FilmArray Respiratory Panel 2 (RP2)

{0} # 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY A. 510(k) Number: K170604 B. Purpose for Submission: New device C. Measurand: Adenovirus, Human Metapneumovirus (hMPV), Influenza A (Flu A), Influenza A subtype H1 (Flu A H1), Influenza A subtype H1- 2009 (Flu A H1-2009), Influenza A subtype H3 (Flu A H3), Influenza B (Flu B), Coronavirus 229E (CoV-229E), Coronavirus HKU1 (CoV-HKU1), Coronavirus NL63 (CoV-NL63), Coronavirus OC43 (CoV-OC43), Parainfluenza virus 1 (PIV 1), Parainfluenza virus 2 (PIV 2), Parainfluenza virus 3 (PIV 3), Parainfluenza virus 4 (PIV 4), Human Rhinovirus/Enterovirus (HRV/HEV), Respiratory Syncytial Virus (RSV), Bordetella parapertussis (IS1001), Bordetella pertussis (ptxP), Chlamydia pneumoniae, and Mycoplasma pneumoniae nucleic acids target sequences. D. Type of Test: A multiplexed nucleic acid test intended for use with the FilmArray® 2.0 or FilmArray® Torch systems for the simultaneous qualitative in vitro detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (NPS) collected in viral transport media and obtained from individuals suspected of respiratory tract infections. E. Applicant: BioFire Diagnostics, LLC F. Proprietary and Established Names: FilmArray Respiratory Panel 2 (RP2) Common Name: FilmArray RP2 G. Regulatory Information: | Product Code | Classification | Regulation Section | Panel | | --- | --- | --- | --- | | OCC | Class II | 21 CFR 866.3980 Respiratory Viral Panel Multiplex Nucleic Acid Assay | Microbiology (83) | | OEM | Class II | 21 CFR 866.3980 Respiratory Viral Panel Multiplex Nucleic Acid Assay | Microbiology (83) | 1 {1} | OEP | Class II | 21 CFR 866.3980 Respiratory Viral Panel Multiplex Nucleic Acid Assay | Microbiology (83) | | --- | --- | --- | --- | | OOU | Class II | 21 CFR 866.3980 Respiratory Viral Panel Multiplex Nucleic Acid Assay | Microbiology (83) | | OTG | Class II | 21 CFR 866.3980 Respiratory Viral Panel Multiplex Nucleic Acid Assay | Microbiology (83) | | OZE | Class II | 21 CFR 866.3980 Respiratory Viral Panel Multiplex Nucleic Acid Assay | Microbiology (83) | | OZX | Class II | 21 CFR 866.3980 Respiratory Viral Panel Multiplex Nucleic Acid Assay | Microbiology (83) | | OZY | Class II | 21 CFR 866.3980 Respiratory Viral Panel Multiplex Nucleic Acid Assay | Microbiology (83) | | OZZ | Class II | 21 CFR 866.3980 Respiratory Viral Panel Multiplex Nucleic Acid Assay | Microbiology (83) | | OOI | Class II | 21 CFR 862.2570 Instrumentation for Clinical Multiplex Test Systems | Clinical Chemistry (75) | | NSU | Class II | 21 CFR 862.2570 Instrumentation for Clinical Multiplex Test Systems | Clinical Chemistry (75) | ### H. Intended Use: 1. Intended use: The FilmArray® Respiratory Panel 2 (RP2) is a multiplexed nucleic acid test intended for use with FilmArray® 2.0 or FilmArray® Torch systems for the simultaneous qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals suspected of respiratory tract infections. The following organism types and subtypes are identified using the FilmArray® RP2: 2 {2} - Adenovirus - Coronavirus 229E - Coronavirus HKU1 - Coronavirus NL63 - Coronavirus OC43 - Human Metapneumovirus - Human Rhinovirus/Enterovirus - Influenza A, including subtypes H1, H1-2009, and H3 - Influenza B - Parainfluenza Virus 1 - Parainfluenza Virus 2 - Parainfluenza Virus 3 - Parainfluenza Virus 4 - Respiratory Syncytial Virus - Bordetella parapertussis (IS1001) - Bordetella pertussis (ptxP) - Chlamydia pneumoniae - 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 3 {3} testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens. 2. Indication for use: Same as Intended Use 3. Special conditions for use statement(s): For prescription use only 4. Special instrument requirements: FilmArray 2.0 or FilmArray Torch systems # I. Device Description: The FilmArray Respiratory Panel 2 (RP2) is designed to simultaneously identify 21 different potential pathogens (see the Intended Use section) of respiratory tract infection from a single NPS specimen in approximately 45 minutes. 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 a 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 FilmArray 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, PCR1. 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, LLC). The solution is then distributed to each well of the array. Array 4 {4} wells contain sets of primers designed specifically to amplify sequences internal to the PCR products generated during the first stage PCR reaction. The second stage PCR, or nested PCR, PCR2, is performed in singleplex fashion in each well of the array. At the conclusion of the second 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 second stage PCR captures fluorescent images of the PCR reactions and software interprets the data. The FilmArray Software automatically interprets the results of each DNA melt curve analysis and combines the data with the results of the internal pouch controls to provide a test result for each organism on the panel. ### Materials Provided with the FilmArray RP2 Kit: Each kit contains sufficient reagents to test 6 samples (6-test kit; RFIT-ASY-0130) or 30 samples (30-test kit; RFIT-ASY-0129): - Individually-packaged FilmArray RP2 pouches - Single-use (1.0 mL) Sample Buffer ampoules - Single-use pre-filled (1.5 mL) Hydration Injection Vials (blue) - Single-use Sample Injection Vials (red) - Individually-packaged Transfer Pipettes ### Materials Needed but Not Provided with the FilmArray RP2 Kit: - 10% bleach solution FilmArray system including: - FilmArray 2.0 or FilmArray Touch and accompanying software - FilmArray Pouch Loading Station ### Interpretation of Results When PCR2 is complete, the FilmArray instrument performs a DNA melting analysis on the PCR products and measures the fluorescence signal generated in each well. The FilmArray Software then performs several analyses and assigns a final assay result. The steps in the analyses are described below. - Analysis of melt curves The FilmArray Software evaluates the DNA melt curve for each well of the PCR2 array to determine if a PCR product was present in that well. If the melt profile indicates the presence of a PCR product, then the analysis software calculates the melting temperature (Tm) of the curve and compares it against the expected Tm range for the assay. If the software determines that the Tm falls inside the assay-specific Tm range, the melt curve is called positive. If the software determines that the melt curve is not in the appropriate Tm range, the melt curve is called negative. - Analysis of replicates Once melt curves have been identified, the software evaluates the three replicates for each assay to determine the assay result. For an assay to be called positive, at least two of the three associated melt curves must be called positive, and the Tm for at least two of the three positive melt curves must be similar (i.e., within 1°C). Assays that do not meet these criteria are called negative. 5 {5} For the following organisms detected by the FilmArray RP2, the organism is reported as “Detected” if a single corresponding assay is positive. - Coronavirus 229E - Coronavirus HKU1 - Coronavirus NL63 - Coronavirus OC43 - Human Metapneumovirus - Human Rhinovirus/Enterovirus - Influenza B - Parainfluenza Virus 1 - Parainfluenza Virus 2 - Parainfluenza Virus 3 - Parainfluenza Virus 4 - Respiratory Syncytial Virus - *Bordetella parapertussis* (IS1001) - *Bordetella pertussis* (ptxP) - *Chlamydia pneumoniae* - *Mycoplasma pneumoniae* The test results for Adenovirus and Influenza A (including subtyping) depend on the interpretation of results from more than one corresponding assay. Interpretation and actions for these results are provided below. - Adenovirus The FilmArray RP2 pouch contains five different assays (Adeno2, Adeno3, Adeno6, Adeno7.1, and Adeno8) for the detection of Adenovirus. The FilmArray Software interprets each of these assays independently and the results are combined as a final test result for the virus. If one or any combination of assays is positive, the test report result will be Adenovirus “Detected”. If all assays are negative, the test report result will be Adenovirus “Not Detected”. - Influenza A and Subtyping The assays in the FilmArray RP2 are designed to both detect Influenza A and to differentiate commonly occurring hemagglutinin subtypes. To accomplish this, the FilmArray RP2 uses two Influenza A assays, FluA-pan-1 and FluA-pan-2, and three subtyping assays, FluA-H1-2, FluA-H1-2009, and FluA-H3, directed at the respective hemagglutinin gene. Each of the individual assays is interpreted independently and the test result reported for Influenza A is based on the combined results of the five assays as outlined in Table 1. 6 {6} **Table 1: Possible Assay Results for Influenza A and Corresponding Interpretation** | Assay Result | FluA-pan Assays (n=2) | FluA-H1-2 | FluA-H1-2009 | FluA-H3 | Action | | --- | --- | --- | --- | --- | --- | | Influenza A Not Detected | Negative | Negative | Negative | Negative | None | | Influenza A H1 | ≥1 positive | Positive | Negative | Negative | | | Influenza A H3 | ≥1 positive | Negative | Negative | Positive | | | Influenza A H1-2009 | ≥1 positive | Any result | Positive | Negative | | | Influenza A H1 Influenza A H3 | ≥1 positive | Positive | Negative | Positive | Multiple infections are possible but rare^{a}, retest to confirm result^{b} | | Influenza A H1-2009 Influenza A H3 | ≥1 positive | Any result | Positive | Positive | | | Influenza A (no subtype detected) | 2 positive | Negative | Negative | Negative | Retest | | Influenza A Equivocal | 1 positive | Negative | Negative | Negative | Retest | | Influenza A H1 Equivocal | Negative | Positive | Negative | Negative | | | Influenza A H3 Equivocal | Negative | Negative | Negative | Positive | | | Influenza A H1-2009 Equivocal | Negative | Any result | Positive | Negative | | $^{a}$ The FilmArray RP2 can simultaneously detect multiple influenza viruses contained in multivalent vaccines. $^{b}$ Repeated multiple positives should be further confirmed by other FDA cleared Influenza subtyping tests. Influenza A (no subtype detected): If both of the FluA-pan assays are positive, but none of the hemagglutinin subtyping assays are positive, then the interpretation is Influenza A (no subtype detected). This result could occur when the titer of the virus in the specimen is low and not detected by the subtyping assays. This result could also indicate the presence of a novel Influenza A strain. In both cases, the sample in question should be retested. If the retest provides a different result, test the sample a third time to ensure the accuracy of the result. If the retest provides the same result, then the function of the RP2 pouches should be verified by testing with appropriate external control materials (known positive samples for Influenza A H1, Influenza A H3 and Influenza A H1-2009), and a negative control should also be run to test for PCR-product contamination. If the FilmArray RP2 accurately identifies the external and negative controls, contact the appropriate public health authorities for confirmatory testing. ### FilmArray RP2 Test Report The FilmArray RP2 test report is automatically displayed upon completion of a run and can be printed or saved as a PDF file. Each report contains a Run Summary, a Result Summary, and a Run Details section. An example of the test report is presented below: 7 {7}  #### - Run Summary The Run Summary section of the test report provides the Sample ID, time and date of the run, control results and an overall summary of the test results. Any organism with a “Detected” result will be listed in the corresponding field of the summary. If all of the organism assays were negative then “None” will be displayed in the Detected field. All Influenza A equivocal results (refer to Table 2) will be displayed in the Equivocal field. Controls are listed as “Passed”, “Failed” or “Invalid”. Table 2 below provides additional information for each of the possible control field results. Table 2: Interpretation of Controls Field on the FilmArray RP2 Test Report | Control Result | Explanation | Action | | --- | --- | --- | | Passed | The run was successfully completed ANDBoth pouch controls were successful. | NoneReport the results provided on the test report | | Failed | The run was successfully completed BUTAt least one of the pouch controls (RNA Process Control and/or PCR2 Control) failed. | Repeat the test using a new pouch.If the error persists, contact Technical Support for further instruction. | 8 {8} | Control Result | Explanation | Action | | --- | --- | --- | | Invalid | The controls are invalid because the run did not complete. (Typically this indicates a software or hardware error). | Note any error codes displayed during the run and the Run Status field in the Run Details section of the report. Refer to the appropriate FilmArray Operator's Manual or contact Technical Support for further instruction. Once the error is resolved, repeat the test or repeat the test using another instrument. | - Results Summary The Result Summary section of the test report lists the result for each target tested by the panel. Possible results for each organism except for Influenza A and subtyping are "Detected", "Not Detected", or "Invalid". Table 3 below provides an explanation for each interpretation and any follow-up necessary to obtain a final result. **Table 3: Reporting of Results and Required Actions** | Result | Explanation | Action | | --- | --- | --- | | Detected^{a} | The run was successfully completed AND The pouch controls were successful (Passed) AND The assay(s) for the organism were POSITIVE (i.e., met the requirements for a positive result described in the Interpretation of Results section above) | Report results. | | Not Detected | The run was successfully completed AND The pouch controls were successful (Passed) AND The assay(s) for the organism were NEGATIVE (i.e., did not meet the requirements for a positive result described in the Assay Interpretation section above) | Report results. | | Equivocal | The run was successfully completed AND The pouch controls were successful (Passed) AND The combination of positive and negative assay results for Influenza A were inconclusive (see Table 2) | Retest the original specimen using a new pouch and report the results of the retest. | | Invalid | The pouch controls were not successful (Failed) OR The run was not successful (Run Status displayed as: Aborted, Incomplete, Instrument Error or Software Error) | See Table 3. | $^{a}$ If four or more organisms are detected in a specimen, retesting is recommended to confirm the polymicrobial result. - Run Details The Run Details section provides additional information about the run including: pouch information (type, lot number, and serial number), Run Status (Completed, 9 {9} Incomplete, Aborted, Instrument Error, Instrument Communication Error, or Software Error), the protocol that was used to perform the test, the identity of the operator that performed the test, and the instrument used to perform the test. # **J. Substantial Equivalence Information:** # 1. Predicate device name(s): FilmArray Respiratory Panel (RP) # 2. Predicate K number(s): K160068 # 3. Comparison with predicate(s): | Similarities or Differences | | | | --- | --- | --- | | Element | FilmArray Respiratory Panel 2 (RP2) (K170604) | FilmArray Respiratory Panel (RP) (K160068) | | Specimen Types | NPS (in transport media) | Same | | Organisms Detected | Adenovirus, Coronavirus 229E, Coronavirus HKU1, Coronavirus NL63, Coronavirus OC43, Human Metapneumovirus, Influenza A, Influenza A subtype H1, Influenza A subtype H3, Influenza A subtype H1-2009, Influenza B, Parainfluenza Virus 1, Parainfluenza Virus 2, Parainfluenza Virus 3, Parainfluenza Virus 4, Human Rhinovirus/Enterovirus, Respiratory Syncytial Virus, *Bordetella parapertussis*, *Bordetella pertussis*, *Chlamydia pneumoniae*, and *Mycoplasma pneumoniae* | Same, except that it does not detect *Bordetella parapertussis* | | Analyte | RNA/DNA | Same | | Technological Principles | Multiplex nucleic acid | Same | | Instrumentation | FilmArray 2.0 or FilmArray Torch | FilmArray, FilmArray 2.0 or FilmArray Torch | | Time to Result | About 45 minutes | About 1 hour | | Reagent Storage | Room temperature | Same | | Test Interpretation | Automated test interpretation and report generation. User cannot access raw data. | Same | | Controls | Two controls are included in each reagent pouch to control for sample processing and both stages of PCR and melt analysis. | Same | 10 {10} # **K. Standard/Guidance Documents Referenced (if applicable):** - • FDA guidance document issued on August 27, 2014, titled “*Highly Multiplexed Microbiological/Medical Countermeasure In Vitro Nucleic Acid Based Diagnostic Devices*” - • FDA guidance document issued on October 9, 2009, titled “*Class II Special Controls Guidance Document: Respiratory Viral Panel Multiplex Nucleic Acid Assay*” - • FDA guidance document issued on October 9, 2009, titled “*Class II Special Controls Guidance Document: Testing for Detection and Differentiation of Influenza A Virus Subtypes Using Multiplex Assays*” - • FDA guidance document issued on October 9, 2009, titled “*Testing for Human Metapneumovirus (hMPV) Using Nucleic Acid Assays*” - • FDA guidance document issued on March 13, 2007, titled “*Statistical Guidance on Reporting Results from Studies Evaluating Diagnostic Tests*” - • FDA guidance document issued on July 15, 2011, titled “*Establishing the Performance Characteristics of In Vitro Diagnostic Devices for the Detection or Detection and Differentiation of Influenza Viruses*” - • FDA guidance document issued on April 25, 2005, titled “*Guidance on Informed Consent for In Vitro Diagnostic Device Studies Using Leftover Human Specimens that are Not Individually Identifiable*” - • FDA guidance document issued on May 11, 2005, titled “*Guidance for the Content of Premarket Submissions for Software Contained in Medical Devices*” - • FDA guidance document issued on September 9, 1999, titled “*Off-The-Shelf Software Use in Medical Devices*” - • FDA guidance document issued on January 11, 2002, titled “*General Principle of Software Validation*” - • FDA guidance document issued on November 30, 2004, titled “*Use of Symbols on Labels and in Labeling of In Vitro Diagnostic Devices Intended for Professional Use*” - • FDA guidance document issued on January 1, 2000, titled “*Guidance for Industry and FDA on Alternative to Certain Prescription Device Labeling Requirements*” - • Interference Testing in Clinical Chemistry; Clinical and Laboratory Standards Institute (CLSI) Approved Guideline – Second Addition, EP7-A2 (2005) - • User Protocol for Evaluation of Qualitative Test Performance; Clinical and Laboratory Standards Institute (CLSI) Approved Guideline – Second Edition, EP12-A2 (January 2008) - • Molecular Diagnostic Methods for Infectious Diseases; Clinical and Laboratory Standards Institute (CLSI) Approved Guideline, MM3-A2 (February 2006) - • Evaluation of Stability of In Vitro Diagnostic Reagents; Clinical and Laboratory Standards Institute (CLSI) Approved Guideline, EP25-A (September 2009) # **L. Test Principle:** The FilmArray instrument, software, and pouch work together to perform sample lysis and purification, amplification, and detection of nucleic acid. One of the primary functions of a FilmArray instrument is to drive the various steps in the testing process by moving liquids to appropriate locations within the pouch. Liquids are moved from the fitment to the blisters of the pouch by means of pneumatic actuators in the 11 {11} instrument piston block which press on the syringe-like plunging devices in the pouch fitment. There are 12 pistons that operate in a specified sequence to deliver reagents to the appropriate blisters in the pouch when they are needed. Within the pouch, liquids are moved by using bladders and hard seals to exert pressure on the exterior of the pouch, such that the instrument is never in contact with the liquids contained in the pouch. The bladders are inflatable elastomeric membranes used to 'squish' the pouch blisters, thus forcing the liquid out of the compressed blister and along any connecting channels. The hard-seals are piston driven actuators used to direct the flow of liquids from the blisters by pinching shut the channels and blocking flow while pinched. Thermal interactions between the pouch and instrument play a crucial role in the amplification of target nucleic acids. Temperature control is driven by a pair of numerically controlled Peltier devices; solid-state thermal control instruments fitted with calibrated temperature sensors and protective circuitry. These Peltier devices heat and cool to perform the first and second stage PCR reactions and carefully control the temperature across the array during DNA melting. The instrument uses a blue LED to illuminate the second stage PCR array and a digital camera to record fluorescence generated in the second stage PCR. The optical system is designed to detect the fluorescence signal generated during DNA melting. The instrument communicates with the computer and the FilmArray software using Ethernet cables. The software provides instructions to the instrument to control each of the steps described above. A detailed explanation of specific steps in the testing process is provided below: # 1. 1. Sample Lysis and Purification Nucleic acid purification occurs in the first four blisters of the pouch using magnetic bead technology. # 1. 1. a. Sample Lysis Prior to loading the sample into the pouch, nucleases are inactivated by mixing the sample with a denaturing buffer (FilmArray Sample Buffer). The sample/sample buffer mixture is then loaded into the pouch via the injection port and pouch vacuum pulls the liquid into the sample well of the pouch fitment. During the addition of the sample/sample buffer mixture, the template for the RNA Process Control is rehydrated and introduced into the reaction mixture. The RNA Process Control targets an mRNA of *Schizosaccharomyces pombe*, which is freeze-dried into the sample well of each pouch. The *S. pombe* is processed in parallel with the patient sample through each step of the test including nucleic acid extraction, reverse transcription (RT), PCR1, PCR2, and DNA melting. A positive result for the RNA Process Control indicates that all steps in the test are functioning properly. The instrument activates a piston located above the fitment to move the sample/sample buffer mixture from the fitment into the trapezoidal sample lysis blister and then heat- 12 {12} seals the fitment to prevent sample loss. The sample lysis blister contains ceramic beads. The instrument then activates the bead beater assembly which rotates a metal bar that strikes the pouch for 60 seconds to lyse organisms in the sample by creating high speed impacts between the sample and beads (bead beating). At the conclusion of the bead beating process, cells and organisms are lysed and the cell contents, including the nucleic acids, are released into the reaction mixture. # b. Nucleic Acid Isolation The instrument inflates the appropriate bladder and moves the lysed sample into the magnetic bead blister. Here, the liberated nucleic acids are captured by adsorption to silica-magnetic beads. The instrument then uses a retractable magnet positioned adjacent to the blister to hold the beads against the inside of the blister while they are washed to remove proteins, cell debris, and other potential PCR inhibitors. The instrument moves the wash buffer from the fitment into the appropriate blisters by exerting pressure on pistons located above the fitment. After the washes are completed, an elution buffer is moved from the fitment to the appropriate blister resulting in the nucleic acids being released from the beads. The instrument then moves the purified nucleic acid solution to the 1st stage PCR blister while the beads and other waste products are pushed back to the trapezoidal blister. # 2. Reverse Transcription and 1st Stage Multiplex PCR In the 1st stage PCR blister, liquid containing the purified nucleic acid rehydrates a freeze-dried reagent pellet containing all of the outer primers. A PCR master mix, containing all components needed for PCR and reverse transcription (RT), is moved from the fitment to an adjoining blister. A Peltier device is in contact with these two blisters and the instrument performs a “hot-start” PCR and RT by preheating the blisters containing the purified sample and the PCR master mix. Once the appropriate temperature is reached, the contents of the two blisters are mixed and the RT step and thermo-cycling is initiated. Since the FilmArray RP2 includes RNA viruses, an RT step is needed to convert the viral RNA into cDNA that can be amplified by PCR. Both the RT and the first stage of the nested PCR reaction are performed using the same outer primers and master mix. # 3. Dilution, 2nd Stage PCR and DNA Melt Analysis Following completion of the RT and 1st stage PCR steps, a second singleplex PCR is carried out. To accomplish this, the instrument dilutes the products of the 1st stage PCR with fresh PCR master mix containing a double stranded DNA binding dye (LC Green® Plus, BioFire Diagnostics, LLC). This solution is distributed over the 2nd stage PCR array, where it rehydrates the dried primers in each well. The individual wells in the array contain primers for different assays (each assay is present in triplicate wells of the array) that target specific nucleic acid sequences from each of the pathogens or control templates. The primers in the PCR2 array are “nested” or internal to the specific PCR 13 {13} products of the 1st stage multiplex reaction. A second Peltier device is responsible for driving the PCR2 reaction and for controlling temperature during DNA melting. The product of the 2nd stage PCR is visualized with the fluorescent LC Green® Plus dye. At the conclusion of PCR2, the temperature of the array is gradually increased and the fluorescence in each well is monitored and analyzed to generate a melt curve. The instrument then transfers images and temperature measurements to the FilmArray software for analysis. The PCR2 array also contains a control assay, called the PCR2 Control, which is comprised of a specific set of PCR2 assay primers along with the corresponding template pre-spotted into three specific wells of the array. Failure of the PCR2 Control invalidates the run and indicates a test failure that is specific to the PCR2 step of the testing process. # 4. Data Analysis and Result Reporting The temperature at which a specific PCR product melts (melting temperature or Tm) is consistent and predictable. The FilmArray software automatically evaluates the results from replicate wells of each assay for the detection of amplicons with a specific Tm, which denotes the presence of specific bacterial or viral targets. The FilmArray software uses the following steps to interpret the melt curve data generated from each FilmArray RP2 assay: # a. Analysis of Melt Curves First, the FilmArray RP2 Melt Detector performs a set of basic calculations on the melt data to determine if a PCR reaction occurred in each well. If the melt profile indicates that a PCR product is present, then the analysis software calculates one or two Tm values, depending on the number of melt curves present in the data, and the Tm values are compared against an expected melt range for the associated assay. If the software determines that the melt is positive and the melt curve falls inside the assay's specific melt range, then the curve is called positive. If the software determines that the melt is negative or that it is not in the appropriate range, then the curve is called negative. # b. Analysis of Replicates Next, the analysis software evaluates the replicates for each assay (target and control) to determine if the assay is positive or negative. To be called positive, at least two of the three wells associated with an assay must have a positive melt curve and the Tm for the positive curves must be similar (i.e., within 1°C). Assays with replicates that do not meet these criteria are called negative. # c. Analysis of Controls Results for control assays are compared to their expected values and assigned a single pass or fail result for each control. Pouch-specific rules define how control failures affect interpretations. The default rule specifies that any control failure invalidates the entire run. For the FilmArray RP2, failure of the RNA Process Control or the PCR2 Control is interpreted as a control failure and all target assays (regardless of the assay result) are assigned a test result of invalid. 14 {14} # d. Interpretation of Assay Results Once the results for the individual assays are determined, the software applies interpretation rules to determine the final test results. For many organisms, the target is determined to be present or absent if a single associated assay is positive or negative, respectively. For these organisms, the final test result is either “Detected” (for positive results), “Not Detected” (for negative results) or “Invalid” (when either control fails or the run fails). The FilmArray RP2 also includes test results that rely on the results of multiple assays. See the Interpretation of Results section for more information on interpreting these test results. # **M. Performance Characteristics (if/when applicable):** # 1. Analytical performance: # a. *Reproducibility Study :* A reproducibility study was conducted at three testing sites on a combination of FilmArray 2.0 and FilmArray Torch systems. The study incorporated a range of potential variation introduced by site (three testing sites), day (five different days), operator (at least two per site), system, instrument or Torch module (at least three per site/sample), and pouch lot (at least three). A total of four contrived NPS samples containing known quantities of various RP2 analytes (Table 4 below) were prepared in simulated NPS in VTM sample matrix$^{1}$. The contrived samples contained various combinations of 12 different FilmArray RP2 analytes$^{2}$, each at three different concentrations, Negative, Low Positive (1×LoD), and Moderate Positive (3×LoD). Note that the negative data were acquired from samples not spiked with a particular analyte (i.e., negative data for the analytes in Sample#1 and #2 were obtained from Sample#3 and #4, and vice-versa). $^{1}$ Note: The simulated NPS in VTM sample matrix and the natural NPS in VTM sample matrix were demonstrated to be equivalent with regard to FilmArray® RP test detectability of analytes in analytical studies in support of the original FDA-clearance of the FilmArray® RP test. Refer to K103175, K110764, and K120267 for additional details of the analytical studies. $^{2}$ Note: Single-spiked and multi-spiked specimens were demonstrated to be equivalent with regard to FilmArray® RP test detectability of analytes in analytical studies in support of the original FDA-clearance of the FilmArray® RP test. Refer to K103175, K110764, and K120267 for additional details of the analytical studies. 15 {15} Table 4: Reproducibility Test Panel for the FilmArray RP2 | Organism | Strain/Serotype | Source/ ID | Limit of Detection (LoD) Concentration | Sample #1 Concentration (1xLoD) | Sample #2 Concentration (3xLoD) | | --- | --- | --- | --- | --- | --- | | Coronavirus OC43 | OC43 | ATCC VR-759 | 5.6E+02 Copies/mL (3.0E+01 TCID_{50}/mL) | 5.6E+02 Copies/mL (3.0E+01 TCID_{50}/mL) | 1.7E+03 Copies/mL (9.0E+01 TCID_{50}/mL) | | Parainfluenza virus 2 | Type 2 | Zeptometrix 0810015CF | 3.0E+01 Copies/mL (5.0E-01 TCID_{50}/mL) | 3.0E+01 Copies/mL (5.0E-01 TCID_{50}/mL) | 9.0E+01 Copies/mL (1.5E+00 TCID_{50}/mL) | | Adenovirus C2 | Species C Serotype 2 | ATCC VR-846 | 3.7E+01 Copies/mL (2.0E+00 TCID_{50}/mL) | 3.7E+01 Copies/mL (2.0E+00 TCID_{50}/mL) | 1.1E+02 Copies/mL (6.0E+00 TCID_{50}/mL) | | Influenza A H3N2 | A/Port Chalmers/1/73 | ATCC VR-810 | 2.1E+01 Copies/mL (1.0E-01 TCID_{50}/mL) | 2.1E+01 Copies/mL (1.0E-01 TCID_{50}/mL) | 6.3E+01 Copies/mL (3.0E-01 TCID_{50}/mL) | | Rhinovirus | Type 1A | Zeptometrix 0810012CFN | 3.8E+01 Copies/mL (1.0E-01 TCID_{50}/mL) | 3.8E+01 Copies/mL (1.0E-01 TCID_{50}/mL) | 1.1E+02 Copies/mL (3.0E-01 TCID_{50}/mL) | | *Bordetella parapertussis* | A747 | Zeptometrix 081461 | 5.8E+01 *IS1001* Copies/mL (4.1E+01 CFU/mL) | 5.8E+01 *IS1001* Copies/mL (4.1E+01 CFU/mL) | 1.7E+02 *IS1001* Copies/mL (1.2E+02 CFU/mL) | | Organism | Strain/Serotype | Source/ ID | Limit of Detection (LoD) | Sample #3 (1xLoD) | Sample #4 (3xLoD) | | *Chlamydia pneumoniae* | TW183 | ATCC VR-92 | 6.6E+01 Copies/mL (1.0E-01 TCID_{50}/mL) | 6.6E+01 Copies/mL (1.0E-01 TCID_{50}/mL) | 2.0E+02 Copies/mL (3.0E-01 TCID_{50}/mL) | | Influenza B | B/FL/04/06 | Zeptometrix 0810255CF^{a} | 3.4E+01 Copies/mL (5.0E+00 TCID_{50}/mL) | 3.4E+01 Copies/mL (5.0E+00 TCID_{50}/mL) | 1.0E+02 Copies/mL (1.5E+01 TCID_{50}/mL) | | Parainfluenza virus 4 | Type 4a | Zeptometrix 0810060CF | 1.6E+03 Copies/mL (5.0E+01 TCID_{50}/mL) | 1.6E+03 Copies/mL (5.0E+01 TCID_{50}/mL) | 4.8E+03 Copies/mL (1.5E+02 TCID_{50}/mL) | | Human Metapneumovirus | Type 16, A1 IA10-2003 | Zeptometrix 0810161CF | 1.2E+03 Copies/mL (1.0E+01 TCID_{50}/mL) | 1.2E+03 Copies/mL (1.0E+01 TCID_{50}/mL) | 3.6E+03 Copies/mL (3.0E+01 TCID_{50}/mL) | | Respiratory Syncytial Virus | Type A | Zeptometrix 0810040ACF | 9.0E+00 Copies/mL (2.0E-02 TCID_{50}/mL) | 9.0E+00 Copies/mL (2.0E-02 TCID_{50}/mL) | 2.7E+01 Copies/mL (6.0E-02 TCID_{50}/mL) | | *Bordetella pertussis*^{b} | A639 | Zeptometrix 0801459 | 1.0E+03 CFU/mL | 1.0E+03 CFU/mL | 3.0E+03 CFU/mL | $^{a}$ Formerly Zeptometrix 0810037CF. $^{b}$ For B. pertussis, the FilmArray RP2 amplifies a single-copy target and commercially available qPCR assays typically target the multi-copy IS481 sequences, therefore RP2 testing was performed based on the CFU/mL and an equivalent Copies/mL was not determined for this analyte. Once prepared, each sample of the reproducibility study panel was tested with the FilmArray RP2 to confirm that it contained the intended analytes at the intended concentration and then divided into single-use aliquots (400 μL) and stored frozen (≤ -70°C) until the day of testing. After being distributed to the sites, six replicates of each sample were tested on five different days on various FilmArray Torch modules (Site A, Site C) or FilmArray 2.0 instruments (Site B, Site C). Sites A and C conducted testing with at least three Torch modules per sample, while sites B and C utilized at least three different FilmArray 2.0 instruments per sample. Note that Site C tested a total of 12 replicates of each sample 16 {16} per day, with six replicates tested on the Torch system and six replicates tested on the FilmArray 2.0 system. Daily testing was performed by at least two different operators per system and site, and three different pouch lots were used on rotating days so that data from all variables were distributed between reagent lots. For any required retest per the Instructions for Use, another aliquot of the same sample was tested on the same day by the same operator using the same system, instrument or Torch module, and pouch lot. Results of the valid retest were used as the final test result for the sample aliquot. In total, 120 data points per sample (over a total of 480 valid runs) were obtained, with 60 data points per sample per system (i.e., FilmArray 2.0 and FilmArray Torch systems), 30 data points per sample per Site A and B, and 60 data points at Site C. Over the course of the reproducibility study, a total of 15 different FilmArray 2.0 instruments and 19 different FilmArray Torch modules (four Torch bases) were used by seven different operators at three different sites. Valid results were obtained from 480 out of the 489 runs that were initiated (480/489, 98.2%). The majority of invalid runs (8/9) were associated with a Control failure, while one invalid run was due to an instrument error (Table 5 below). Table 5: Performance of the FilmArray Systems and RP2 Controls during the Reproducibility Study | | Runs | Control Failure (Percentage) | Instrument Errors (Percentage) | Software Errors (Percentage) | | --- | --- | --- | --- | --- | | FilmArray 2.0 | 247 | 6(2.4%) | 1(0.4%) | 0(0.0%) | | FilmArray Torch | 242 | 2(0.8%) | 0(0.0%) | 0(0.0%) | | Total | 489 | \( 8^a \)(1.6%) | 1(0.2%) | 0(0.0%) | \( ^{a} \) Seven control failures occurred on pouch lot 349116, while one occurred on pouch lot 347416. A summary of the reproducibility study results, percent (%) agreement with the expected Detected or Not Detected result, for each analyte (by site and system) is provided in Table 6 below. 17 {17} Table 6: Reproducibility of FilmArray RP2 Results on FilmArray Torch and FilmArray 2.0 Systems | Analyte | Concentration Tested | Expected Result | Agreement with 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 | | | Viruses | | | | | | | | | | | Adenovirus | Moderate Positive (3× LoD) 1.1E+02 Copies/mL (6.0E+00 TCID_{50}/mL) | Detected | 30/30 100% | 29/30 96.7% | 59/60 98.3% | 29/30 96.7% | 30/30 100% | 59/60 98.3% | 118/120 98.3% (94.1%-99.8%) | | | Low Positive (1× LoD) 3.7E+01 Copies/mL (2.0E+00 TCID_{50}/mL) | Detected | 30/30 100% | 30/30 100% | 60/60 100% | 30/30 100% | 29/30 96.7% | 59/60 98.3% | 119/120 99.2% (95.4%-100%) | | | None (no analyte) | Not Detected | 60/60 100% | 60/60 100% | 120/120 100% | 60/60 100% | 60/60 100% | 120/120 100% | 240/240 100% (98.5%-100%) | | Coronavirus 229E | None (no analyte) | Not Detected | 120/120 100% | 120/120 100% | 240/240 100% | 120/120 100% | 120/120 100% | 240/240 100% | 480/480 100% (99.2%-100%) | | Coronavirus HKU1 | None (no analyte) | Not Detected | 120/120 100% | 120/120 100% | 240/240 100% | 120/120 100% | 120/120 100% | 240/240 100% | 480/480 100% (99.2%-100%) | | Coronavirus OC43 | Moderate Positive (3× LoD) 1.7E+03 Copies/mL (9.0E+01 TCID_{50}/mL) | Detected | 29/30 96.7% | 29/30 96.7% | 58/60 96.7% | 29/30 96.7% | 30/30 100% | 59/60 98.3% | 117/120 97.5% (92.9%-99.5%) | | | Low Positive (1× LoD) 5.6E+02 Copies/mL (3.0E+01 TCID_{50}/mL) | Detected | 30/30 100% | 30/30 100% | 60/60 100% | 30/30 100% | 27/30 90.0% | 57/60 95.0% | 117/120 97.5% (92.9%-99.5%) | | | None (no analyte) | Not Detected | 60/60 100% | 60/60 100% | 120/120 100% | 60/60 100% | 60/60 100% | 120/120 100% | 240/240 100% (98.5%-100%) | | Coronavirus NL63 | None (no analyte) | Not Detected | 120/120 100% | 120/120 100% | 240/240 100% | 120/120 100% | 120/120 100% | 240/240 100% | 480/480 100% (99.2%-100%) | 18 {18} | Analyte | Concentration Tested | Expected Result | Agreement with 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 | | | Human Metapneumovirus | Moderate Positive (3× LoD) 3.6E+03 Copies/mL (3.0E+01 TCID50/mL) | Detected | 30/30 100% | 30/30 100% | 60/60 100% | 30/30 100% | 30/30 100% | 60/60 100% | 120/120 100% (97.0%-100%) | | | Low Positive (1× LoD) 1.2E+03 Copies/mL (1.0E+01 TCID50/mL) | Detected | 30/30 100% | 30/30 100% | 60/60 100% | 28/30 93.3% | 30/30 100% | 58/60 96.7% | 118/120 98.3% (94.1%-99.8%) | | | None (no analyte) | Not Detected | 60/60 100% | 60/60 100% | 120/120 100% | 60/60 100% | 60/60 100% | 120/120 100% | 240/240 100% (98.5%-100%) | | Human Rhinovirus/ Enterovirus | Moderate Positive (3× LoD) 1.1E+02 Copies/mL (3.0E-01 TCID50/mL) | Detected | 30/30 100% | 30/30 100% | 60/60 100% | 28/30 93.3% | 30/30 100% | 58/60 96.7% | 118/120 98.3% (94.1%-99.8%) | | | Low Positive (1× LoD) 3.8E+01 Copies/mL (1.0E-01 TCID50/mL) | Detected | 30/30 100% | 30/30 100% | 60/60 100% | 30/30 100% | 30/30 100% | 60/60 100% | 120/120 100% (97.0%-100%) | | | None (no analyte) | Not Detected | 60/60 100% | 60/60 100% | 120/120 100% | 60/60 100% | 60/60 100% | 120/120 100% | 240/240 100% (98.5%-100%) | | Influenza A H3 | Moderate Positive (3× LoD) 6.3E+01 Copies/mL (3.0E-01 TCID50/mL) | Detected | 30/30 100% | 30/30 100% | 60/60 100% | 29/30 96.7% | 30/30 100% | 59/60 98.3% | 119/120 99.2% (95.4%-100%) | | | Low Positive (1× LoD) 2.1E+01 Copies/mL (1.0E-01 TCID50/mL) | Detected | 30/30 100% | 30/30 100% | 60/60 100% | 30/30 100% | 30/30 100% | 60/60 100% | 120/120 100% (97.0%-100%) | | | None (no analyte) | Not Detected | 60/60 100% | 60/60 100% | 120/120 100% | 60/60 100% | 60/60 100% | 120/120 100% | 240/240 100% (98.5%-100%) | 19 {19} | Analyte | Concentration Tested | Expected Result | Agreement with 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 | | | Influenza A H1-2009 | None (no analyte) | Not Detected | 120/120 100% | 120/120 100% | 240/240 100% | 120/120 100% | 120/120 100% | 240/240 100% | 480/480 100% (99.2%-100%) | | Influenza A H1 | None (no analyte) | Not Detected | 120/120 100% | 120/120 100% | 240/240 100% | 120/120 100% | 120/120 100% | 240/240 100% | 480/480 100% (99.2%-100%) | | Influenza B | Moderate Positive (3× LoD) 1.0E+02 Copies/mL (1.5E+01 TCID_{50}/mL) | Detected | 30/30 100% | 30/30 100% | 60/60 100% | 30/30 100% | 30/30 100% | 60/60 100% | 120/120 100% (97.0%-100%) | | | Low Positive (1× LoD) 3.4E+01 Copies/mL (5.0E+00 TCID_{50}/mL) | Detected | 30/30 100% | 30/30 100% | 60/60 100% | 30/30 100% | 30/30 100% | 60/60 100% | 120/120 100% (97.0%-100%) | | | None (no analyte) | Not Detected | 60/60 100% | 60/60 100% | 120/120 100% | 60/60 100% | 60/60 100% | 120/120 100% | 240/240 100% (98.5%-100%) | | Parainfluenza Virus 1 | None (no analyte) | Not Detected | 120/120 100% | 120/120 100% | 240/240 100% | 120/120 100% | 120/120 100% | 240/240 100% | 480/480 100% (99.2%-100%) | | Parainfluenza Virus 2 | Moderate Positive (3× LoD) 9.0E+01 Copies/mL (1.5E+00 TCID_{50}/mL) | Detected | 30/30 100% | 29/30 96.7% | 59/60 98.3% | 29/30 96.7% | 30/30 100% | 59/60 98.3% | 118/120 98.3% (94.1%-99.8%) | | | Low Positive (1× LoD) 3.0E+01 Copies/mL (5.0E-01 TCID_{50}/mL) | Detected | 30/30 100% | 29/30 96.7% | 59/60 98.3% | 30/30 100% | 27/30 90.0% | 57/60 95.0% | 116/120 96.7% (91.7%-99.1%) | | | None (no analyte) | Not Detected | 60/60 100% | 60/60 100% | 120/120 100% | 60/60 100% | 60/60 100% | 120/120 100% | 240/240 100% (98.5%-100%) | | Parainfluenza Virus 3 | None (no analyte) | Not Detected | 120/120 100% | 120/120 100% | 240/240 100% | 120/120 100% | 120/120 100% | 240/240 100% | 480/480 100% (99.2%-100%) | 20 {20} | Analyte | Concentration Tested | Expected Result | Agreement with 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 | | | Parainfluenza Virus 4 | Moderate Positive (3× LoD) 4.8E+03 Copies/mL (1.5E+02 TCID50/mL) | Detected | 30/30 100% | 30/30 100% | 60/60 100% | 30/30 100% | 30/30 100% | 60/60 100% | 120/120 100% (97.0%-100%) | | | Low Positive (1× LoD) 1.6E+03 Copies/mL (5.0E+01 TCID50/mL) | Detected | 30/30 100% | 29/30 96.7% | 59/60 98.3% | 29/30 96.7% | 30/30 100% | 59/60 98.3% | 118/120 98.3% (94.1%-99.8%) | | | None (no analyte) | Not Detected | 60/60 100% | 60/60 100% | 120/120 100% | 60/60 100% | 60/60 100% | 120/120 100% | 240/240 100% (98.5%-100%) | | Respiratory Syncytial Virus | Moderate Positive (3× LoD) 2.7E+01 Copies/mL (6.0E-02 TCID50/mL) | Detected | 30/30 100% | 30/30 100% | 60/60 100% | 30/30 100% | 30/30 100% | 60/60 100% | 120/120 100% (97.0%-100%) | | | Low Positive (1× LoD) 9.0E+00 Copies/mL (2.0E-02 TCID50/mL) | Detected | 29/30 96.7% | 30/30 100% | 59/60 98.3% | 30/30 100% | 29/30 96.7% | 59/60 98.3% | 118/120 98.3% (94.1%-99.8%) | | | None (no analyte) | Not Detected | 60/60 100% | 60/60 100% | 120/120 100% | 60/60 100% | 60/60 100% | 120/120 100% | 240/240 100% (98.5%-100%) | | Bacteria | | | | | | | | | | | Bordetella parapertussis | Moderate Positive (3× LoD) 1.8E+02 IS1001 Copies/mL (1.2E+02 CFU/mL) | Detected | 30/30 100% | 30/30 100% | 60/60 100% | 29/30 96.7% | 30/30 100% | 59/60 98.3% | 119/120 99.2% (95.4%-100%) | | | Low Positive (1× LoD) 6.0E+01 IS1001 Copies/mL (4.1E+01 CFU/mL) | Detected | 24/30a 80.0% | 29/30 96.7% | 53/60a 88.3% | 29/30 96.7% | 30/30 100% | 59/60 98.3% | 112/120 93.3% (87.3%-97.1%) | | | None (no analyte) | Not Detected | 60/60 100% | 60/60 100% | 120/120 100% | 60/60 100% | 60/60 100% | 120/120 100% | 240/240 100% (98.5%-100%) | 21 {21} | Analyte | Concentration Tested | Expected Result | Agreement with 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 | | | *Bordetella pertussis* | Moderate Positive (3× LoD) 3.0E+03 CFU/mL | Detected | 30/30 100% | 30/30 100% | 60/60 100% | 30/30 100% | 30/30 100% | 60/60 100% | 120/120 100% (97.0%-100%) | | | Low Positive (1× LoD) 1.0E+03 CFU/mL | Detected | 28/30 93.3% | 30/30 100% | 58/60 96.7% | 30/30 100% | 30/30 100% | 60/60 100% | 118/120 98.3% (94.1%-99.8%) | | | None (no analyte) | Not Detected | 60/60 100% | 60/60 100% | 120/120 100% | 60/60 100% | 60/60 100% | 120/120 100% | 240/240 100% (98.5%-100%) | | *Chlamydia pneumoniae* | Moderate Positive (3× LoD) 2.0E+02 Copies/mL (3.0E-01 TCID_{50}/mL) | Detected | 30/30 100% | 30/30 100% | 60/60 100% | 30/30 100% | 30/30 100% | 60/60 100% | 120/120 100% (97.0%-100%) | | | Low Positive (1× LoD) 6.6E+01 Copies/mL (1.0E-01 TCID_{50}/mL) | Detected | 28/30 93.3% | 30/30 100% | 58/60 96.7% | 29/30 96.7% | 30/30 100% | 59/60 98.3% | 117/120 97.5% (92.9%-99.5%) | | | None (no analyte) | Not Detected | 60/60 100% | 60/60 100% | 120/120 100% | 60/60 100% | 60/60 100% | 120/120 100% | 240/240 100% (98.5%-100%) | | *Mycoplasma pneumoniae* | None (no analyte) | Not Detected | 120/120 100% | 120/120 100% | 240/240 100% | 120/120 100% | 120/120 100% | 240/240 100% | 480/480 100% (99.2%-100%) | | **Overall Agreement with the Expected Result** **All Analytes and All Test Levels** **(95% Confidence Interval)** | | | **9,562/9,600** **99.6%** **(99.5% – 99.7%)** | | | | | | | a Data from Site A were further reviewed by the unique site-specific variables including test day, Torch module, and operator. No correlation could be found between the Not Detected results and any one or more of these variables. The Not Detected results at Site A were found to be statistically non-significant (p>0.05 by Fisher's exact test) and therefore do not appear to indicate a site- or system-dependent variance in precision of the FilmArray RP2 Bordetella parapertussis (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 each assay of ± 0.5°C³ or less observed within and between the FilmArray 2.0 and FilmArray Torch systems (Table 7 below). 3 Note: The Tm window for an assay is determined based on a mathematical model of known sequences and empirical data. An observed standard deviation in Tm of 0.5°C or less is believed to be adequately ensure that Tm variability contributed by the system (rather than amplicon sequence) will not lead to inaccurate results. 22 {22} Table 7: Reproducibility of Tm (°C) For Select FilmArray RP2 Assays on FilmArray Torch and FilmArray 2.0 Systems | Analyte | FilmArray RP2 Assay | Tm Observed (°C)\( ^a \) | | | | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | FilmArray Torch | | | | FilmArray 2.0 | | | | All Sites/Systems | | | | | Site A | | Site C | | Site B | | Site C | | | | | | | Mean | StDev | Mean | StDev | Mean | StDev | Mean | StDev | Mean | StDev | | Controls | Yeast RNA | 82.3 | ±0.3 | 82.1 | ±0.2 | 82.2 | ±0.3 | 82.0 | ±0.2 | 82.1 | ±0.3 | | | PCR2 | 76.1 | ±0.2 | 75.9 | ±0.2 | 76.0 | ±0.2 | 75.8 | ±0.2 | 75.9 | ±0.2 | | VIRUSES | | | | | | | | | | | | | Adenovirus | Adeno2 | 89.0 | ±0.2 | 88.8 | ±0.1 | 88.8 | ±0.3 | 88.7 | ±0.2 | 88.8 | ±0.2 | | | Adeno6 | 89.6 | ±0.2 | 89.4 | ±0.2 | 89.4 | ±0.3 | 89.2 | ±0.2 | 89.4 | ±0.3 | | Coronavirus OC43 | CoV-OC43-2 | 80.7 | ±0.2 | 80.6 | ±0.1 | 80.6 | ±0.3 | 80.5 | ±0.2 | 80.6 | ±0.2 | | Human Metapneumovirus | hMPV | 78.2 | ±0.3 | 78.0 | ±0.2 | 78.0 | ±0.3 | 77.8 | ±0.2 | 78.0 | ±0.3 | | Rhinovirus | 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 | | | FluA-pan2 | 78.9 | ±0.2 | 78.9 | ±0.1 | 78.9 | ±0.2 | 78.8 | ±0.2 | 78.9 | ±0.2 | | | FluA-H3 | 82.1 | ±0.2 | 81.9 | ±0.2 | 82.0 | ±0.3 | 81.9 | ±0.2 | 82.0 | ±0.2 | | Influenza B | FluB | 80.4 | ±0.3 | 80.3 | ±0.2 | 80.4 | ±0.2 | 80.2 | ±0.2 | 80.3 | ±0.2 | | Parainfluenza virus 2 | PIV2 | 83.2 | ±0.2 | 83.1 | ±0.2 | 83.2 | ±0.2 | 83.0 | ±0.2 | 83.1 | ±0.2 | | Parainfluenza virus 4 | PIV4 | 77.1 | ±0.2 | 77.0 | ±0.3 | 77.2 | ±0.3 | 77.0 | ±0.2 | 77.1 | ±0.3 | | Respiratory Syncytial Virus | RSV | 81.2 | ±0.2 | 81.1 | ±0.2 | 81.1 | ±0.2 | 81.0 | ±0.2 | 81.1 | ±0.2 | | BACTERIA | | | | | | | | | | | | | Bordetella parapertussis | IS1001 | 87.7 | ±0.2 | 87.6 | ±0.2 | 87.6 | ±0.3 | 87.5 | ±0.2 | 87.6 | ±0.2 | | Bordetella pertussis | ptxP | 88.6 | ±0.2 | 88.5 | ±0.2 | 88.5 | ±0.3 | 88.2 | ±0.2 | 88.4 | ±0.3 | | Chlamydia pneumoniae | Cpne | 79.6 | ±0.3 | 79.5 | ±0.2 | 79.5 | ±0.3 | 79.3 | ±0.2 | 79.5 | ±0.3 | \( ^{a} \) Mean Tm values are calculated from a combination of Tm values obtained at the \( 3 \times \) LoD and \( 1 \times \) LoD concentrations. b. Linearity/assay reportable range: Not applicable, qualitative assay c. Traceability, Stability, Expected values (controls, calibrators, or methods): ### Assay Controls Two process controls are included in each pouch: #### RNA Process Control The RNA Process Control assay targets an RNA transcript from the yeast Schizosaccharomyces pombe. The yeast is present in the pouch in a freeze-dried form and becomes rehydrated when sample is loaded. The control material is carried through all stages of the test process, including lysis, nucleic acid purification, reverse transcription, PCR1, dilution, PCR2, and DNA melting. A positive control result indicates that all steps carried out in the FilmArray RP2 pouch were successful. 23 {23} ### PCR2 Control The PCR2 Control assay detects a DNA target that is dried into wells of the array along with the corresponding primers. A positive result indicates that PCR2 was successful. Both control assays must be positive for the test run to pass. If the controls fail, the sample should be retested using a new pouch. The FilmArray Software automatically fails the run if the melting temperature (Tm) for either the RNA Process Control or the PCR2 Control is outside of an acceptable range (i.e., 80.3-84.3°C for the RNA Process Control and 73.8-77.8°C for the PCR2 Control). If required by local, state, or accrediting organization quality control requirements, users can monitor the system by trending Tm values for the control assays and maintaining records according to standard laboratory quality control practices. ### External Controls External controls are not provided with the FilmArray RP2. However, five frozen (-70°C) external control mixes (ECMs) (see Table 8 below) were prepared and provided to the clinical study sites for testing during the prospective clinical trial and the clinical study testing contrived specimens. FilmArray operators were required to complete a valid ECM run (correct results obtained) on each day of clinical specimen testing (tested on a rotating basis). Table 8: External Control Mixes (ECMs) Utilized in the Clinical Evaluations | External Control Mixes | Expected Calls | | --- | --- | | ECM 1 | Adenovirus, Coronavirus 229E, Human Metapneumovirus, Influenza A H1, Influenza B, and Mycoplasma pneumoniae | | ECM 2 | Coronavirus NL63, Influenza A H3, Parainfluenza Virus 3, Respiratory Syncytial Virus and Bordetella pertussis (ptxP) | | ECM 3 | Coronavirus HKU1, Influenza A H1-2009, Parainfluenza Virus 4, and Bordetella parapertussis (IS1001) | | ECM 4 | Coronavirus OC43, Human Rhinovirus/Enterovirus, Parainfluenza Virus 1, Parainfluenza Virus 2, and Chlamydia pneumoniae | | ECM 5 | Negative (Not Detected) | The sponsor is also recommending the following in the product package insert: “Good laboratory practice recommends running external positive and negative controls regularly. Transport media can be used as an external negative control. Previously characterized positive samples or negative samples spiked with well characterized organisms can be used as external positive controls. External controls should be used in accordance with the appropriate accrediting organization requirements, as applicable.” ### Specimen Stability FilmArray RP2 testing requires approximately 0.3 mL of NPS in Viral Transport Medium (VTM) specimen, collected according to a standard technique. Samples in VTM should be tested as soon as possible, though they may be stored at room 24 {24} temperature (15-25°C) for up to four hours, under refrigeration (2-8°C) for up to three days, or frozen (≤-15 °C or ≤-70°C) for up to 30 days. Detailed documentation concerning NPS in VTM sample storage and transport was provided in the original FilmArray RP submissions (K103175, K110764, and K120267). The results of the analytical study conducted for the original FilmArray RP submissions supported the claim that NPS specimens in Viral Transport Medium (VTM) can be stored for up to 4 hours at room temperature (18-30°C), 3 days in the refrigerator (2-8°C) or 30 days in the freezer (< -15°C) without affecting the accuracy of FilmArray RP test results. The specimen stability claims for the FilmArray RP2 remain unchanged. ### Fresh vs. Frozen Study Detailed documentation concerning fresh vs. frozen NPS in VTM specimens was provided in the original FilmArray RP submissions (K103175, K110764, and K120267). The results of the analytical study conducted for the original FilmArray RP submissions demonstrated that preservation of samples (by freezing at ≤-70°C) does not affect the accuracy of test results compared to freshly collected or freshly prepared samples. Therefore, it is appropriate to utilize frozen archived prospective and retrospective clinical samples in the evaluation of FilmArray RP2 to supplement the prospective (fresh) clinical study data, and to use frozen simulated samples in analytical studies for this submission. ### Single-Spiked vs. Multi-Spiked Specimen Study Detailed documentation concerning analytical study results used to determine whether the presence of multiple organisms in a specimen would affect the system LoD was provided in the original FilmArray RP submissions (K103175, K110764, and K120267). The results of the analytical study demonstrated that the LoD for each target analyte from testing single-spiked and multi-spiked specimens were comparable. Therefore, it is appropriate to utilize the multi-spiked approach in conducting the analytical studies and the clinical study testing contrived clinical specimens in support of this submission. ### Simulated vs. Natural NPS in VTM Specimen Study Detailed documentation concerning analytical study results used to determine whether the simulated NPS in VTM sample matrix and the natural NPS in VTM sample matrix were equivalent with regard to FilmArray RP test analytical sensitivity was provided in the original FilmArray RP submissions (K103175, K110764, and K120267). Result of the analytical study demonstrated that the simulated NPS in VTM sample matrix and the natural NPS in VTM sample matrix were equivalent with regard to FilmArray RP test ability to detect target analytes. Therefore, it is appropriate to utilize the simulated NPS in VTM sample matrix in conducting the analytical studies in support of this submission. 25 {25} # Quantification of Nucleic Acids Derived from Viral and Bacterial Cultures The majority of the FilmArray RP2 analytes require specific technique-dependent methods for culturing and quantification. Consistency in the quantification method of isolates and strains within and between species is important to mitigate inconsistencies in molecular detection that may be caused by variability of quantification methods based on infectivity or viability of microorganisms. Therefore, an analytical study was conducted to determine the nucleic acids concentration (copies/mL) of bacterial and virus stocks used in the analytical and clinical studies of the FilmArray RP2 using commercially available quantitative real-time PCR (qPCR) assay kits. # d. Detection limit: Limit of detection (LoD) estimation and confirmation studies were carried out with contrived samples in simulated NPS in VTM (Remel M4 transport medium) matrix designed to resemble a natural clinical NPS in VTM specimen. An equivalence study was performed previously which demonstrated that the simulated matrix was equivalent to the natural clinical NPS matrix and did not impact FilmArray RP test performance. Refer to the “Simulated vs. Natural NPS in VTM Specimen Study” section. Representative isolates of respiratory viruses and bacteria were selected to make contrived samples in order to obtain positive results for every assay on the panel. In some cases, testing of more than one isolate/strain per analyte was performed to assess LoD for clinically important species or variants, specifically when more than one assay was needed to detect the expected diversity of an analyte (e.g. Adenovirus). An estimated LoD concentration for each analyte was first determined by testing contrived samples serially diluted ten-fold to span at least four concentrations (10× to 0.01×) bracketing the anticipated LoD. Five replicates were tested at each dilution, with additional dilutions tested, if needed, to reach a concentration at which loss in detection could be observed. The estimated LoD concentration was subsequently confirmed by testing 20 replicates on the FilmArray 2.0 and 20 replicates on the FilmArray Torch, for a total of 40 replicates at 1×LoD. An additional 20 replicates per system were tested at a 10-fold lower concentration, for a total of 40 replicates at 0.1×LoD. The required criteria for confirmation of LoD was a detection rate of at least 95% at 1×LoD (≥19/20 per system) and a detection rate of less than 95% below LoD (≤19/20 detected per system at 0.1×LoD). A multi-spiked approach was employed in both the LoD estimation and the LoD confirmation studies where samples spiked with up to five analytes at various concentrations were tested in the LoD studies. An equivalence study was performed previously which demonstrated that the multi-spiked samples were equivalent to the single-spiked samples and employing the multi-spiking approach did not impact FilmArray RP test performance. Refer to the “Single-Spiked vs. Multi-Spiked 26 {26} Specimen Study” section. LoD confirmation criteria were met for each analyte on both the FilmArray 2.0 and FilmArray Torch systems at the same concentration. The confirmed LoD concentrations are shown in Table 9 below with detected results at the 1× (shaded) and 0.1× concentrations presented separately for each system and also combined. The LoD concentration for most analytes is reported in Copies/mL, as determined primarily by commercially-available quantitative PCR and RT-PCR assays. Refer to the “Quantification of Nucleic Acids Derived from Viral and Bacterial Cultures” section. In addition, viable units for the specific cultures tested (50% tissue culture infectious dose (TCID₅₀/mL) or colony forming unit (CFU/mL)) based on the values obtained from the respective Certificate of Analysis (CoA) are also presented. Table 9: Limit of Detection Confirmation Testing Results for Each RP2 Analyte at 1× LoD and 0.1× LoD on FilmArray 2.0, FilmArray Torch, and Combined | RP2 Analyte | Isolate/Strain | Concentration | FilmArray 2.0 | FilmArray Torch | Total | | --- | --- | --- | --- | --- | --- | | Adenovirus | Adenovirus Species A, Serotype 18 ATCC VR-19 | 7.6E+03 Copies/mL (5.0E+00 TCID₅₀/mL) | 20/20 | 19/20 | 39/40 | | | | | 100% | 95% | 98% | | | | 7.6E+02 Copies/mL (5.0E-01 TCID₅₀/mL) | 15/20 | 17/20 | 32/40 | | | | | 75% | 85% | 80% | | | Adenovirus Species B, Serotype 7A Zeptometrix 0810021CF | 3.9E+01 Copies/mL (5.0E-02 TCID₅₀/mL) | 20/20 | 20/20 | 40/40 | | | | | 100% | 100% | 100% | | | | 3.9E+00 Copies/mL (5.0E-03 TCID₅₀/mL) | 13/20 | 13/20 | 26/40 | | | | | 65% | 65% | 65% | | | Adenovirus Species C, Serotype 2 ATCC VR-846 | 3.7E+01 Copies/mL (2.0E+00 TCID₅₀/mL) | 19/20 | 20/20 | 39/40 | | | | | 95% | 100% | 98% | | | | 3.7E+00 Copies/mL (2.0E-01 TCID₅₀/mL) | 13/20 | 13/20 | 26/40 | | | | | 65% | 65% | 65% | | | Adenovirus Species D, Serotype 37 Zeptometrix 0810119CF | 9.0E+00 Copies/mL (5.0E-02 TCID₅₀/mL) | 20/20 | 20/20 | 40/40 | | | | | 100% | 100% | 100% | | | | 9.0E-01 Copies/mL (5.0E-03 TCID₅₀/mL) | 13/20 | 10/20 | 23/40 | | | | | 65% | 50% | 58% | | | Adenovirus Species E, Serotype 4a S. Carolina/2004, UIRF | 3.0E+03 Copies/mL (1.0E+01 TCID₅₀/mL) | 19/20 | 19/20 | 38/40 | | | | | 95% | 95% | 95% | | | | 3.0E+02 Copies/mL (1.0E+00 TCID₅₀/mL) | 1/20 | 6/20 | 7/40 | | | | | 5% | 30% | 18% | | | Adenovirus Species F, Serotype 41 Tak, ATCC VR-930 | 1.2E+02 Copies/mL (1.0E+00 TCID₅₀/mL) | 20/20 | 20/20 | 40/40 | | | | | 100% | 100% | 100% | | | | 1.2E+01 Copies/mL (1.0E-01 TCID₅₀/mL) | 12/20 | 7/20 | 19/40 | | | | | 60% | 35% | 48% | | Coronavirus 229E | Coronavirus 229E ATCC VR-740 | 6.5E+01 Copies/mL (4.0E-01 TCID₅₀/mL) | 20/20 | 20/20 | 40/40 | | | | | 100% | 100% | 100% | | | | 6.5E+00 Copies/mL (4.0E-02 TCID₅₀/mL) | 14/20 | 17/20 | 31/40 | | | | | 70% | 85% | 78% | 27 {27} | Coronavirus HKU1 | Coronavirus HKU1 Clinical specimen a | 2.0E+03 RNA Copies/mL | 20/20 | 20/20 | 40/40 | | --- | --- | --- | --- | --- | --- | | | | | 100% | 100% | 100% | | | | 2.0E+02 RNA Copies/mL | 11/20 | 13/20 | 24/40 | | | | | 55% | 65% | 60% | | Coronavirus NL63 | Coronavirus NL63 BEI NR-470 | 5.4E+01 Copies/mL (2.5E-01 TCID\( _{50} \)/mL) | 20/20 | 20/20 | 40/40 | | | | | 100% | 100% | 100% | | | | 5.4E+00 Copies/mL (2.5E-02 TCID\( _{50} \)/mL) | 12/20 | 10/20 | 22/40 | | | | | 60% | 50% | 55% | | Coronavirus OC43 | Coronavirus OC43 ATCC VR-759 | 5.6E+02 Copies/mL (3.0E+01 TCID\( _{50} \)/mL) | 19/20 | 20/20 | 39/40 | | | | | 95% | 100% | 98% | | | | 5.6E+01 Copies/mL (3.0E+00 TCID\( _{50} \)/mL) | 14/20 | 11/20 | 25/40 | | | | | 70% | 55% | 63% | | Human Metapneumovirus | Human Metapneumovirus 16, Type A1 IA10-2003 Zeptometrix | 1.2E+03 Copies/mL (1.0E+01 TCID\( _{50} \)/mL) | 20/20 | 20/20 | 40/40 | | | | | 100% | 100% | 100% | | | | 1.2E+02 Copies/mL (1.0E+00 TCID\( _{50} \)/mL) | 16/20 | 17/20 | 33/40 | | | | | 80% | 85% | 83% | | Human Rhinovirus/ Enterovirus | Enterovirus Species D, Serotype 68 ATCC VR-1823 | 2.6E+01 Copies/mL (3.0E+02 TCID\( _{50} \)/mL) | 20/20 | 20/20 | 40/40 | | | | | 100% | 100% | 100% | | | | 2.6E+00 Copies/mL (3.0E+01 TCID\( _{50} \)/mL) | 12/20 | 11/20 | 23/40 | | | | | 60% | 55% | 58% | | | Human Rhinovirus Type 1A Zeptometrix 0810012CFN | 3.8E+01 Copies/mL (1.0E-01 TCID\( _{50} \)/mL) | 20/20 | 20/20 | 40/40 | | | | | 100% | 100% | 100% | | | | 3.8E+00 Copies/mL (1.0E-02 TCID\( _{50} \)/mL) | 11/20 | 13/20 | 24/40 | | | | | 55% | 65% | 60% | | Influenza A H1 | Influenza A H1N1 A/New Caledonia/20/99 Zeptometrix 0810036CF | 1.4E+02 Copies/mL (1.0E+03 TCID\( _{50} \)/mL) | 20/20 | 20/20 | 40/40 | | | | | 100% | 100% | 100% | | | | 1.4E+01 Copies/mL (1.0E+02 TCID\( _{50} \)/mL) | 9/20 | 12/20 | 21/40 | | | | | 45% | 60% | 53% | | Influenza A H1-2009 | Influenza A H1N1pdm09 A/Swine/NY/03/2009 Zeptometrix | 3.3E+02 Copies/mL (5.0E-01 TCID\( _{50} \)/mL) | 20/20 | 20/20 | 40/40 | | | | | 100% | 100% | 100% | | | | 3.3E+01 Copies/mL (5.0E-02 TCID\( _{50} \)/mL) | 8/20 | 15/20 | 23/40 | | | | | 40% | 75% | 58% | | Influenza A H3 | Influenza H3N2 A/Port Chalmers/1/73 ATCC VR-810 | 2.1E+01 Copies/mL (1.0E-01 TCID\( _{50} \)/mL) | 20/20 | 20/20 | 40/40 | | | | | 100% | 100% | 100% | | | | 2.1E+00 Copies/mL (1.0E-02 TCID\( _{50} \)/mL) | 8/20 | 9/20 | 17/40 | | | | | 40% | 45% | 43% | | Influenza B | Influenza B B/FL/04/06 Zeptometrix 0810255CF | 3.4E+01 Copies/mL (5.0E+00 TCID\( _{50} \)/mL) | 20/20 | 20/20 | 40/40 | | | | | 100% | 100% | 100% | | | | 3.4E+00 Copies/mL (5.0E-01 TCID\( _{50} \)/mL) | 14/20 | 9/20 | 23/40 | | | | | 70% | 45% | 58% | | Parainfluenza Virus 1 | Parainfluenza Virus 1 Type 1 Zeptometrix 0810014CF | 1.0E+03 Copies/mL (5.0E+00 TCID\( _{50} \)/mL) | 20/20 | 20/20 | 40/40 | | | | | 100% | 100% | 100% | | | | 1.0E+02 Copies/mL (5.0E-01 TCID\( _{50} \)/mL) | 16/20 | 15/20 | 31/40 | | | | | 80% | 75% | 78% | 28 {28} | Parainfluenza Virus 2 | Parainfluenza Virus 2 Type 2 Zeptometrix 0810015CF | 3.0E+01 Copies/mL (5.0E-01 TCID\( _{50} \)/mL) | 20/20 | 20/20 | 40/40 | | --- | --- | --- | --- | --- | --- | | | | | 100% | 100% | 100% | | | | 3.0E+00 Copies/mL (5.0E-02 TCID\( _{50} \)/mL) | 3/20 | 9/20 | 12/40 | | | | | 15% | 45% | 30% | | Parainfluenza Virus 3 | Parainfluenza Virus 3 Type 3 Zeptometrix 0810016CF | 3.8E+01 Copies/mL (2.5E+00 TCID\( _{50} \)/mL) | 19/20 | 20/20 | 39/40 | | | | | 95% | 100% | 98% | | | | 3.8E+00 Copies/mL (2.5E-01 TCID\( _{50} \)/mL) | 9/20 | 10/20 | 19/40 | | | | | 45% | 50% | 48% | | Parainfluenza Virus 4 | Parainfluenza Virus 4 Type 4a Zeptometrix 0810060CF | 1.6E+03 Copies/mL (5.0E+01 TCID\( _{50} \)/mL) | 20/20 | 20/20 | 40/40 | | | | | 100% | 100% | 100% | | | | 1.6E+02 Copies/mL (5.0E+00 TCID\( _{50} \)/mL) | 14/20 | 12/20 | 26/40 | | | | | 70% | 60% | 65% | | Respiratory Syncytial Virus | Respiratory Syncytial Virus Type A Zeptometrix 0810040ACF | 9.0E+00 Copies/mL (2.0E-02 TCID\( _{50} \)/mL) | 19/20 | 20/20 | 39/40 | | | | | 95% | 100% | 98% | | | | 9.0E-01 Copies/mL (2.0E-03 TCID\( _{50} \)/mL) | 7/20 | 8/20 | 15/40 | | | | | 35% | 40% | 38% | | Bordetella parapertussis (IS1001) | B. parapertussis A747 Zeptometrix 0801461 | 6.0E+01 IS1001 Copies/mL\( ^b \)(4.1E+01 CFU/mL) | 20/20 | 19/20 | 39/40 | | | | | 100% | 95% | 98% | | | | 6.0E+00 IS1001 Copies/mL\( ^b \)(4.1E+00 CFU/mL) | 9/20 | 7/20 | 16/40 | | | | | 45% | 35% | 40% | | Bordetella pertussis (pxP) | B. pertussis A639 Zeptometrix 0801459 | 1.0E+03 CFU/mL | 20/20 | 20/20 | 40/40 | | | | | 100% | 100% | 100% | | | | 1.0E+02 CFU/mL | 14/20 | 10/20 | 24/40 | | | | | 70% | 50% | 60% | | Chlamydia pneumoniae | C. pneumoniae TW183 ATCC VR-2282 | 6.6E+01 Copies/mL (1.0E-01 TCID\( _{50} \)/mL) | 20/20 | 19/20 | 39/40 | | | | | 100% | 95% | 98% | | | | 6.6E+00 Copies/mL (1.0E-02 TCID\( _{50} \)/mL) | 11/20 | 10/20 | 21/40 | | | | | 55% | 50% | 53% | | Mycoplasma pneumoniae | M. pneumoniae M129 Zeptometrix 0801579 | 4.6E+02 Copies/mL\( ^c \)(1.0E+01 TCID\( _{50} \)/mL) | 20/20 | 20/20 | 40/40 | | | | | 100% | 100% | 100% | | | | 4.6E+01 Copies/mL\( ^c \)(1.0E+00 TCID\( _{50} \)/mL) | 14/20 | 10/20 | 24/40 | | | | | 70% | 50% | 60% | \( ^{a} \) A cultured isolate of Coronavirus HKU1 was not available for testing. LoD for Coronavirus HKU1 was therefore determined by testing dilutions of a clinical NPS specimen known to contain the virus. The amount of viral RNA in the specimen (in RNA copies/mL) was determined by real-time RT-PCR against a standard curve. \( ^{b} \) IS1001 sequences can be present in more than one copy per cell, so the relationship between CFU/mL and copies/mL may vary from strain to strain and culture to culture. LoD was determined based on the copy number of IS1001 measured by an independent quantitative real-time PCR assay and for this culture. \( ^{c} \) 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. ### e. Analytical Reactivity: The analytical reactivity/inclusivity of the FilmArray RP2 for each reported analyte was initially assessed by in silico analysis and subsequently by testing unique strains or isolates. Isolate/strain selection emphasized variants of known human clinical 29 {29} significance, including relevant species, serotypes, genotypes, isolates collected at various times and from different locations, recently circulating or newly emerging variants, or any strains of particular interest based on sequence alignments, as available. For more genetically diverse analytes a larger collection of isolates were selected in order to better demonstrate and characterize the analytical reactivity of the assay(s) with the expected diversity of the analyte. Each isolate/strain was prepared and tested as a contrived sample in Remel M4 Transport Medium at a concentration near the LoD (3×LoD) in triplicate, with one replicate on each of three different reagent lots. The majority of isolates were tested based on molecular quantification in units of copies/mL, though a few were tested in viable units of TCID$_{50}$/mL or CFU/mL. Any isolate that was not reliably detected in 3/3 or 4/5 replicates at the 3×LoD level, was tested again at 10×LoD or a higher concentration, as needed, until detection was observed or a limitation on assay analytical reactivity with that isolate was identified. Analytical reactivity wet testing results are summarized in Table 10 to Table 21 below. **Table 10: Adenovirus Isolates Tested and Detected by FilmArray RP2** | Species ^{a} | Serotype | Isolate ID/Source | [Strain/Location/Year] | xLoD Detected | Result | | --- | --- | --- | --- | --- | --- | | A | 12 | ATCC VR-863 | [Huie/Massachusetts] | 3x | Adenovirus Detected | | | 18 | ATCC VR-19 | [Washington DC/1954] | 1x | | | | 31 | Zeptometrix 0810073CF | - | 3x | | | B | 3 | Zeptometrix 0810062CF | - | 3x | | | | 7A | Zeptometrix 0810021CF | - | 1x | | | | 7d/d2 | Univ of Iowa Research Foundation | [Iowa/2001] | 3x | | | | 7h | Univ of Iowa Research Foundation | [Iowa/1999] | 3x | | | | 11 | Univ of Iowa Research Foundation | [Wisconsin/2005] | 3x | | | | 14 | Univ of Iowa Research Foundation | [Missouri/2005] | 3x | | | | 16 | ATCC VR-17 | [CH.79/Saudi Arabia/1955] | 3x | | | | 21 | Univ of Iowa Research Foundation | [Missouri/2005] | 3x | | | | 34 | ATCC VR-716 | [Compton/1972] | 3x | | | | 35 | ATCC VR-718 | [Holden] | 3x | | | | 50 | ATCC VR-1602 | [Wan/Amsterdam/1988] | 3x | | | C | 1 | Zeptometrix 0810050CF | - | 3x | | | | 2 | ATCC VR-846 | [Adenoid 6] | 1x | | | | 5 | Zeptometrix 0810020CF | - | 3x | | | | 6 | ATCC VR-6 | [Tonsil 99/Washington DC] | 3x | | | D | 8 | Zeptometrix 0810069CF | - | 3x | | | | 20 | Zeptometrix 0810115CF | - | 3x | | | | 37 | Zeptometrix 0810119CF | - | 1x | | 30 {30} | E | 4a | Univ of Iowa Research Foundation | [S Carolina/2004] | 1x | | | --- | --- | --- | --- | --- | --- | | | 4 | Zeptometrix 0810070CF | - | 3x | | | F | 40 | Zeptometrix 0810084CF | - | 3x | | | | | NCPV 0101141v | - | 3x | | | | 41 | ATCC VR-930 | [Tak/73-3544/Netherlands/1973] | 1x | | | | | Zeptometrix 0810085CF | - | 3x | | \( ^{a} \) In silico analysis of available sequences predicts that the FilmArray RP2 will also react with Adenovirus B55, C57, species D serotypes, and G52. Table 11: Coronavirus Isolates/Specimens Tested and Detected by FilmArray RP2 | Coronavirus Type | Isolate ID/Source | [Location/Year] | xLoD Detected | Result | | --- | --- | --- | --- | --- | | 229E | ATCC VR-740 | - | 1x | Coronavirus 229E Detected | | | Zeptometrix 0810229CF | - | 3x | | | HKU1 | Clinical Specimen | [Utah/2015] | 1x | Coronavirus HKU1 Detected | | | Clinical Specimen | [Utah/2015] | 3x | | | | Clinical Specimen | [Utah/2015] | 3x | | | | Clinical Specimen | [S. Carolina/2010] | 3x | | | | Clinical Specimen | [Detroit/2010] | 3x | | | NL63 | BEI NR-470a | [Amsterdam/2003] | 1x | Coronavirus NL63 Detected | | | Zeptometrix 0810228CF | - | 3x | | | OC43 | ATCC VR-759b | - | 1x | Coronavirus OC43 Detected | | | Zeptometrix 0810024CF | - | 3x | | \( ^{a} \) Organism obtained through the NIH Biodefense and Emerging Infections Research Resources Repository, NIAID, NIH: Human Coronavirus NL63, NR-470. \( ^{b} \) Discontinued part number; see ATCC VR-1558. Table 12: Human Metapneumovirus Isolates Tested and Detected by FilmArray RP2 | Genotype | Serotype | Isolate ID/Source | [Location/Year] | xLoD Detected | Result | | --- | --- | --- | --- | --- | --- | | A1 | 16 | Zeptometrix 0810161CF | [Iowa10/2003] | 1x | Human Metapneumovirus Detected | | | 9 | Zeptometrix 0810160CF | [Iowa3/2002] | 3x | | | A2 | 20 | Zeptometrix 0810163CF | [Iowa14/2003] | 3x | | | | 27 | Zeptometrix 0810164CF | [Iowa27/2004] | 3x | | | B1 | 3 | Zeptometrix 0810156CF | [Peru2/2002] | 3x | | | | 5 | Zeptometrix 0810158CF | [Peru3/2003] | 3x | | | | 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 | U…