key: cord-0916805-6n3lk7z1 authors: Johnson, Grant; Zubrzycki, Arek; Henry, Michele; Ranadheera, Charlene; Corbett, Cindi; Meyers, Adrienne F.A.; Sandstrom, Paul A.; Becker, Michael G. title: Clinical Evaluation of the GeneXpert® Xpert® Xpress SARS-CoV-2/Flu/RSV Combination Test date: 2021-04-12 journal: Journal of clinical virology plus DOI: 10.1016/j.jcvp.2021.100014 sha: 42bccb66eb23301ba6529d6e9324b6b7719eda40 doc_id: 916805 cord_uid: 6n3lk7z1 The Cepheid Xpert Xpress SARS-CoV-2/Flu/RSV combination test received emergency use authorization approval by the United States Food and Drug Administration in December 2020, and Health Canada approval in January 2021. The performance characteristics of the GeneXpert Xpert Xpress SARS-CoV-2/Flu/RSV combination test were assessed at Lakeridge Health Oshawa and the National Microbiology Laboratory of Canada. The combination test was compared to the Xpert SARS-CoV-2 and Xpert Flu/RSV assays, and the BioFire FilmArray Respiratory Panel 2.1 (RP2.1) test kit. Materials evaluated were serial dilutions of chemically-inactivated SARS-CoV-2 and remnant clinical specimens (nasal or nasopharyngeal swabs) collected from patients. The limit of detection (LOD) for the SARS-CoV-2 component of the Xpert SARS-CoV-2/Flu/RSV combination test was determined to be <100 viral copies/mL when using chemically-inactivated SARS-CoV-2. In total, 86 clinical positive and 51 clinical negative samples were used for this study, with mixtures of clinical positives being used to mimic coinfection and screen for competitive inhibition. The combination test showed a high percent agreement with the Xpert SARS-CoV-2 and Xpert Flu/RSV tests, as well as the BioFire FilmArray RP2.1. Based on the findings from this study and a growing body of research, the Xpert SARS-CoV-2/Flu/RSV combination test will serve as an effective replacement for the Xpert SARS-CoV-2 and Xpert Flu/RSV assays. The coronavirus disease 2019 (COVID-19) pandemic has created an unprecedented demand for diagnostic testing. In response, many SARS-CoV-2 rapid tests have been approved by the United States Food and Drug Administration (FDA) and Health Canada in 2020, including the Cepheid ® GeneXpert ® Xpert ® Xpress SARS-CoV-2 Assay. The GeneXpert performs rapid, fully-automated, and self-contained multiplex RT-qPCR tests with run times of <50 minutes. The Xpert SARS-CoV-2 assay targets two SARS-CoV-2 genome regions, the envelope (E) and the nucleocapsid (N), and returns a cycle threshold (Ct) value if the target is detected within 45 amplification cycles. In response to the COVID-19 pandemic, existing platforms that support multiplex respiratory assays, such as the BioMérieux BioFire ® FilmArray ® , have expanded their panels to include SARS-CoV-2. Consistent with this trend, Cepheid has developed a new formulation of their Xpert Flu/RSV (Respiratory Syncytial Virus) assay that includes targets for SARS-CoV-2. This new multiplex Xpert ® SARS-CoV-2/Flu/RSV test will effectively replace existing Xpert SARS-CoV-2 and Xpert Flu/RSV assays that have been widely implemented across North America. The current Xpert Xpress SARS-CoV-2 and Xpert Xpress Flu/RSV assays have been demonstrated to be highly-sensitive tests in numerous analytical and clinical studies. Cepheid reports 100% sensitivity (n=35) at 250 copies (cp)/mL for its SARS-CoV-2 assay, while independent studies have reported a limit of detection (LOD) ranging from 8.3 -60 cp/mL [1] [2] [3] . The assay has shown excellent agreement with the Roche Cobas 6800 system [4] [5] [6] [7] [8] and laboratory-developed RT-qPCR tests [3, 6] . Similarly, the GeneXpert Flu/RSV assay has shown a high level of agreement (~97%) with established laboratory-developed tests [9, 10] and the BioFire FilmArray [11] . As a rapid near-point-of-care device, the GeneXpert is currently used as a testing solution to improve turnaround times in major health centres. As an example of its utility in urban health centres, one hospital study found the GeneXpert Flu/RSV test significantly improved turnaround time and decreased the time until isolation from an average of 21 hours to 4 hours in comparison with their standard laboratory-developed Influenza test [12] . The GeneXpert has also been utilized in Northern, Remote, and Isolated (NRI) communities throughout Canada during the COVID-19 pandemic to support rapid on-site testing and to significantly decrease the test turnaround time. As the GeneXpert is being widely used in a variety of settings, evaluation of the newly-developed SARS-CoV-2/Flu/RSV combination assay is essential. In this evaluation study, remnant clinical samples were used to evaluate the SARS-CoV- Clinical Specimens: To investigate the percent positive agreement of the Xpert SARS-CoV-2/Flu/RSV combination test, 300 µL of transport media from nasal or nasopharyngeal swabs was tested in parallel using the Xpert SARS-CoV-2, Xpert Flu/RSV, and Xpert SARS-CoV-2/Flu/RSV assays. To mimic coinfection, clinical samples containing SARS-CoV-2, Influenza, or RSV were combined in equal volumes to represent coinfection with two or three viruses, and tested across all three Xpert assays. For a subset of these clinical specimens, including the combined samples used to mimic coinfection, 300 µL of transport media was also tested on the BioFire FilmArray as per the manufacturer's instructions. In addition, GeneXpert tests performed at Lakeridge Health Oshawa were timed beginning from when the cartridge was loaded onto the device and ending with cartridge ejection. Commercially available, chemically-inactivated SARS-CoV-2 was used to compare the LOD performance of the Xpert SARS-CoV-2/Flu/RSV assay and the Xpert SARS-CoV-2 assay ( Table 1) . Both assays detected SARS-CoV-2 at an input concentration of 64 cp/mL but only the Xpert SARS-CoV-2 assay detected the virus at 32 cp/mL; however, the sample size for determining LOD was limited. Note that only one Ct value is provided for SARS-CoV-2 on the SARS-CoV-2/Flu/RSV assay, as both E and N2 targets are detected on the same channel and cannot be read separately. Table 1 ). Similar side-by-side comparisons of the Xpert Flu/RSV and Xpert SARS-CoV-2/Flu/RSV assays were performed (Table 3; Figure 1B-D) . Overall, there was a high level of concordance between assays, with 99.0% agreement for Influenza A and Influenza B, and 100% agreement for RSV (Table 3) . Table 1 ). Two high Ct value Influenza A specimens (Ct values 40.5 and 40.6) detected by the laboratory-based test failed to be detected by any assays performed on either the GeneXpert or Biofire FilmArray. A laboratory and clinical evaluation of the Xpert SARS-CoV-2/Flu/RSV combination test was performed by comparing it to existing Xpert assays for SARS-CoV-2 and Flu/RSV as well as the BioFire FilmArray RP2.1 Panel. This study required a total of 151 clinical samples (including mixtures) that were processed at two independent institutions. When used as a multiplex assay, the new Xpert SARS CoV-2/Flu/RSV combination assay has a run time of approximately 36.8 minutes as opposed to run times of 40.6 minutes for a positive and 48.9 minutes for a negative SARS-CoV-2 result when utilizing the Xpert SARS-CoV-2 assay. Cepheid reports that when the combination test is run as a single assay for only SARS-CoV-2, an early termination of the test can be applied which can reduce the test run time to as short as 25 minutes (as per the package insert). BioMérieux reports a run time of approximately 45 minutes, consistent with our observations during this study. The combination test reports on a single channel for SARS-CoV-2, but otherwise utilizes the same test architecture as the Xpert SARS-CoV-2 and Xpert Flu/RSV assays. This evaluation has shown that the Xpert SARS-CoV-2/Flu/RSV combination test is highly concordant with existing Xpert assays and the BioFire RP2.1 Panel. Mixtures of clinical samples were used to identify potential issues that are known to affect multiplex assays such as competitive inhibition. The presence of multiple targets did not have a notable effect on agreement between assays nor reported Ct values. These findings are consistent with other recent research comparing the Xpert SARS-CoV-2/Flu/RSV to other Xpert assays [13, 14] or the BioFire FilmArray RP2.1 panel [13] where a high percent agreement (>98%) was also observed. Together, these studies provide strong evidence that the Xpert Flu/RSV assays currently in use across North America. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 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