-48680.

º Obtained through BEI Resources, NIAID, NIH: Human Parainfluenza Virus 3, NIH 47885, NR-3233.

	Table 18. Respiratory Syncytial Virus Isolates Tested and Detected by FilmArray RP2

Type	Source	[Strain/Location/Year]	xLoDDetected	Result
A	Zeptometrix 0810040ACF	[2006]	1x
A	ATCC VR-26	[Long/Maryland/1956]	3x
A	ATCC VR-1540	[A2/Melbourne/1961]	3x	RespiratorySyncytial Virus
B	Zeptometrix 0810040CF	[Ch-93 (18)-18]	3x
B	ATCC VR-1400	[WV/14617/1985]	3x
B	ATCC VR-955	[9320/Massachusetts/1977]	3x
B	ATCC VR-1580	[18537/Washington DC/1962]	10x

{24}------------------------------------------------

Species	Source	[Strain/Location/Year]	xLoDDetected	Result
Bordetella parapertussis	Zeptometrix 0801461	[A747]	1x
Bordetella parapertussis	Zeptometrix 0801462	[E595]	3x
Bordetella parapertussis	ATCC 15237	[NCTC 10853]	3x	Bordetellaparapertussis(IS1001)
Bordetella parapertussis	ATCC 15311	[NCTC 5952]	3x	Bordetellaparapertussis(IS1001)
Bordetella parapertussis	ATCC BAA-587	[12822/Germany/1993]	3x	Bordetellaparapertussis(IS1001)
Bordetella bronchiseptica(containing IS1001)	NRRL B-59909	[MBORD849/Pig/Netherlands]	3x

Table 19. Bordetella parapertussis (and Bordetella bronchiseptica) Isolates Tested and Detected by FilmArray RP2

2 Readivity with IS1001 sequences in B. bronchiseptica reactivity of the assay, however the analyte will be inacurately reported as B. parapertussis. The assay does not react with IS 1001-like sequences in B. holmesil (see Analytical Reactivity).

Table 20. Bordetella pertussis Isolates Tested and Detected by FilmArray RP2

Isolate ID/Source	[Strain]	xLoD Detected	Result
Zeptometrix 0801459	[A639]	1x
Zeptometrix 0801460	[E431]	3x
ATCC 8467	[F]	3x
ATCC 9340	[5,17921]	3x
ATCC 9797	[18323/NCTC 10739]	3x	Bordetella
ATCC 10380	[10-536]	3x	pertussis (ptxP)
ATCC 51445	[CNCTC Hp 12/63,623]	3x
ATCC BAA-589	[Tohama]	3x
ATCC BAA-1335	[MN2531]	3x

Table 21. Chlamydia pneumoniae Isolates Tested and Detected by FilmArray RP2

Isolate ID/Source	[Strain/Location/Year]	xLoD Detected	Result
ATCC VR-2282	[TW-183/Taiwan/1965]	1x	Chlamydiapneumoniae
ATCC VR-1310	[CWL-029]	3x
ATCC VR-1360	[CM-1/Georgia]	3x
ATCC 53592	[AR-39/Seattle/1983]	3x

	Table 22. Mycoplasma pneumoniae Isolates Tested and Detected by FilmArray RP2
--	--------------------------------------------------------------------------------------	--	--

Type	Isolate ID/Source	[Strain]	xLoD Detected	Result
1	Zeptometrix 0801579	[M129]	1x	Mycoplasmapneumoniae
	ATCC 29342	[M129-B7]	3x
	ATCC 29085	[PI 1428]	3x
2	ATCC 15531	[FH strain of Eaton Agent [NCTC 10119]	3x
	ATCC 15492	[Mac]	3x
unknown	ATCC 15293	[M52]	3x
	ATCC 15377	[Bru]	3x
	ATCC 39505	[Mutant 22]	3x
		ATCC 49894	[UTMB-10P]	3x

Exclusivity

The potential for non-specific amplification and detection by the FilmArray RP2 assays was evaluated in silico by sequence analysis and testing of high concentrations of organisms in contrived samples. A total of 22 on-panel organisms were tested to assess the potential for intrapanel cross-reactivity and 50 off-panel organisms were tested to evaluate panel specificity. Offpanel organisms included normal respiratory flora and pathogens that may be present in NPS

{25}------------------------------------------------

specimens as well as near-neighbors or species genetically related to the organisms detected by the FilmArray RP2. The on-panel and off-panel organisms tested are shown in Table 23.

The final concentration of analyte in the sample (typically at least 1×106 CFU/mL for bacteria and fungi and at least 1×10 TCID<0/mL for viruses) represented levels ~70 - 400,000 fold higher than the LoD of the FilmArray RP2 assays. Some potential for intra-panel cross-reactivity with Bordetella species and Influenza A subtypes of swine origin was predicted by sequence analysis and observed when tested at high concentrations, as summarized in Table 24.

	On-Panel Viruses		On-Panel Bacteria
Adenovirus	Human Rhinovirus	Influenza B	Bordetella parapertussis
Coronavirus 229 E	Human Metapneumovirus	Parainfluenza Virus 1	Bordetella pertussis
Coronavirus HKU1ª	Influenza A H1N1	Parainfluenza Virus 2	Chlamydia (Chlamydophila)pneumoniae
Coronavirus NL63	Influenza A H3N2	Parainfluenza Virus 3	Mycoplasma pneumoniae
Coronavirus OC43	Influenza A H1N1pdm09	Parainfluenza Virus 4
Enterovirus (Echovirus 6)	Influenza A Hsw1N1	Respiratory Syncytial Virus
	Off-Panel Bacteria		Off-Panel Viruses
Acinetobacter calcoaceticus	Enterobacter aerogenes	Neisseria gonorrhoeae	Bocavirus
Bordetella avium	Escherichia coli	Neisseria meningitidis	Cytomegalovirus (CMV)
Bordetella bronchiseptica	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	Middle East RespiratorySyndrome Coronavirusb
Legionella feeleii	Moraxella catarrhalis	Stenotrophomonas maltophilia
Legionella longbeacheae	Mycoplasma genitalium	Streptococcus pneumoniae	Off-Panel 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 diphtheriae	Neisseria elongata	Ureaplasma urealyticum	Aspergillus flavus

Table 23. Organisms Tested for Evaluation of FilmArray RP2 Analytical Specificity Organisms with the potential for non-specific amplification by a FilmArray RP2 assay are shown in bold.

a Two different clinical specimens containing up to 8.9×10° RNA copies/mL of Coronavirus HKU1.

b Heat-inactivated viral culture obtained through BEI Resources, NIAID, NIH: Middle East Respiratory Syndrome Coronavirus (MERS-CoV), EMC/2012, Heat-Inactivated, NR-50171.

{26}------------------------------------------------

Cross-reactive Organism(s)	FilmArrayRP2 Result	Description
Non-pertussis Bordetella species(e.g., Bordetella parapertussisBordetella bronchisepticaa)	Bordetella pertussis (ptxP) b,c	The Bordetella pertussis (ptxP) assay can amplifypertussis toxin pseudogene sequences in B.bronchiseptica and B. parapertussis primarilywhen present at high concentration ((≥1.2E+09CFU/mL).
Bordetella bronchisepticaa(with IS1001 sequences)	Bordetella parapertussis (IS1001)	Some strains of B. bronchiseptica carry IS1001insertion sequences identical to those carried byB. parapertussis. These sequences will beefficiently amplified by the IS1001 assay andreported by FilmArray RP2 as Bordetellaparapertussis (IS1001).
Bordetella pertussisBordetella parapertussisBordetella bronchiseptica	Human Rhinovirus/Enterovirusd,e	The Human Rhinovirus/Enterovirus assay mayamplify off-target sequences found in strains of B.pertussis, B. bronchiseptica, and B. parapertussiswhen present at high concentration. Cross-reactivitywith B. pertussis was observed at a concentration of4.5E+07 CFU/mL or higher.
Influenza A H1N1(swine origin)	Influenza A H1-2009f	The Influenza A H1-2009 assay may react withH1 hemagglutinin gene sequences from viruses ofswine origin.FilmArray RP2 will report either Influenza A H1or Influenza A H1-2009, depending on the strainand concentration in the samplef.

Table 24. Predicted and Observed Cross-Reactivity of the FilmArray RP2

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).

6 Cross-reactivity between the Bordetella pertussis (ptxP) assay and B. parapertussis will be reported as a co-detection (Bordetella parapertussis (IS/001) Detected and Bordetella pertussis (pt.rP) Detected); while cross-reactivity with most strains of B. bronchissprice (that do not carry IS/00/) 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.

6 Cross-reactivity between the Human Rhinovirus assays and B. pertussis will be reported as a cedection (Bordetella pertussis (pt.P) Detected and Human Rhinovirus/Enterovins Detected parapertussis (15/00/) Detected and Human Rhinovirus Detected); while cross-reactivity with most strains of B. bronchiseptical (that do not carry IS/100) will be reported (falsely) only as Human Rhinovirus/Enterovirus Detected.

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

Interference

Potentially interfering substances that could be present in NPS specimens or introduced during specimen collection and testing were evaluated for their effect on FilmArray RP2 performance. Substances included endogenous substances that may be found in specimens at normal or elevated levels (e.g. blood, mucus/mucin, human genomic DNA), various commensal or infectious microorganisms, medications, washes or topical applications for the nasal passage, various swabs and transport media for specimen collection, and substances used to clean, decontaminate, or disinfect work areas.

Each substance was added to contrived samples containing representative organisms at concentrations near (2-3×) LoD. The concentration of substance added to the samples (Table 25) was equal to or greater than the highest level expected to be in NPS specimens.

None of the substances were shown to interfere with the FilmArray RP2 function. However, it was observed that exposure of samples to bleach prior to testing could damage the

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organisms/nucleic acids in the sample, leading to inaccurate FilmArray RP2 test results (lack of analyte detection). The effect of bleach was dependent on the concentration and/or length of time the bleach was allowed to interact with the sample.

Substance Tested	Concentration Tested	Result
Endogenous Substances
Human Whole Blood	10% v/v	No Interference
Human Mucus (Sputum)	1 swab/mL sample	No Interference
Human Genomic DNA	20 ng/µL	No Interference
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	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	1.2E+06 TCID50/mL	No Interference
Exogenous Substancesa
Tobramycin (systemic antibiotic)	0.6 mg/mL	No Interference
Mupirocin(active ingredient in anti-bacterial ointment)	2% w/v	No Interference
Saline Nasal Spray with Preservatives(0.65% NaCl, Phenylcarbinol, Benzalkonium chloride)	1% v/v	No Interference
Nasal Decongestant Spray(Oxymetazoline HCl 0.05%, Benzalkonium chloride, phosphate)	1% v/v	No Interference
Analgesic ointment (Vicks®VapoRub®)	1% w/v	No Interference
Petroleum Jelly (Vaseline®)	1% w/v	No Interference
Snuff (Tobacco)	1% w/v	No Interference
Disinfecting/Cleaning Substances
Bleach	1% and 2% v/v[up to 1024 ppm chlorine]	Interferenceb
Disinfecting wipes (ammonium chloride)	½ in²	No Interference
Ethanol	7% v/v	No Interference
DNAZap (Ambion™ AM9891G & AM9892G)	1% v/v	No Interference
RNaseZap (Ambion™ AM9782)	1% v/v	No Interference
Specimen Collection Materials
Rayon Swabs (Copan 168C)	N/A	No Interference
Nylon Flocked Swabs (Copan 553C)	N/A	No Interference
Polyester Swabs (Copan 175KS01)	N/A	No Interference
Calcium Alginate Swabs (Puritan 25-801 A 50)	N/A	No Interference
Substance Tested	Concentration Tested	Result
M4® Transport Medium (Remel)	100%	No Interference
M4-RT® Transport Medium (Remel)	100%	No Interference
M5® Transport Medium (Remel)	100%	No Interference
M6™ Transport Medium (Remel)	100%	No Interference
Universal Viral Transport vial (BD)	100%	No Interference
Sigma-Virocult™ Viral Collection and Transport System(Swab and Transport Medium)	100%	No Interference
Copan ESwab™ Sample Collection and Delivery System(Swab and Liquid Amies Medium)	100%	No Interference

Table 25. Evaluation of Potentially Interfering Substances on the FilmArray RP2

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4 Nasal influenza vaccines (e.g. FluMis) were not evaluated to be reactive with the FilmArray RP2 Influenza A (subtype) and Influenza B assays.

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

Reproducibility

Reproducibility testing of contrived samples was performed at three test sites on a combination of FilmArray 2.0 and FilmArray Torch systems. The study incorporated a range of potential variation introduced by site, day, operator (at least two per site), system, instrument or Torch module (at least three per site/sample), and pouch lot (at least three). The samples contained various combinations of twelve different FilmArray RP2 analytes, each at three different concentrations (Negative, Low Positive (1×LoD), and Moderate Positive (3×LoD)). Frozen samples were repeatedly tested on five different days for 120 data points per sample (60 per system) over 480 total valid runs.

A summary of results (percent (%) agreement with the expected Detected or Not Detected result) for each analyte (by site and system) is provided in Table 26.

			Agreement with Expected Result
Analyte	ConcentrationTested	ExpectedResult	FilmArray TorchSite A	FilmArray TorchSite C	FilmArray TorchSystem Sub-Total	FilmArray 2.0Site B	FilmArray 2.0Site C	FilmArray 2.0SystemSub-Total	All Sites/Systems(95% CI)
			Viruses
AdenovirusC2ATCC VR-846	Moderate Positive3× LoD6.0E+00 TCID50/mL(1.1E+02 copies/mL)	Detected	30/30100%	29/3096.7%	59/6098.3%	29/3096.7%	30/30100%	59/6098.3%	118/12098.3%(94.1%-99.8%)
AdenovirusC2ATCC VR-846	Low Positive1× LoD2.1E+00 TCID50/mL(3.7E+01 copies/mL)	Detected	30/30100%	30/30100%	60/60100%	30/30100%	29/3096.7%	59/6098.3%	119/12099.2%(95.4%-100%)
AdenovirusC2ATCC VR-846	None(no analyte)	NotDetected	60/60100%	60/60100%	120/120100%	60/60100%	60/60100%	120/120100%	240/240100%(98.5%-100%)
Coronavirus 229E	None(no analyte)	NotDetected	120/120100%	120/120100%	240/240100%	120/120100%	120/120100%	240/240100%	480/480100%(99.2%-100%)
Coronavirus HKU1	None(no analyte)	NotDetected	120/120100%	120/120100%	240/240100%	120/120100%	120/120100%	240/240100%	480/480100%(99.2%-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
Coronavirus OC43ATCC VR-759	Moderate Positive3x LoD9.0E+01 TCID50/mL(1.7E+03 copies/mL)	Detected	29/3096.7%	29/3096.7%	58/6096.7%	29/3096.7%	30/30100%	59/6098.3%	117/12097.5%(92.9%-99.5%)
	Low Positive1x LoD3.0E+01 TCID50/mL(5.6E+02 copies/mL)	Detected	30/30100%	30/30100%	60/60100%	30/30100%	27/3090.0%	57/6095.0%	117/12097.5%(92.9%-99.5%)
	None(no analyte)	NotDetected	60/60100%	60/60100%	120/120100%	60/60100%	60/60100%	120/120100%	240/240100%(98.5%-100%)
Coronavirus NL63	None(no analyte)	NotDetected	120/120100%	120/120100%	240/240100%	120/120100%	120/120100%	240/240100%	480/480100%(99.2%-100%)
HumanMetapneumovirusType 16, A1IA10-2003Zeptometrix0810161CF	Moderate Positive3x LoD3.0E+01 TCID50/mL(3.6E+03 copies/mL)	Detected	30/30100%	30/30100%	60/60100%	30/30100%	30/30100%	60/60100%	120/120100%(97.0%-100%)
	Low Positive1x LoD1.0E+01 TCID50/mL(1.2E+03 copies/mL)	Detected	30/30100%	30/30100%	60/60100%	28/3093.3%	30/30100%	58/6096.7%	118/12098.3%(94.1%-99.8%)
	None(no analyte)	NotDetected	60/60100%	60/60100%	120/120100%	60/60100%	60/60100%	120/120100%	240/240100%(98.5%-100%)
HumanRhinovirus/EnterovirusRhinovirus 1AZeptometrix0810012CFN	Moderate Positive3x LoD3.0E-01 TCID50/mL(1.1E+02 copies/mL)	Detected	30/30100%	30/30100%	60/60100%	28/3093.3%	30/30100%	58/6096.7%	118/12098.3%(94.1%-99.8%)
	Low Positive1x LoD1.0E-01 TCID50/mL(3.8E+01 copies/mL)	Detected	30/30100%	30/30100%	60/60100%	30/30100%	30/30100%	60/60100%	120/120100%(97.0%-100%)
	None(no analyte)	NotDetected	60/60100%	60/60100%	120/120100%	60/60100%	60/60100%	120/120100%	240/240100%(98.5%-100%)
Influenza A H3Influenza A H3N2A/PortChalmers/1/73ATCC VR-810	Moderate Positive3x LoD3.0E-01 TCID50/mL(6.3E+01 copies/mL)	Detected	30/30100%	30/30100%	60/60100%	29/3096.7%	30/30100%	59/6098.3%	119/12099.2%(95.4%-100%)
	Low Positive1x LoD1.0E-01 TCID50/mL(2.1E+01 copies/mL)	Detected	30/30100%	30/30100%	60/60100%	30/30100%	30/30100%	60/60100%	120/120100%(97.0%-100%)
	None(no analyte)	NotDetected	60/60100%	60/60100%	120/120100%	60/60100%	60/60100%	120/120100%	240/240100%(98.5%-100%)
Influenza AH1-2009	None(no analyte)	NotDetected	120/120100%	120/120100%	240/240100%	120/120100%	120/120100%	240/240100%	480/480100%(99.2%-100%)
		ExpectedResult	Agreement with Expected Result						All Sites/Systems(95% CI)
Analyte	ConcentrationTested		FilmArray Torch			FilmArray 2.0
			Site A	Site C	System Sub-Total	Site B	Site C	SystemSub-Total
Influenza A H1	None(no analyte)	NotDetected	120/120100%	120/120100%	240/240100%	120/120100%	120/120100%	240/240100%	480/480100%(99.2%-100%)
Influenza BB/FL/04/06Zeptometrix0810255CF	Moderate Positive3x LoD1.5E+01 TCID50/mL(1.0E+02 copies/mL)	Detected	30/30100%	30/30100%	60/60100%	30/30100%	30/30100%	60/60100%	120/120100%(97.0%-100%)
	Low Positive1x LoD5.0E+00 TCID50/mL(3.4E+01 copies/mL)	Detected	30/30100%	30/30100%	60/60100%	30/30100%	30/30100%	60/60100%	120/120100%(97.0%-100%)
	None(no analyte)	NotDetected	60/60100%	60/60100%	120/120100%	60/60100%	60/60100%	120/120100%	240/240100%(98.5%-100%)
Parainfluenza Virus1	None(no analyte)	NotDetected	120/120100%	120/120100%	240/240100%	120/120100%	120/120100%	240/240100%	480/480100%(99.2%-100%)
Parainfluenza Virus2Type 2Zeptometrix0810015CF	Moderate Positive3x LoD1.5E+00 TCID50/mL(9.0E+01 copies/mL)	Detected	30/30100%	29/3096.7%	59/6098.3%	29/3096.7%	30/30100%	59/6098.3%	118/12098.3%(94.1%-99.8%)
	Low Positive1x LoD5.0E-01 TCID50/mL(3.0E+01 copies/mL)	Detected	30/30100%	29/3096.7%	59/6098.3%	30/30100%	27/3090.0%	57/6095.0%	116/12096.7%(91.7%-99.1%)
	None(no analyte)	NotDetected	60/60100%	60/60100%	120/120100%	60/60100%	60/60100%	120/120100%	240/240100%(98.5%-100%)
Parainfluenza Virus3	None(no analyte)	NotDetected	120/120100%	120/120100%	240/240100%	120/120100%	120/120100%	240/240100%	480/480100%(99.2%-100%)
Parainfluenza Virus4Type 4aZeptometrix0810060CF	Moderate Positive3x LoD1.5E+02 TCID50/mL(4.8E+03 copies/mL)	Detected	30/30100%	30/30100%	60/60100%	30/30100%	30/30100%	60/60100%	120/120100%(97.0%-100%)
	Low Positive1x LoD5.0E+01 TCID50/mL(1.6E+03 copies/mL)	Detected	30/30100%	29/3096.7%	59/6098.3%	29/3096.7%	30/30100%	59/6098.3%	118/12098.3%(94.1%-99.8%)
	None(no analyte)	NotDetected	60/60100%	60/60100%	120/120100%	60/60100%	60/60100%	120/120100%	240/240100%(98.5%-100%)
RespiratorySyncytial VirusType AZeptometrix0810040ACF	Moderate Positive3x LoD6.0E-02 TCID50/mL(2.7E+01 copies/mL)	Detected	30/30100%	30/30100%	60/60100%	30/30100%	30/30100%	60/60100%	120/120100%(97.0%-100%)
	Low Positive1x LoD2.0E-02 TCID50/mL(9.0E+00 copies/mL)	Detected	29/3096.7%	30/30100%	59/6098.3%	30/30100%	29/3096.7%	59/6098.3%	118/12098.3%(94.1%-99.8%)
Analyte	ConcentrationTested	ExpectedResult	FilmArray Torch			FilmArray 2.0			All Sites/Systems(95% CI)
			Site A	Site C	System Sub-Total	Site B	Site C	SystemSub-Total
None(no analyte)	None(no analyte)	NotDetected	60/60100%	60/60100%	120/120100%	60/60100%	60/60100%	120/120100%	240/240100%(98.5%-100%)
Bacteria
Bordetellaparapertussis(IS1001)A747Zeptometrix0801461	Moderate Positive3x LoD1.8E+02 IS1001copies/mL(1.2E+02 CFU/mL)	Detected	30/30100%	30/30100%	60/60100%	29/3096.7%	30/30100%	59/6098.3%	119/12099.2%(95.4%-100%)
	Low Positive1x LoD6.0E+01 IS1001copies/mL (4.1E+01CFU/mL)	Detected	24/30a80.0%	29/3096.7%	53/60188.3%	29/3096.7%	30/30100%	59/6098.3%	112/12093.3%(87.3%-97.1%)
	None(no analyte)	NotDetected	60/60100%	60/60100%	120/120100%	60/60100%	60/60100%	120/120100%	240/240100%(98.5%-100%)
Bordetella pertussis(ptxP)A639Zeptometrix0801459	Moderate Positive3x LoD3.0E+03 CFU/mL	Detected	30/30100%	30/30100%	60/60100%	30/30100%	30/30100%	60/60100%	120/120100%(97.0%-100%)
	Low Positive1x LoD1.0E+03 CFU/mL	Detected	28/3093.3%	30/30100%	58/6096.7%	30/30100%	30/30100%	60/60100%	118/12098.3%(94.1%-99.8%)
	None(no analyte)	NotDetected	60/60100%	60/60100%	120/120100%	60/60100%	60/60100%	120/120100%	240/240100%(98.5%-100%)
Chlamydia(Chlamydophila)pneumoniaeTW183ATCC VR-2282	Moderate Positive3x LoD3.0E-01 TCID50/mL(2.0E+02 copies/mL)	Detected	30/30100%	30/30100%	60/60100%	30/30100%	30/30100%	60/60100%	120/120100%(97.0%-100%)
	Low Positive1x LoD1.0E-01 TCID50/mL(6.6E+01 copies/ml)	Detected	28/3093.3%	30/30100%	58/6096.7%	29/3096.7%	30/30100%	59/6098.3%	117/12097.5%(92.9%-99.5%)
	None(no analyte)	NotDetected	60/60100%	60/60100%	120/120100%	60/60100%	60/60100%	120/120100%	240/240100%(98.5%-100%)
Mycoplasmapneumoniae	None(no analyte)	NotDetected	120/120100%	120/120100%	240/240100%	120/120100%	120/120100%	240/240100%	480/480100%(99.2%-100%)
Overall Agreement with the Expected ResultAll Analytes and All Test Levels(95% Confidence Interval)			9,562/9,60099.6%(99.5% - 99.7%)

Table 26. Reproducibility of FilmArray RP2 Results on FilmArray Torch and FilmArray 2.0 Systems

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Data from Site A were further reviewed by the uniables including test day, Toch 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 increatent variance in precision of the FilmArray RP2 Bordeella parapertusis (IS1001) results.

The reproducibility (standard deviation) of melting temperature (Tm) for the amplification products generated by the FilmArray RP2 was also evaluated, with a Tm standard deviation for

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each assay of ± 0.5℃ or less observed within and between the FilmArray 2.0 and FilmArray Torch systems (Table 27).

		Site A		Site C		Site B		Site C		All Sites/Systems
Analyte	Assay	Mean	StDev	Mean	StDev	Mean	StDev	Mean	StDev	Mean	StDev
Controls	yeastRNA	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 C2	Adeno2	89.0	± 0.2	88.8	± 0.1	88.8	± 0.3	88.7	± 0.2	88.8	± 0.2
(ATCC VR-846)	Adeno6	89.6	± 0.2	89.4	± 0.2	89.4	± 0.3	89.2	± 0.2	89.4	± 0.3
Coronavirus OC43(ATCC VR-759)	CoV-OC43-2	80.7	± 0.2	80.6	± 0.1	80.6	± 0.3	80.5	± 0.2	80.6	± 0.2
Human Metapneumovirus(Zeptometrix 0810161CF)	hMPV	78.2	± 0.3	78.0	± 0.2	78.0	± 0.3	77.8	± 0.2	78.0	± 0.3
Rhinovirus(Zeptometrix 0810012CFN)	HRV/EV	84.3	± 0.2	84.2	± 0.2	84.3	± 0.3	84.1	± 0.2	84.2	± 0.2
Influenza A H3N2	FluA-pan1	84.2	± 0.2	84.0	± 0.1	84.0	± 0.3	83.8	± 0.2	84.0	± 0.2
(ATCC VR-810)	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(Zeptometrix 0810255CF)	FluB	80.4	± 0.3	80.3	± 0.2	80.4	± 0.2	80.2	± 0.2	80.3	± 0.2
Parainfluenza virus 2(Zeptometrix 0810015CF)	PIV2	83.2	± 0.2	83.1	± 0.2	83.2	± 0.2	83.0	± 0.2	83.1	± 0.2
Parainfluenza virus 4(Zeptometrix 0810060CF)	PIV4	77.1	± 0.2	77.0	± 0.3	77.2	± 0.3	77.0	± 0.2	77.1	± 0.3
Respiratory Syncytial Virus(Zeptometrix 0810040ACF)	RSV	81.2	± 0.2	81.1	± 0.2	81.1	± 0.2	81.0	± 0.2	81.1	± 0.2
BACTERIA
Bordetella parapertussis(Zeptometrix 0801461)	IS 1001	87.7	± 0.2	87.6	± 0.2	87.6	± 0.3	87.5	± 0.2	87.6	± 0.2
Bordetella pertussis(Zeptometrix 0801459)	ptxP	88.6	± 0.2	88.5	± 0.2	88.5	± 0.3	88.2	± 0.2	88.4	± 0.3
Chlamydia pneumoniae(ATCC VR-2282)	Cpne	79.6	± 0.3	79.5	± 0.2	79.5	± 0.3	79.3	± 0.2	79.5	± 0.3

Table 27. Reproducibility of Tm (°C) For Select FilmArray Torch and FilmArray Torch and FilmArray 2.0 Systems Mean Tm values are calculated from a combination of Tm values obtained at the 3x LoD and 1x LoD concentrations.

4 Calculated from results at the 3× LoD and 1× LoD test concentrations combined.

Regulation Number and Section

§ 866.3980 Respiratory viral panel multiplex nucleic acid assay.

(a)
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.
(b)
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.