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The BioFire Respiratory Panel 2.1 (RP2.1) is a PCR-based multiplexed nucleic acid test intended for use with the BioFire FilmArray 2.0 or BioFire 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, including COVID-19.

The following organism types and subtypes are identified using the BioFire RP2.1:

• Adenovirus, ●
• Coronavirus 229E. ●
• Coronavirus HKU1, ●
• Coronavirus NL63, ●
• Coronavirus OC43.
• Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2), ●
• 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, and ●
• Mycoplasma pneumoniae ●

Nucleic acids from the respiratory viral and bacterial organisms identified by this test are generally detectable in NPS specimens during the acute phase of infection. The detection and identification of specific viral and bacterial nucleic acids from individuals exhibiting signs and/or symptoms of respiratory infection is indicative of the presence of the identified microorganism and 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 an NPS specimen. Positive results do not rule out coinfection with other organisms. The agent(s) detected by the BioFire RP2.1 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.

The BioFire Respiratory Panel 2.1 is designed to simultaneously identify 22 different potential pathogens of the respiratory tract infection, including the novel coronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), from a single NPS specimen in transport medium or saline. BioFire RP2.1 is compatible with BioFire's PCR-based in vitro diagnostic BioFire FilmArray 2.0 and BioFire FilmArray Torch systems for infectious disease testing. A specific software module (i.e., BioFire RP2.1 Pouch Module Software) is used to perform BioFire RP2.1 testing on these systems.

The RP2.1 reagent kit contains all the materials required to complete tests and includes the RP2.1 pouch, hydration solution, sample buffer, and sample handling components such as transfer pipettes. The RP2.1 pouches are used to test patient samples and is a closed-system disposable that stores all the necessary reagents for sample preparation reverse transcription. polymerase chain reaction (PCR), and detection in order to isolate, amplify, and detect nucleic acid from multiple pathogens within a single NPS specimen. The rigid plastic component ("fitment") of the pouch contains reagents in freeze-dried form. The flexible plastic portion of the pouch is divided into discrete segments ("blisters") where the required chemical processes are carried out. After sample collection, the user injections hydration solution and sample combined with BioFire Sample Buffer into the pouch, places the pouch into a FilmArray instrument, and starts the run. All other operations are automated.

The FilmArray instruments (FilmArray 2.0 and FilmArray Torch systems) interact with the pouch mechanically, thermally, and optically to drive the multi-step chemical process required for purification and detection of specific nucleic acid targets from the patient sample. FilmArray instruments follow a protocol defined in the BioFire RP2.1 Pouch Module Software that is downloaded from the host computer prior to runtime. The instrument protocol defines the specific sequence of the testing process, including the times and temperatures, as the instrument performs bead-based extraction/isolation/purification of nucleic acids, performs reverse transcription and a 2-stage nested PCR reaction, executes DNA melt and fluorescent signal detection, and monitors system performance in real time, and communicates results and errors to the user via software. The primary difference between the FilmArray 2.0 and FilmArray Torch systems is the external configuration of multiple modules in a system. Up to eight FilmArray 2.0 modules can be connected to one computer and pouch loading station, while up to 12 FilmArray Torch modules can be connected to one system base in a vertical stack to a computer and pouch loading station. In addition, the pouches are front-loaded via an automated mechanism for the Torch system whereas the pouches are manually inserted, removed, and there is pouch and lid sensing in the FilmArray 2.0.

Once a test run is completed, the software automatically interprets the results and displays a test report. The report can be printed and/or saved as a file. The test report is a single page containing three sections: Run Summary, Result Summary, and Run Details. An additional section, Change Summary, is present in specific situations. The overall layout of the report was previously described in the BioFire RP2 510(k) [K170604] and remains unchanged for the BioFire RP2.1.

Here's a breakdown of the acceptance criteria and study proving the BioFire Respiratory Panel 2.1 (RP2.1) meets those criteria, based on the provided text.

Acceptance Criteria and Reported Device Performance

The acceptance criteria are generally implied by the successful performance demonstrated in the various analytical and clinical studies. For qualitative tests like this, the key performance metrics are sensitivity (Positive Percent Agreement - PPA) and specificity (Negative Percent Agreement - NPA). The reproducibility study also defines implicit acceptance for consistent detection.

Table of Acceptance Criteria and Reported Device Performance (Summary for SARS-CoV-2 - a key new analyte)

Study Details

2. Sample Size and Data Provenance

• Test Set Sample Sizes:

  • Prospective Clinical Study: 524 valid NPS specimens.
  • Retrospective Clinical Study: 50 SARS-CoV-2 positive archived clinical specimens + 50 NPS specimens collected before Dec 2019 (expected negative for SARS-CoV-2) = 100 total specimens (98 evaluable due to instrument errors).
  • Contrived Clinical Specimen Study: 50 contrived clinical specimens (spiked with inactivated SARS-CoV-2) + 10 non-spiked specimens = 60 total specimens.
  • Clinical Comparison Study (RP2 vs RP2.1): 210 archived clinical NPS specimens.
  • Analytical Reproducibility: For each analyte tested, 120 valid runs per sample (total 360 valid runs for the entire study, across multiple analytes and concentrations).
  • Analytical LoD (SARS-CoV-2): 20 replicates each for 1x LoD and 0.1x LoD for inactivated virus; 20 replicates each for 1x LoD and 0.1x LoD for infectious virus.

• Data Provenance:

  • Prospective Clinical Study: Prospectively collected NPS specimens from three collection sites in the U.S. (fresh/recent collection).
  • Retrospective Clinical Study: Retrospective, archived clinical specimens from March and April 2020 (U.S.) and NPS specimens collected before December 2019 (U.S. from three geographically distinct laboratories).
  • Contrived Clinical Specimen Study: Unique NPS specimens collected before December 2019 (expected negative for SARS-CoV-2).
  • Clinical Comparison Study: Archived nasopharyngeal swab (NPS) specimens collected prior to December 2019, obtained from BioFire specimen repository and external sources.
  • Analytical Studies (Reproducibility, LoD, Reactivity, Specificity): Primarily used contrived samples with known concentrations of isolates/strains. Provenance of source materials (isolates) is provided (e.g., ATCC, Zeptometrix, BEI, WHO International Standard).

3. Number of Experts and Qualifications for Ground Truth

• The document does not explicitly state the number of experts or their specific qualifications (e.g., "radiologist with 10 years of experience") used to establish the ground truth for the clinical test sets.
• Ground Truth Determination for Clinical Studies:
  • Prospective Clinical Study (SARS-CoV-2): Ground truth was established using a composite comparator method of three U.S. FDA EUA tests. "Concordance for two out of three of the EUA tests were considered the final result for the comparator." This implies the "experts" were the results of these FDA EUA assays.
  • Retrospective Clinical Study (SARS-CoV-2): Ground truth (known positive/negative status) was based on previous characterization using the respective Emergency Use Authorization (EUA) assay employed at the collection site. This is also a comparator method.
  • Contrived Clinical Specimen Study (SARS-CoV-2): Ground truth was based on the known presence and concentration of the spiked inactivated SARS-CoV-2 isolate and the known SARS-CoV-2 negative status of the base clinical specimens. This is a known sample composition ground truth.
  • Clinical Comparison Study: Ground truth for analytes was based on results from the BioFire RP2 panel (for positive agreement) and the expected negative status for SARS-CoV-2 (for specificity).

4. Adjudication Method for the Test Set

• For the Prospective Clinical Study, the "composite comparator" method described an adjudication-like process:
  • Rule 1: Pos/Pos/Any -> Positive (if 2 of 3 EUA tests are positive, regardless of the third)
  • Rule 2: Neg/Neg/Any -> Negative (if 2 of 3 EUA tests are negative, regardless of the third)
  • Rule 3: Pos/Neg/Inv -> Specimen excluded (no clear consensus)
  • Rule 4: Inv/Inv/Any -> Specimen excluded (no clear consensus)
• No explicit human expert adjudication method is described. The adjudication relies on the consensus of the comparator FDA EUA tests.

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

• No MRMC comparative effectiveness study was done or reported.
• This device is an automated in vitro diagnostic (IVD) test, not an AI-assisted diagnostic tool for human readers. Therefore, there's no "human readers improve with AI vs without AI assistance" effect size to report. The comparison studies are on the device's performance against comparator devices or known ground truth.

6. Standalone (Algorithm Only) Performance

• Yes, standalone performance was done. The entire evaluation described in the "Performance Characteristics" section (analytical performance, comparison studies, and clinical studies) represents the standalone performance of the BioFire RP2.1 device, as it is an automated system that provides a qualitative result without human interpretation of raw signals. The software within the FilmArray instruments interprets the melt curve data and applies interpretation rules to determine the final test results.

7. Type of Ground Truth Used

• For Clinical Studies (SARS-CoV-2):
  • Prospective Clinical Study: Comparator Gold Standard (composite of three U.S. FDA EUA tests).
  • Retrospective Clinical Study: Comparator Gold Standard (previous EUA assay results at collection sites).
  • Contrived Clinical Specimen Study: Known Sample Composition / Definitive Ground Truth (spiked inactivated virus at known concentrations, or known negative specimens).
• For Analytical Studies (e.g., LoD, Reactivity, Specificity): Known Sample Composition / Definitive Ground Truth (contrived samples with specific isolates/strains at known concentrations).

8. Sample Size for the Training Set

• The document does not explicitly state the sample size for the training set.
• As an in vitro diagnostic (IVD) device, the development process typically involves internal analytical characterization and optimization (which can be considered analogous to "training" for a traditional assay, albeit not a machine learning model in the modern sense), followed by formal verification and validation studies. The analytical performance data (LoD, reactivity, specificity, etc.) are gathered on well-characterized samples to confirm the assay's performance.

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

• The concept of a "training set" with ground truth in the context of this traditional IVD device (not an AI/ML model) refers to the development and optimization of the assays themselves.
• The ground truth for these developmental/training phases would be established through a combination of:
  • Defined concentrations of specific viral/bacterial isolates or nucleic acids: This is seen in the LoD and analytical reactivity studies where concentrations are precisely measured (e.g., copies/mL, TCID50/mL, CFU/mL) using established quantification methods (e.g., ddPCR, qPCR, plate counting).
  • Known sample compositions: Creating samples with specific organisms present or absent at controlled levels.
  • Previous BioFire RP2 data: The document notes that the RP2.1 builds upon the previous RP2 panel, meaning much of the "training" (i.e., assay design, optimization of reaction conditions, primer selection for common pathogens) for the shared analytes was likely carried over and validated.
  • In silico analysis: Used for SARS-CoV-2 primer design and reactivity prediction against sequence databases (NCBI, GISAID) as a form of "training" or initial validation for robustness against known variants. This informs assay design.

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