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The BioFire® COVID-19 Test 2 is a qualitative nested multiplexed RT-PCR in vitro diagnostic test intended for use with the BioFire® FilmArray® 2.0 and BioFire® FilmArray® Torch Systems. The BioFire COVID-19 Test 2 detects nucleic acids from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swabs (NPS) from symptomatic individuals suspected of COVID-19 by their healthcare provider.

Results are for the identification of SARS-CoV-2 RNA. The SARS-CoV-2 RNA is generally detectable in NPS specimens during the acute phase of infection. Positive results are indicative of the presence of SARS-CoV-2 RNA; clinical correlation with patient history and other diagnostic information is necessary to determine patient infection status. Positive results do not rule out co-infection with other pathogens.

Results are meant to be used in conjunction with other clinical, epidemiologic, and laboratory data, in accordance with the guidelines provided by the relevant public health authorities. The BioFire COVID-19 Test 2 is intended for use by trained medical and laboratory professionals in a laboratory setting or under the supervision of a trained laboratory professional.

For In Vitro Diagnostic Use.

The BioFire COVID-19 Test 2 is a multiplexed nucleic acid-based test for the detection of SARS-CoV-2 RNA from nasopharyngeal swabs (NPS) eluted in either transport medium or saline. The test was originally described and cleared in K211079. The BioFire COVID-19 Test 2 uses BioFire FilmArray technology and is for use with BioFire FilmArray 2.0 and BioFire FilmArray Torch instruments. Once the sample is injected into the FilmArray pouch is loaded into the Film Array instrument which performs all aspects of testing including nucleic acid extraction, reverse-transcription, and nested PCR with melt analysis. The currently cleared version of the test uses three SARS-CoV-2 assays and returns a 'SARS-CoV-2 Detected' call if one or more of the SARS-CoV-2 assays are positive.

The purpose of this submission is to display results for four additional SARS-CoV-2 assays which are currently present on the test, but for which results are masked through software. The assays are being unmasked as a mitigation against the risk of future SARS-CoV-2 variants affecting the sensitivity of the BioFire COVID-19 Test 2 due to mutations in assay primer regions. Note that to date BioFire Defense has not identified any variants that are predicted to affect the performance of the three-assay version of BioFire COVID-19 Test 2 described in K211079. These changes are being requested preemptively. The calling scheme when using the seven total SARS-CoV-2 assays will remain unchanged: one or more positive SARS-CoV-2 assay results will return an overall 'SARS-CoV-2 Detected' result.

Here's a breakdown of the acceptance criteria and the study details for the BioFire COVID-19 Test 2 based on the provided document:

1. Table of Acceptance Criteria and Reported Device Performance

The provided document describes a Special 510(k) submission where the primary change is the unmasking of four additional SARS-CoV-2 assays within an already cleared device (BioFire COVID-19 Test 2, K211079). Therefore, the "acceptance criteria" are implied by demonstrating equivalence to the predicate device's performance. The re-analysis of prior studies with the modified software is used to show this equivalence.

Since this is a submission for a software modification to unmask existing assays and not a de novo submission establishing new performance benchmarks, the acceptance criteria are largely defined by matching or showing no significant degradation from the predicate device's performance.

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

The document states that no additional testing was performed for this submission. Instead, all study data previously submitted in K211079 were re-analyzed using the updated software.

Therefore, the "test set" for this specific submission is the re-analysis of data from the original K211079 (and potentially EUA200044) studies.

• Clinical Testing (DF-SDY-030617):
  • Sample Size: 69 positive samples, 454 negative samples. (Total = 523 samples)
  • Data Provenance: Prospective Clinical Evaluation. The document does not explicitly state the country of origin, but generally, FDA submissions for predicate devices are expected to include data from diverse geographic regions within the US, or from regions with comparable patient populations.
• Bench Testing: Sample sizes vary per study. For example:
  • LoD studies (DF-SDY-029904, DF-SDY-030331) involve serial dilutions and replicates.
  • Specificity/Exclusivity (DF-SDY-029903, DF-SDY-030333) involve testing a panel of organisms/viruses.
  • Reproducibility (DF-SDY-030398) involves testing replicates across multiple sites.

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

The ground truth for the clinical samples (DF-SDY-030617) would typically be established by a reference method, most commonly another FDA-authorized RT-PCR assay. The document does not specify the number or qualifications of experts involved in determining the ground truth for the clinical samples. For molecular diagnostics, "expert consensus" is less common for ground truth than established reference tests.

For bench testing studies (e.g., LoD, inclusivity, exclusivity), the ground truth is based on the known concentration of spiked analytes or the known identity of the assayed organisms/viruses, which does not typically involve human experts establishing ground truth in the same way as clinical image interpretation.

4. Adjudication Method for the Test Set

The document does not describe an adjudication method for the test set regarding human interpretation, as the device is an in vitro diagnostic (IVD) molecular test for direct detection of SARS-CoV-2 RNA. Results are determined by the instrument and its software.

For the clinical study, the reference method used to establish positive/negative status for the clinical samples would be the "adjudication." However, the method (e.g., comparison to a composite reference standard, or another cleared test) is not detailed here.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was Done

No, an MRMC comparative effectiveness study was not done. This device is an in vitro diagnostic (IVD) device that performs a laboratory test. It does not involve human readers interpreting images or data with or without AI assistance. The output is a "SARS-CoV-2 Detected" or "SARS-CoV-2 Not Detected" result.

6. If a Standalone (Algorithm Only Without Human-in-the-Loop Performance) was done

Yes, the performance presented is standalone/algorithm only. The BioFire COVID-19 Test 2 is an IVD automated system. The results are generated directly by the device's software based on its internal processes (nucleic acid extraction, RT-PCR, melt analysis) without human interpretation of raw data. The study validates the device's ability to detect SARS-CoV-2 RNA independently. The phrasing "Software uses results from 7 assays" further confirms this.

7. The Type of Ground Truth Used

• Clinical Testing (DF-SDY-030617): The ground truth for the prospective clinical evaluation samples would most commonly be established by a highly sensitive and specific reference molecular assay (e.g., another FDA-cleared or EUA RT-PCR test for SARS-CoV-2), possibly combined with clinical diagnosis, but the document does not explicitly state this.
• Bench Testing:
  • LoD, Reactivity (Inclusivity): Ground truth is based on the known concentration and identity of specific SARS-CoV-2 strains/genomic material spiked into negative matrix.
  • Specificity (Exclusivity): Ground truth is based on the known identity of other respiratory pathogens or commensals tested.
  • Interfering Substances: Ground truth is based on the known presence of potential interfering substances without SARS-CoV-2.
• In Silico Analysis: Ground truth is based on publicly available genetic sequence databases (e.g., GISAID for SARS-CoV-2 variants).

8. The Sample Size for the Training Set

The document does not mention a separate training set for this submission. The purpose of this submission is to unmask existing assays on an already established device. The performance data presented are from validation and verification studies (effectively "test sets") previously conducted for the predicate device. For the original development of the BioFire COVID-19 Test 2 assays, various internal optimization and calibration steps (which could be considered analogous to "training") would have occurred, but these details are not part of this 510(k) summary.

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

As no specific "training set" is described for this 510(k) modification, this question is not directly applicable. For the initial development of the assays, the ground truth would have been established through a combination of:

• Bioinformatics: Designing primers and probes based on known SARS-CoV-2 sequences.
• Analytical studies: Testing synthetic targets and cultured virus at known concentrations.
• Clinical studies: Initial evaluation with patient samples where SARS-CoV-2 status was determined by a reference method.

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The BioFire COVID-19 Test 2 is a qualitative nested multiplexed RT-PCR in vitro diagnostic test intended for use with the BioFire FilmArray 2.0 and BioFire FilmArray Torch Systems. The BioFire COVID-19 Test 2 detects nucleic acids from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swabs (NPS) from symptomatic individuals suspected of COVID-19 by their healthcare provider.

Results are for the identification of SARS-CoV-2 RNA. The SARS-CoV-2 RNA is generally detectable in NPS specimens during the acute phase of infection. Positive results are indicative of the presence of SARS-CoV-2 RNA; clinical correlation with patient history and other diagnostic in necessary to determine patient infection status. Positive results do not rule out co-infection with other pathogens.

Results are meant to be used in conjunction with other clinical, epidemiologic, and laboratory data, in accordance with the guidelines provided by the relevant public health authorities. The BioFire COVID-19 Test 2 is intended for use by trained medical and laboratory professionals in a laboratory setting or under the supervision of a trained laboratory professional.

The BioFire COVID-19 Test 2 is a multiplexed nucleic acid-based test designed to be used with BioFire® FilmArray® Systems (BioFire® FilmArray® 2.0 or BioFire® FilmArray® Torch). The BioFire COVID-19 Test 2 consists of the BioFire COVID-19 Test 2 pouch which contains freeze-dried reagents to perform nucleic acid purification and nested, multiplexed polymerase chain reaction (PCR) with DNA melt analysis. The BioFire COVID-19 Test 2 conducts three independent tests for the detection of SARS-CoV-2 RNA in nasopharyngeal swabs (NPS) eluted in transport medium or saline. Results from the BioFire COVID-19 Test 2 are available in about 45 minutes.

A test is initiated by loading Hydration Solution into one port of the pouch and a NPS specimen mixed with the provided Sample Buffer into the other port of the pouch. The pouch contains all the reagents required for specimen testing and analysis in a freeze-dried format; the addition of Hydration Solution and the Sample Buffer rehydrates the reagents. After the pouch is prepared, the FilmArray Software on the FilmArray System guides the user through the steps of placing the pouch into the instrument, scanning the pouch barcode, entering the sample identification, selecting the appropriate protocol, and initiating the run on the FilmArray System.

The FilmArray instruments contain 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 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 subsequent melt.

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, a nested multiplexed PCR is executed in two stages. During the first stage, a single, large volume, multiplexed reverse transcription PCR (rt-PCR) reaction is performed. 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 Defense, LLC). The solution is then distributed to each well of the arrav. 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 and melt, or nested PCR, is performed 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 array captures fluorescent images of the PCR2 reactions The FilmArray software automatically analyzes the results of each DNA melt curve and the results of the internal pouch controls to provide a final test interpretation.

Here's a summary of the acceptance criteria and the study that proves the device meets them, based on the provided text:

1. Table of Acceptance Criteria and Reported Device Performance

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

• Clinical Performance Test Set:

  • Sample Size: 523 specimens (after exclusions). Initially 534 were assigned a study code. 11 excluded (7 for not meeting storage/comparator result, 4 for insufficient volume/no BioFire COVID-19 Test 2 result).
  • Data Provenance: Prospective clinical study conducted at three study sites in the United States over four months (July-October 2020) during the COVID-19 pandemic. Specimens were residual after standard of care (SoC) testing.

• Analytical Performance Test Sets:

  • Limit of Detection (LoD): 20 replicates for each LoD concentration tested (USA-WA1/2020 infectious and heat-inactivated). Contrived samples.
  • NPS in Saline Sensitivity Validation: 20 contrived samples.
  • FDA SARS-CoV-2 Reference Panel: Blinded samples from the FDA Reference Panel.
  • Inclusivity: Triplicate samples for each of 5 SARS-CoV-2 variants. Spiked into pooled NPS specimens.
  • Exclusivity: Panel of 77 viruses and organisms.
  • Interference: Samples with SARS-CoV-2 at 1x LoD, plus potentially interfering substances.
  • Reproducibility: 6 replicates per sample (moderate positive, low positive, negative), tested at 3 locations over 5 days (total 90 replicates per sample, 270 valid test results).
  • Specimen Storage: 10 contrived samples per storage condition/time point.

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

• Clinical Performance Study: The ground truth was established by comparison with a BioFire RP2.1 comparator result. This predicate device was authorized under EUA202392 and later granted de novo classification DEN200031. The text does not specify the number or qualifications of experts involved in determining the ground truth for SoC or BioFire RP2.1 results.

4. Adjudication Method for the Test Set

• Clinical Performance Study: The text describes the comparison method: "A BioFire COVID-19 Test 2 result ('Detected' or 'Not Detected') was considered True Positive (TP) or True Negative (TN) only when it agreed with the BioFire RP2.1 comparator result." This indicates a direct comparison to a single reference (BioFire RP2.1). There is no mention of an adjudication process (e.g., 2+1, 3+1) involving multiple human readers for discrepancies.

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

• No, an MRMC comparative effectiveness study was not done. The study focuses on the diagnostic performance of the device itself (standalone) compared to a predicate device, rather than the effect of the device on human reader performance.

6. Standalone Performance Study

• Yes, a standalone performance study was done. The entire clinical and analytical performance evaluation described focuses on the BioFire COVID-19 Test 2 algorithm's performance without specific mention of human-in-the-loop assistance influencing the reported performance metrics. The device automates nucleic acid purification and PCR, with the FilmArray software analyzing results to provide a final interpretation.

7. Type of Ground Truth Used

• Clinical Performance Study: The ground truth for the clinical performance was established using results from a legally marketed predicate device, the BioFire RP2.1 (which itself was authorized under EUA and later de novo classified). Additionally, for one FN and one FP case, the text mentions "SoC and Central Reference Lab (CRL) testing" as further evidence, suggesting a form of expert or established laboratory consensus for those specific cases.
• Analytical Studies: The ground truth for analytical studies (LoD, inclusivity, exclusivity, interference, reproducibility, specimen storage) was based on known concentrations of spiked SARS-CoV-2 material, known panels of other organisms/viruses, and known concentrations of interfering substances.

8. Sample Size for the Training Set

• The document does not provide information on the sample size used for the training set for the BioFire COVID-19 Test 2 algorithm. This information is typically proprietary to the manufacturer and not usually included in a 510(k) summary unless specifically requested or if the device utilizes machine learning that requires detailed training data descriptions.

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

• The document does not provide information on how the ground truth for the training set was established. As with the training set size, this detail is typically not openly disclosed in a 510(k) summary. For molecular diagnostic tests using RT-PCR, algorithm development often relies on a combination of known positive and negative samples, synthetic constructs, and potentially early clinical samples, with ground truth established through well-characterized reference methods (e.g., sequencing, highly sensitive RT-PCR assays, or expert consensus on clinical samples).

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