








key: cord-332723-rz1iilsv
authors: Creager, Hannah M.; Cabrera, Barbara; Schnaubelt, Andy; Cox, Jesse L.; Cushman-Vokoun, Allison M.; Shakir, Salika M.; Tardif, Keith D.; Huang, Meei-Li; Jerome, Keith R.; Greninger, Alexander L.; Drobysheva, Daria; Spaulding, Usha; Rogatcheva, Margarita; Bourzac, Kevin M.; Hinrichs, S.H.; Broadhurst, M.J.; Fey, P.D.
title: Clinical evaluation of the BioFire® Respiratory Panel 2.1 and detection of SARS-CoV-2
date: 2020-07-06
journal: J Clin Virol
DOI: 10.1016/j.jcv.2020.104538
sha: 
doc_id: 332723
cord_uid: rz1iilsv

We evaluated the performance of the BioFire® Respiratory Panel 2.1 (RP2.1) in the detection of SARS CoV-2 in comparison against three other SARS CoV-2 EUA assays. In these studies, the RP2.1 panel had 98% positive percent agreement (48/49) and 100% negative percent agreement (49/49). Since 30% of nasopharyngeal swab specimens have a SARS CoV-2 Ct >30 and thus detection of virus in low titers is clinically relevant, a sample with a high titer was diluted and each 10 fold dilution was tested in triplicate and compared against 6 other EUA approved SARS CoV-2 assays. These data suggested that the BioFire® RP2.1 panel, along with four other SARS CoV-2 assays (Roche cobas, Cepheid Xpert Xpress, BioFire® Defense COVID19, and NECoV19), consistently detected viral RNA at the 10-7 dilution. Overall, these studies suggest that the BioFire® RP2.1 assay can be used to detect acute cases of SARS CoV2 in addition to patients with low viral titer later in disease presentation.

The gold standard for SARS-CoV-2 diagnosis is detection of viral RNA in nasopharyngeal (NP) swab specimens. Sample-to-answer nucleic acid amplification assays for the detection of SARS-CoV-2 RNA are available for a limited number of high-throughput diagnostic platforms including the Roche cobas 6800/8800 (1, 2) , the Hologic Panther and Panther Fusion, (3) (4) (5) (6) and the Abbott m2000 (7) . High-throughput platforms are mostly utilized in larger reference laboratories, state public health laboratories, and academic medical centers, but these assays are not well-suited to use in other settings that lack large testing volumes or the capacity to perform high complexity tests. This has led to the centralization of SARS-CoV-2 testing, meaning that turnaround time may be prolonged by the need to transport specimens over long distances.

As SARS-CoV-2 prevalence increases, decentralized testing capability is needed to facilitate rapid identification of SARS-CoV-2 cases. To date, the BioFire COVID-19, Cepheid Xpert Xpress SARS-CoV-2 (8), DiaSorin Simplexa (9) and the GenMark ePlex SARS-CoV-2 tests (6) have emerged as rapid COVID19 testing platforms that fill this niche.

The BioFire ® FilmArray ® System (BioFire Diagnostics, LLC, Salt Lake City, UT, "BioFire") is another testing platform that is widely used in multiple laboratory environments. This multiplex, 

Residual natural nasopharyngeal swab in transport media (NPS) specimens leftover from SARS-CoV-2 testing performed as part of patient care were collected during March and April of 2020 at the University of Washington (Seattle, WA), University of Nebraska Medical Center (Omaha, NE), and ARUP laboratories (Salt Lake City, UT). Original specimen testing for SARS-CoV-2 was conducted according to manufacturer's instructions at ARUP laboratories using the Hologic Panther Fusion SARS-CoV-2 assay (FDA EUA), at the University of Nebraska Medical Center using the Roche cobas SARS-CoV-2 assay (FDA EUA), or at the University of Washington using a laboratory developed test based on the CDC N1 and N2 SARS-CoV-2 assays (Washington EUA) conducted as described in Perchetti et al. (10) . Specimens were frozen upon study enrollment to allow for storage and shipping. Additional NPS specimens collected before December 2019 and therefore presumed to be negative for SARS-CoV-2 were provided by BioFire Diagnostics.

Ten-fold serial dilutions of a natural nasopharyngeal swab specimen with known high positivity for SARS-CoV-2 RNA (E gene detected at a cycle threshold (Ct) of 16.6 by the cobas SARS-CoV-2 assay) were prepared with a diluent of pooled NPS. Diluent was prepared from samples that tested negative for SARS-CoV-2 using an assay developed at Nebraska Medicine (NECoV19; FDA EUA) and was confirmed to be PCR negative prior to use. These samples were not tested for other respiratory viruses prior to pooling. On two separate subsequent occasions, an aliquot of the 10 -4 or 10 -5 diultions was thawed and added to newly generated pools of NPS to create additional intermediate dilutions between 10 -6 and 10 -8 . Single-use aliquots of each dilution were stored at -80°C and thawed immediately prior to use. 

Testing by commercial assays (BioFire RP2. performed according to manufacturer's instructions with the exception of the Abbot ID NOW. The instructions for use for this assay have been revised and now limit testing to swab specimens that can be used to directly inoculate the sample cup. Because comparison between platforms required use of a liquid specimen, we followed the instructions for use associated with the original J o u r n a l P r e -p r o o f EUA for the Abbot ID NOW assay and used transfer pipettes from the kit to add 200 μL of NP swab specimens in transport medium to the sample cup. Table 1 ). The remaining fifty specimens were expected to test negative for SARS-CoV-2 because they were collected prior to December 2019. Testing of one positive and one negative specimen yielded invalid results due to instrument errors; these could not be retested according to the instructions for use due to insufficient specimen volume. These specimens were excluded from the analyses, resulting in reduction of sample size to 49 valid positive and 49 valid negative specimens.

The 49 negative specimens tested negative for SARS-CoV-2 on the BioFire RP2.1 assay (100% negative percent agreement, Table 1 ). The BioFire RP2.1 assay detected SARS-CoV-2 in 48/49 positive specimens (98% positive percent agreement, Table 1 Performance of the SARS-CoV-2 assay on the BioFire RP2.1 was comparable to that of NECoV19, cobas, GeneXpert, and BioFire Defense assays ( Table 2, Supplemental Table 1 ) with detection of SARS-CoV-2 in all replicates down to the 10 -7 dilution. Applying Ct values from the LDT to a standard curve generated from extracted SARS-CoV-2 quantitated RNA standard showed that this dilution contains approximately 10 3 copies/mL. Virus detection was inconsistent at lower concentrations. SARS-CoV-2 detection dropped off below the 10 -6 dilution and 10 -5 J o u r n a l P r e -p r o o f dilutions for the Hologic Aptima assay and Abbott ID NOW assay, respectively (Table 2,   Supplemental Table 1 ).

Our studies show that the BioFire RP2.1 has similar performance to high throughput shedding at levels unlikely to result in transmission, a false negative result would be less consequential (13, 14) .

The addition of a SARS-CoV-2 test to a commonly used multiplex PCR panel will expand the number of laboratories able to test for SARS-CoV-2 and will allow detection of coinfection as well as of alternative diagnoses. Once the northern hemisphere respiratory season arrives, the ability to test for influenza, RSV, and SARS-CoV-2 simultaneously on the BioFire RP2.1 will greatly benefit hospitals as an important infection control management tool. 

The Corresponding author has acted as a consultant and received grant funding from BioFire Diagnostics.

Ct values are shown for each assay used for characterizing clinical specimens, as indicated on the X axis. Horizontal bars represent Ct median values for each assay.

J o u r n a l P r e -p r o o f 

The Detection of SARS-CoV-2 using the Cepheid Xpert Xpress SARS-CoV-2 and Roche cobas SARS-CoV-2 Assays

Comparison of SARS-CoV-2 Detection from Nasopharyngeal Swab Samples by the Roche cobas(R) 6800 SARS-CoV-2 Test and a Laboratory-Developed Real-Time RT-PCR test

Rapid random access detection of the novel SARScoronavirus-2 (SARS-CoV-2, previously 2019-nCoV) using an open access protocol for the Panther Fusion

Comparison of the Panther Fusion and a laboratory-developed test targeting the envelope gene for detection of SARS-CoV-2

Comparison of Commercially Available and Laboratory Developed Assays for in vitro Detection of SARS-CoV-2 in Clinical Laboratories

Comparison of Four Molecular In Vitro Diagnostic Assays for the Detection of SARS-CoV-2 in Nasopharyngeal Specimens

Comparison of Abbott ID Now and Abbott m2000 methods for the detection of SARS-CoV-2 from nasopharyngeal and nasal swabs from symptomatic patients

Multicenter Evaluation of the Cepheid Xpert Xpress SARS-CoV-2 Test

Rapid and sensitive detection of SARS-CoV-2 RNA using the Simplexa COVID-19 direct assay

Validation of SARS-CoV-2 detection across multiple specimen types

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

Research Use Only 2019-Novel Coronavirus (2019-nCoV) Real-time RT-PCR Primers and Probes

Virological assessment of hospitalized patients with COVID-2019

This work was funded by a grant from BioFire Diagnostics to PDF. We would like to thank 

