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The FilmArray Respiratory Panel 2 plus (RP2plus) is a multiplexed nucleic acid test intended for use with FilmArray 2.0 or FilmArray Torch systems for the simultaneous qualitative detection and identification of nucleic acids from Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and multiple common viral and bacterial respiratory pathogens in nasopharyngeal swabs (NPS) obtained from individuals meeting MERS-CoV clinical and/or epidemiological criteria.

Testing with the FilmArray RP2plus should not be performed unless the patient meets clinical and/or epidemiologic criteria for testing suspected MERS-CoV specimens. This includes: clinical signs and symptoms associated with MERS-CoV infection, contact with a probable or confirmed MERS-CoV case, history of travel to geographic locations where MERS-CoV cases were detected, or other epidemiological links for which MERS-CoV testing may be indicated.

The FilmArray RP2plus identifies:

• Middle East Respiratory Syndrome Coronavirus (MERS-CoV) ●
  And the following viral and bacterial respiratory pathogen types and subtypes:

• Adenovirus

• Coronavirus 229E ●

• Coronavirus HKU1 ●

• Coronavirus NL63

• Coronavirus OC43 ●

• Human Metapneumovirus

• Human Rhinovirus/Enterovirus ●

• Influenza A. including subtypes H1. H1-2009, and H3 ●

• Influenza B

• Parainfluenza Virus 1

• Parainfluenza Virus 2

• Parainfluenza Virus 3 ●

• Parainfluenza Virus 4

• Respiratory Syncytial Virus ●

• Bordetella parapertussis (IS 1001) ●

• Bordetella pertussis (ptxP)

• . Chlamydia pneumoniae

• . Mycoplasma pneumoniae

The detection and identification of specific viral and bacterial nucleic acids from MERS-CoV and other respiratory pathogens in individuals meeting MERS-CoV clinical and/or epidemiological criteria aids in the differential diagnosis of MERS-CoV infection, if used in conjunction with other clinical and epidemiological information in accordance with the guidelines provided by the appropriate public health authorities.

FilmArray RP2plus MERS-CoV positive results are for the presumptive identification of MERS-CoV. The definitive identification of MERS-CoV requires additional testing and confirmation procedures in consultation with the appropriate public health authorities (e.g., local or state public health departments, etc.) for whom reporting is necessary. The diagnosis of MERS-CoV infection must be made based on history, signs, symptoms, exposure likelihood, and other laboratory evidence in addition to the identification of MERS-CoV.

FilmArray RP2plus MERS-CoV negative results, even in the context of a FilmArray RP2plus positive result for one or more of the common respiratory pathogens, do not preclude MERS-CoV infection and should not be used as the sole basis for patient management decisions. The levels of MERS-CoV that would be present in NPS specimens from individuals with early infection and from asymptomatic MERS-CoV carriers are not well understood. The FilmArray RP2plus MERS-CoV negative results may also be due to lower respiratory tract infection with MERS-CoV that may not be detected by an NPS specimen. In this context, collection of lower respiratory and serum specimens (if possible) for MERS-CoV testing using other laboratory tests is highly recommended in addition to testing for MERS-CoV RNA in NPS specimens (i.e., upper respiratory specimens) using the FilmArray RP2plus. A negative FilmArray RP2plus MERS-CoV result in an asymptomatic individual does not rule out the possibility of future illness and does not demonstrate that the individual is not infectious.

Viral culture should not be attempted in the cases of positive FilmArray RP2plus results for MERS-CoV unless a BSL 3 facility is available to receive and culture specimens.

Negative FilmArray RP2plus results in the setting of a respiratory illness may be due to infection with pathogens that are not detected by this test, or other pathogens that may not be detected by an NPS specimen. Positive FilmArray RP2plus results do not rule out coinfection with other organisms: the agent(s) detected by the FilmArray RP2plus may not be the definite cause of disease.

Due to the genetic similarity between Human Rhinovirus and Enterovirus, the FilmArrav RP2plus cannot reliably differentiate them. A positive FilmArray RP2plus Rhinovirus/Enterovirus result should be followed up using an alternate method (e.g., cell culture or sequence analysis) if differentiation is required.

Performance characteristics for Influenza A were established when Influenza A H1-2009, A H1, and A H3 were the predominant Influenza A viruses in circulation. Performance of detecting Influenza A may vary if other Influenza A strains are circulating or a novel Influenza A virus emerges. If infection with a novel Influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent Influenza viruses and sent to state or local health departments for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.

The FilmArray Respiratory Panel 2 plus (RP2plus) is designed to simultaneously detect and identify MERS-CoV and 21 different common pathogens (see the Indications for Use section) of respiratory tract infection from a single NPS specimen in a time frame (~45 minutes) that may allow the test results to be used as an aid in determining appropriate patient treatment and management. FilmArray RP2plus is compatible with BioFire Diagnostics' (BioFire) PCR-based in vitro diagnostic FilmArray 2.0 and FilmArray Torch systems for infectious disease testing. A specific software module (i.e., FilmArray RP2plus pouch module) is used to perform FilmArray RP2plus testing on these systems.

A test is initiated by loading Hydration Solution into one port of the FilmArray pouch and a NPS sample (in transport media) mixed with the provided Sample Buffer into the other port of the FilmArray RP2plus pouch and placing it in a FilmArray instrument. The FilmArray pouch contains all the reagents required for specimen testing and analysis in a freeze-dried format; the addition of Hydration Solution and Sample/Buffer Mix rehydrates the reagents. After the pouch is prepared, the FilmArray Software guides the user though the steps of placing the pouch into the instrument, scanning the pouch barcode, entering the sample identification, and initiating the run.

The FilmArray instrument contains a coordinated system of inflatable bladders and seal points, which act on the pouch to control the movement of liquid between the pouch blisters. When a bladder is inflated over a reagent blister, it forces liquid from the blister into connecting channels. Alternatively, when a seal is placed over a connecting channel it acts as a valve to open or close a channel. In addition, electronically-controlled pneumatic pistons are positioned over multiple plungers in order to deliver the rehydrated reagents into the blisters at the appropriate times. Two Peltier devices control heating and cooling of the pouch to drive the PCR reactions and the melt curve analysis.

Nucleic acid extraction occurs within the FilmArray pouch using mechanical and chemical lysis followed by purification using standard magnetic bead technology. After extracting and purifying nucleic acids from the unprocessed sample, the FilmArray performs a nested multiplex PCR that is executed in two stages. During the first stage, the FilmArray performs a single, large volume, highly multiplexed reverse transcription PCR (RT-PCR) reaction, PCR1. The products from first stage PCR are then diluted and combined with a fresh, primerfree master mix and a fluorescent double stranded DNA binding dye (LC Green Plus, BioFire Diagnostics, LLC). The solution is then distributed to each well of the array. Array wells contain sets of primers designed specifically to amplify sequences internal to the PCR products generated during the first stage PCR reaction. The 2nd stage PCR, or nested PCR, PCR2. is performed in singleplex fashion in each well of the array. At the conclusion of the 2nd stage PCR, the array is interrogated by melt curve analysis for the detection of signature amplicons denoting the presence of specific targets. A digital camera placed in front of the 2nd stage PCR captures fluorescent images of the PCR reactions and software interprets the data.

The FilmArray Software automatically interprets the results of each DNA melt curve analysis and combines the data with the results of the internal pouch controls to provide a test result for each organism on the panel.

Table of Acceptance Criteria and Reported Device Performance:

2. Sample sizes used for the test set and the data provenance:

• Reproducibility Study Test Set: 120 data points per sample, across 4 contrived NPS samples, totaling 480 valid runs. The data was from a combined total of 15 different FilmArray 2.0 instruments and 19 different FilmArray Torch modules at three testing sites. The samples were contrived NPS samples in simulated VTM.
• LoD Confirmation Test Set: For each analyte, 40 replicates (20 on FilmArray 2.0, 20 on FilmArray Torch) at 1xLoD, and 40 replicates at 0.1xLoD. Samples were contrived in simulated NPS in VTM.
• Analytical Reactivity Test Set: Each isolate/strain was tested in triplicate (one replicate on each of three different reagent lots) near 3xLoD. Tested at 10xLoD or higher if not reliably detected. Samples were contrived in Remel M4 Transport Medium.
• Analytical Specificity/Cross-Reactivity Test Set: Each organism tested in triplicate on three different lots of FilmArray RP2plus pouches at high concentrations (typically ≥1.0E+06 CFU/ml for bacteria and fungi and ≥1.0E+05 TCID50/mL for viruses). Samples were in Remel M4 transport medium.
• Interfering Substances Test Set: Each test substance added to a contrived sample containing a mix of five analytes (each near LoD).
• Comparator Assay for B. parapertussis: Validation testing of the PCR followed by sequencing assays used samples (nature and number not specified beyond demonstrating equivalent analytical sensitivity to RP2plus).
• MERS-CoV Comparative Analytical Sensitivity: A MERS-CoV proficiency test panel (QCMD, U.S. government contracted) containing MERS-CoV samples and common coronaviruses.
• Prospective Clinical Study Test Set: 1612 residual NPS specimens in VTM after exclusions. Data provenance: Three geographically distinct U.S. study sites (Salt Lake City, UT; Chicago, IL; Columbus, OH) during portions of the 2015-2016 and 2016-2017 respiratory illness seasons. Samples were retrospective (archived/frozen) and prospective (fresh).
• Retrospective Clinical Study - Common Respiratory Pathogens Test Set: A total of 214 archived NPS in VTM specimens after exclusions. These were preselected specimens previously tested positive at the source laboratory. Data provenance: BioFire Diagnostics (testing site), specimens were archived from various source laboratories (presumably US-based).
• Retrospective Clinical Study - MERS-CoV Test Set: 3 archived retrospective NPS in VTM specimens from confirmed cases of MERS-CoV infection. Data provenance: Laboratory in South Korea during the 2015 MERS-CoV outbreak.
• Contrived Clinical Specimens Test Set: 50 MERS-CoV positive contrived specimens, 48 Influenza A H1 positive contrived specimens, and 50 un-spiked negative specimens. Samples were prepared using unique residual NPS specimens that previously tested negative from a clinical site. Data provenance: One of the three clinical sites from the prospective evaluation.

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts:

• Analytical Studies (Reproducibility, LoD, Analytical Reactivity, Analytical Specificity, Interfering Substances, MERS-CoV Comparative Analytical Sensitivity): The document does not specify a number of "experts" for ground truth outside of the internal process for developing the melt detector algorithm. Ground truth for these was established using known concentrations of cultured isolates, molecular quantification (qPCR/RT-PCR), or known positive/negative status of contrived samples. The melt detector algorithm's performance was compared to "expert annotation," but the number and qualifications of these experts are not explicitly stated.
• Prospective Clinical Study: Ground truth was established by an FDA-cleared multiplexed respiratory pathogens panel (for most analytes) and two analytically-validated PCR followed by bidirectional sequencing assays (for B. parapertussis). For MERS-CoV, the ground truth was the expected negative result in this population. The performance of these comparator methods, acting as the reference standard, generally implies expert oversight in their validation and historical use, but no specific human experts are mentioned for establishing the ground truth for this particular study.
• Retrospective Clinical Study - Common Respiratory Pathogens: Ground truth was established by the same comparator methods as the prospective study (FDA-cleared multiplexed respiratory pathogens panel and B. parapertussis PCR/sequencing).
• Retrospective Clinical Study - MERS-CoV: Ground truth was established by standard of care methods, specifically two rRT-PCR assays targeting upE and ORF1A (based on WHO primer/probe sequences) used during the 2015 MERS-CoV outbreak. These methods would have been operated by trained laboratory personnel, implying expertise, but no specific number or qualification of experts is provided for creating the ground truth for this study.
• Contrived Clinical Specimens: Ground truth was based on the known spiking of negative specimens with quantified isolates of Influenza A H1 and MERS-CoV.

4. Adjudication method (e.g. 2+1, 3+1, none) for the test set:

• Analytical Studies: Adjudication methods are not explicitly described for discrepancies in analytical tests. Deviations from expected results (e.g., non-detection at 3xLoD) led to retesting at higher concentrations. The Melt Detector algorithm validation involved comparison to "expert annotation" without a specified adjudication protocol.
• Clinical Studies (Prospective and Retrospective - Common Respiratory Pathogens): Discrepant results (FilmArray RP2plus vs. comparator method) were subjected to further investigation. The discrepancy investigation was mainly conducted by performing independent molecular methods with primers different from the FilmArray RP2plus and/or comparator method retesting. This indicates a form of adjudication, where a third, independent method or re-run of the comparator served to resolve the discrepancy, but a specific "X+Y" voting method (like 2+1) is not detailed.
• Retrospective Clinical Study - MERS-CoV: This study involved only three positive samples, and the FilmArray RP2plus showed 100% agreement. No discrepancies were observed, so no adjudication method was needed or described.
• Contrived Clinical Specimens: Ground truth was defined by the known spiking protocol. Any deviations from expected results were analyzed for causes (e.g., presence of a specific strain of Influenza A/Weiss/43 leading to equivocal result).

5. If a multi-reader multi-case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance?

This document describes the evaluation of a fully automated in vitro diagnostic (IVD) device (FilmArray Respiratory Panel 2 plus), which is a nucleic acid amplification test. It is not an AI-assisted diagnostic device where human readers interact with AI. Therefore, a multi-reader multi-case (MRMC) comparative effectiveness study evaluating human reader performance with and without AI assistance was not applicable and not performed. The device itself performs the analysis and provides a result without human interpretation of raw data.

6. If a standalone (i.e., algorithm only without human-in-the-loop performance) was done?

Yes, a standalone performance evaluation was done. The entire submission details the performance of the FilmArray RP2plus, which is described as a "multiplexed nucleic acid test intended for use with FilmArray 2.0 or FilmArray Torch systems for the simultaneous qualitative detection and identification of nucleic acids..." (Section D). The device "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" (Section I). This clearly indicates an algorithm-only (standalone) performance. The clinical studies compare the device's output directly to comparator methods, not to human interpretations that the device might aid.

7. The type of ground truth used (expert consensus, pathology, outcomes data, etc):

• Analytical Studies (Reproducibility, LoD, Analytical Reactivity, Analytical Specificity, Interfering Substances): The ground truth was primarily based on known concentrations of cultured isolates, molecular quantification (e.g., qPCR/RT-PCR), or the inherent design of contrived samples. For the internal Melt Detector validation, it was compared to "expert annotation."
• Prospective Clinical Study: The ground truth was established using a combination of FDA-cleared multiplexed respiratory pathogen panels (molecular comparator devices) and analytically-validated PCR followed by bidirectional sequencing assays for B. parapertussis. For MERS-CoV, the ground truth was the expected negative result in the general study population. Discrepancies were further investigated using independent molecular methods.
• Retrospective Clinical Study - Common Respiratory Pathogens: The ground truth was established by the same comparator methods as the prospective study (FDA-cleared multiplexed respiratory pathogen panels and B. parapertussis PCR/sequencing).
• Retrospective Clinical Study - MERS-CoV: The ground truth was established by standard of care reference molecular methods (two rRT-PCR assays based on WHO primer/probe sequences) used during the 2015 MERS-CoV outbreak, confirming positive MERS-CoV infection.
• Contrived Clinical Specimens: The ground truth was based on the known positive status due to spiking with quantified isolates and the known negative status of un-spiked specimens.

8. The sample size for the training set:

• Melt Detector Algorithm: The "Melt Detector" algorithm was "tuned against a large data set comprising typical and atypical melting curves... with expert annotation (positive or negative calls) during the development of the original FilmArray RP." A specific number for this training set is not explicitly provided in the given text, but it's described as "large" and stemming from the development of the original FilmArray RP.
• Other aspects of the FilmArray RP2plus development: The document hints at iterative development and testing, but a distinct "training set" in the context of machine learning for all aspects of the device is not detailed. The performance evaluations presented are essentially "test set" evaluations against defined ground truths.

9. How the ground truth for the training set was established:

• Melt Detector Algorithm Training: The ground truth for the "large data set comprising typical and atypical melting curves" used to tune the Melt Detector algorithm was established through "expert annotation (positive or negative calls)." The qualifications of these experts are not explicitly stated.

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