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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.

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.

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.

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.

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