key: cord-312849-vgzvpwz9
authors: Eckbo, Eric J.; Locher, Kerstin; Caza, Melissa; Li, Lisa; Lavergne, Valery; Charles, Marthe
title: Evaluation of the BioFire® COVID-19 Test and Respiratory Panel 2.1 for Rapid Identification of SARS-CoV-2 in Nasopharyngeal Swab Samples
date: 2020-11-10
journal: Diagn Microbiol Infect Dis
DOI: 10.1016/j.diagmicrobio.2020.115260
sha: 
doc_id: 312849
cord_uid: vgzvpwz9

The BioFire® COVID-19 Test and Respiratory Panel 2.1 (RP2.1) are rapid, fully automated assays for the detection of SARS-CoV-2 in nasopharyngeal swabs. In the case of the RP2.1, an additional 21 viral and bacterial pathogens can be detected. Both tests have received emergency use authorization from the U.S. Food & Drug Administration and Interim Order authorization from Health Canada for use in clinical laboratories. We evaluated the performance characteristics of these tests in comparison to a laboratory-developed real-time PCR assay targeting the viral RdRP and E genes. A total of 78 tests were performed using the BioFire COVID-19 Test, including 30 clinical specimens and 48 tests in a limit of detection (LoD) study; 57 tests were performed using the RP2.1 for evaluation of SARS-CoV-2 detection, including 30 clinical specimens and 27 tests for LoD. Results showed 100% concordance between the BioFire assays and the laboratory-developed test for all clinical samples tested, and acceptable performance of both BioFire assays at their stated limits of detection. Conclusively, the BioFire COVID-19 Test and RP2.1 are highly sensitive assays that can be effectively used in the clinical laboratory for rapid SARS-CoV-2 testing.

Health Canada (2, 3) . The Infectious Diseases Society of America (IDSA) guidelines on the diagnosis of COVID-19, released in May 2020, advocate for nucleic acid amplification testing of all symptomatic individuals even when clinical suspicion is low (4) . The guidelines highlight the fact that a knowledge gap currently exists regarding the use of rapid tests (defined as turnaround time ≤ 1 hour) versus standard RNA tests in terms of performance characteristics, and therefore makes no recommendations for or against them.

The BioFire tests are fully automated multiplexed PCR assays that detect multiple SARS-CoV-2 regions.

The tests offer a rapid turn-around time while requiring very little hands-on time. The assays have both received EUA authorization in the U.S. and Interim Order authorization in Canada for use with nasopharyngeal (NP) swabs. The COVID-19 Test was developed with funding from the U.S. Department of Defense, and detects three targets within the open reading frame (ORF) region of the viral genome (5) . As a result of the contractual agreement with the Department of Defense, the RP2.1 panel was developed with two novel genome targets -the spike (S) and membrane (M) proteins. The RP2.1 builds upon the RP2 panel, which includes 21 viral and bacterial targets. This panel was extensively evaluated in a prospective study analyzing 1,612 nasopharyngeal swabs, with 33,843 results generated; the evaluation demonstrated excellent analytical sensitivity and specificity of the multiplex panel (6) .

This report describes the results of an independent evaluation of the performance characteristics of the BioFire COVID-19 Test and the RP2.1 for detection of SARS-CoV-2. The assays were compared to our laboratory-developed test (LDT) targeting the SARS-CoV-2 RNA-dependent RNA polymerase (RdRP) and envelope protein (E) genes, for which the average turnaround time, from receipt to result, is 5 -9 hours.

In the first part of the evaluation, a retrospective analysis of 25 SARS-CoV-2 positive and 5 negative patient specimens was performed using both BioFire assays on the FilmArray Torch system. In the second part of the evaluation, viral transport media from nasopharyngeal swab collection devices was spiked with reference material containing inactivated whole virus of known concentrations, and tested in parallel using BioFire and our LDT. the BioFire FilmArray Torch instrument and run using the provided software. The COVID-19 Test is a combination of 3 independent and non-overlapping assays, 2 of which have genomic targets in the ORF1ab region and 1 with a target in the ORF8 region. SARS-CoV-2 was interpreted by the software as "detected" when at least 2 out of the 3 assays were positive based on melt curve analysis. In the event of a single positive target, interpretation will be deemed "equivocal" and the user will be prompted to retest the original sample. The tentative limit of detection (LoD) of the test is 330 genomic copies per millilitre, as published by the manufacturer (9). The RP2.1 contains two independent assays for SARS-CoV-2 with targets in the S and M protein genomic regions. SARS-CoV-2 is reported as "detected" if one or both targets are detected. The LoD is reported as 500 genomic copies per millilitre for heat inactivated virus, and 160 genomic copies per millilitre for infectious virus (10) . Quality control was performed using the BioFire External Positive Control as per the manufacturer protocol prior to study testing. 

The 25 previously positive specimens all tested concordantly positive by the BioFire COVID-19 Test, representing a positive percent agreement of 100% between the two assays (Table 1) . Three samples had an initial LDT E gene Ct value between 38 and 40; of those, 2 were positive for all three targets included in the BioFire COVID-19 Test, whereas one sample was positive for two targets (ORF1ab) and negative for the third (ORF8). This result was interpreted as a positive result by the BioFire software. Similarly, the RP2.1 reported all 25 SARS-CoV-2 positive specimens as detectable. Five previously confirmed SARS-CoV-2 negative NP swabs were concordantly negative for the virus by both BioFire assays, and the RP2.1 accurately detected the alternate viral pathogens in all 5 specimens. There were no discordant results observed, for an overall agreement of 100% for both assays. 

All replicates with concentrations at or slightly above the LoD were positive by the BioFire COVID-19

Test, and both gene targets were detected in the LDT ( The Ct value for SARS-CoV-2 genomic targets obtained during testing is dependent on the initial concentration of nucleic acid in the specimen; consequently, this value has been used as semiquantitative means of tracking viral burden over time (11 -13) and has been shown to be correlated with disease severity (14) . As the next influenza season approaches, it will become increasingly important for laboratories to rapidly detect and differentiate co-circulating respiratory pathogens. The flexibility to redesign and adapt existing multiplex assays to expediently match changes in epidemiology is a highly innovative feature, and could offer extensive benefits to infection control and public health domains. The BioFire COVID-19 Test and Respiratory Panel 2.1 are easy-to-use, highly sensitive, and rapid assays for the detection of SARS-CoV-2 in nasopharyngeal swab specimens. This evaluation demonstrates that the assays perform comparably to our laboratory developed real-time PCR assay, with 100% agreement in testing results for clinical specimens and acceptable performance at their stated limits of detection. The

BioFire FilmArray platform is designed for use in a clinical laboratory and is not approved as a point -of-care test in Canada; it does, however, still maintain the benefits of rapid turnaround time and random access (no batching required). Additionally, it does not require any technical expertise on the part of the user, which is a significant benefit when highly skilled laboratory staff are limited. The BioFire assays are viable alternatives to traditional real-time SARS-CoV-2 PCR assays for rapid results in the laboratory, including in remote areas where higher complexity assays are not feasible. Although the future of the BioFire COVID-19 Test remains unclear with the subsequent introduction of the RP2.1, this singlepathogen test may prove useful for pooling of patient specimens as resources become more limited during the pandemic.

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