Clinical performance of Roche cobas 6800, Luminex ARIES, MiRXES Fortitude Kit 2.1, Altona RealStar, and Applied Biosystems TaqPath for SARS‐CoV‐2 detection in nasopharyngeal swabs

Abstract We compared the performance of five assays for severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) detection on nasopharyngeal swab samples: Roche “cobas,” Luminex “ARIES,” MiRXES “Fortitude,” Altona “RealStar,” and Thermo Fisher Scientific “TaqPath.” A total of 94 nasopharyngeal swab samples were obtained from 80 confirmed coronavirus disease 2019 cases in the first 2 weeks of illness (median, 7 days; range, 2–14 days) and 14 healthy controls. After collection, all samples were transported to the hospital clinical laboratory within 24 h. These samples were tested on all five assays within 3 days of sample receipt. Of the 94 samples, 69 yielded the same result on all platforms, resulting in an agreement of 73.4% (69 of 94). Of these, 14 were the healthy control swabs which all tested negative, demonstrating good specificity across all platforms. The ARIES assay had the lowest detection rate (68.8%), followed by Fortitude (85.0%), RealStar (86.3%), cobas (95.0%), and TaqPath (100%). Statistically significant differences were observed for ARIES, Fortitude, and RealStar when compared against the best performing TaqPath using McNemar's χ 2 test. A consensus result was established based on the results obtained by the cobas, Fortitude, RealStar, and TaqPath. Six discrepancies had failed to reach a consensus and were adjudicated using the Cepheid Xpert Xpress SARS‐CoV‐2. Overall, the TaqPath and cobas assays were the most sensitive at detecting their designated SARS‐CoV‐2 gene targets. On the other hand, the ARIES assay was the least sensitive, thus warranting the need for assay re‐optimization before go‐live at the testing laboratory.


| INTRODUCTION
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, Family Coronaviridae, genus Betacoronavirus, species Severe acute respiratory syndrome-related coronavirus) is the causative agent for coronavirus disease 2019 . Since the beginning of the COVID-19 pandemic in January 2020, 1 multiple commercial molecular diagnostic assays have become available for its diagnosis. 2 Here, we compare the performance of five kits for SARS-CoV-2 RNA detection (Table 1) Of the five platforms selected for this comparison study, only the cobas and the ARIES are sample-to-result platforms providing a fully automated and complete walk-away solution. All testing for the cobas and the ARIES was performed according to the manufacturers' instructions.
The cobas targets the open reading frame 1ab (ORF1ab) nonstructural region for specific SARS-CoV-2 detection and a conserved region of the structural protein envelope (E) gene for pan-sarbecovirus detection while the ARIES targets both the ORF1ab and the nucleoprotein (N) genes for specific SARS-CoV-2 detection ( Figure S1).  Figure S1).

| RESULTS
We then performed more complex concordance analyses involving the establishment of a consensus result which was defined as the result obtained by at least three of the four assays. The four assays comprised the cobas, Fortitude, RealStar, and TaqPath. The ARIES was excluded as it had shown poor agreement when compared against the others. A total of six discrepancies had failed to reach a consensus but were subsequently adjudicated using the Xpert assay. Of the six discrepancies, five were reclassified as positives (detected: cobas, TaqPath, and Xpert; not detected: Fortitude and RealStar) and one was reclassified as negative (detected: cobas and TaqPath; not detected: Fortitude, RealStar, and Xpert). Other cross-comparison results are shown in Table 1.    (Table 2).
Overall, the TaqPath and cobas assays were the most sensitive at detecting their designated SARS-CoV-2 gene targets (Table 1).
On the other hand, the ARIES assay was the least sensitive, warranting the need for further assay re-optimization before golive at the testing laboratory.

| DISCUSSION
SARS-COV-2 subgenomic RNAs are required for efficient viral protein production and these are generated through discontinuous RNA synthesis, linking the 5' leader sequence with the appropriate open reading frames. 3 As such, in cells with replicating SARS-COV-2, the abundance of copies will trend towards the 3' end of the genome. In terms of relative abundance within the replicating cells, there will be TaqPath COVID-19 Combo Kit (Thermo Fisher Scientific "TaqPath")  In the current study, Figure S3 compares  detecting 69 of 80 and 75 of 80, respectively, followed by the assays using the S and ORF1ab gene targets (see Figure S2). Within each assay, similar trends were also seen with TaqPath N gene target being more sensitive than TaqPath S gene target and the cobas E gene target being more sensitive than the ORF1ab gene target. Even in the problematic ARIES kit, this trend was also observed.
Although numerous rapid tests have now been produced since the beginning of the COVID-19 pandemic, 5,6 "gold standard" laboratorybased assays are still required to reliably confirm the presence or absence of SARS-CoV-2 infection in patients, particularly now with the emergence of reinfection cases defined both clinically and in the laboratory, 7-9 to guide and optimize public health interventions to control the ongoing spread of COVID-19.

ACKNOWLEDGMENTS
We would like to thank Biomed Global, SDT Molecular Pte Ltd, and Thermo Fisher Scientific for sponsoring the laboratory testing kits used in this study.