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Clinical Comparison of Three Sample-to-Answer Systems for Detecting SARS-CoV-2 in B.1.1.7 Lineage Emergence

Purpose Accurate molecular diagnostic assays for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, are needed for epidemiology studies and to support infection-control measures. We evaluated the analytical sensitivity and clinical performance of three sample-to-answer molecular-diagnostics systems for detecting SARS-CoV-2 using 325 nasopharyngeal swab clinical samples from symptomatic patients. Methods The BioFire Respiratory Panel 2.1 (RP2.1), cobas Liat SARS-CoV-2 and Influenza A/B, and Cepheid Xpert Xpress SARS-CoV-2/Flu/RSV platforms, which have been granted emergency-use authorization by the US FDA, were tested and compared. Results The positive percent agreement, negative percent agreement, and overall percent agreement among the three point of care testing systems were 98–100%, including for the wild-type SARS-CoV-2 (non-B.1.1.7) and a variant of concern (B.1.1.7). Notably, the BioFire RP2.1 may fail to detect the SARS-CoV-2 S gene in the B.1.1.7 lineage because of the spike protein mutation. Conclusion All three point of care testing platforms provided highly sensitive, robust, and almost accurate results for rapidly detecting SARS-CoV-2. These automated molecular diagnostic assays can increase the effectiveness of control and prevention measures for infectious diseases.


Introduction
A cluster of pneumonia cases of unknown etiology was reported in December of 2019 and was confirmed to be the novel coronavirus 2019 (2019-nCoV). 1-3 By July 2021, more than 200 million reported cases of coronavirus disease , caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), were reported and were associated with over four million deaths globally (https:// covid19.who.int/). Accurate and reliable molecular-diagnostic assays for detecting SARS-CoV-2 may help clinicians better understand the etiology of suspected SARS-CoV-2 infection. 4 Nucleic acid-amplification tests are highly sensitive and specific and are considered as the gold standard for SARS-CoV-2 diagnosis. [5][6][7] A correct SARS-CoV-2 diagnosis contributes to disease treatment and regimens. Although many molecular diagnostic platforms have become available to meet the enormous demands during the COVID-19 pandemic, [8][9][10] the clinical performance of these diagnostic methods has not been thoroughly evaluated. Affordable point of care testing (POCT) kits serve as alternative diagnostic methods but are limited by their availability, although they are widely used to test the general public. 11,12 Recently, a SARS-CoV-2 test was added to the Liat SARS-CoV-2 and Influenza A/B (Roche Molecular Systems, Inc., Pleasanton, CA, USA), Cepheid Xpert Xpress SARS-CoV-2/Flu/RSV (Cepheid, Sunnyvale, CA, USA), and BioFire Respiratory Panel 2.1 (RP2.1; BioFire Diagnostics, LLC; Salt Lake City, UT, USA) tests, which are commonly used multiplex PCR panels for diagnosing upper respiratory tract infections by multiple co-existing respiratory viruses. Importantly, new SARS-CoV-2 variants of concern (VOCs) may enhance virus transmissibility and/or disease severity, as well as diagnostic and/or treatment failure. 13 SARS-CoV-2 lineages carrying the amino acid substitution N501Y spread rapidly in the United Kingdom in late autumn 2020. 14 No relevant research exploring the performance and accuracy of POCT platforms for detecting SARS-CoV -2 VOCs has been performed.
Here, we evaluated the analytical and clinical performance of three sample-to-answer POCTs in terms of their abilities to qualitatively detect SARS-CoV-2 RNA from wild-type (non-B.1.1.7) or VOC (B.1.1.7) strains. These POCTs include the BioFire RP2.1 test, Liat SARS-CoV-2 and Influenza A/B, and Cepheid Xpert Xpress SARS-CoV-2/ Flu/RSV tests. We independently evaluated the performance characteristics of the three platforms for detecting SARS-CoV-2, particularly with respect to the VOC B.1.1.7 lineage.

Study Design and Clinical Specimens
This study was registered on February 8, 2021 and approved by the Tri-Service General Hospital Institutional Review Board (approval number C202005041). We tested 325 deidentified nasopharyngeal swab specimens collected from patients suspected of having COVID-19 using LIBO #1 NP: nasopharyngeal swab #2 Wild-type: No mutation on N501Y nor del 69-70 on the spike gene. #3 Variant of concern: spike gene mutations del 69-70 and

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Specimen Collection and Transport Swab Kits with Universal Transport Medium (New Taipei City, Taiwan). Residual viral transport medium was collected and stored at −80°C. The sample collection periods were between June and September 2020 and May and June 2021. Figure 1 depicts the study design. All SARS-CoV-2 testing results were confirmed by RT-PCR developed by us as described previously. 15 Briefly, the SARS-CoV-2 assay simultaneously detected SARS-CoV-2 E and ORF1ab gene along with the human RP gene to monitor the quality of nucleic acid. Results were interpreted as positive or negative based on the detection of E and ORF1ab or lack of detection of those genes, respectively. Briefly, 300 μL of sample was mixed with sample buffer and injected into a test pouch containing all necessary reagents for nucleic extraction, PCR amplification, and detection of the respective targets. The RP2.1 test contains two independent assays targeting the spike (S) and membrane (M) genes in the SARS-CoV-2 genome. The results were interpreted using the BioFire system software, which interprets each assay independently. If either one or both of the assays are positive, the test will show that SARS-CoV-2 was detected. If both assays are negative, the test report result will show that SARS-CoV-2 was not detected.

Analytical Tests
The limit of detection (LoD) values for SARS-CoV-2 were determined using AMPLIRUN SARS-CoV-2 RNA controls (Vircell, Granada, Spain), which contained purified genomic RNA from the indicated viruses. These controls were used for absolute quantification. The controls were used to prepare a serial-dilution panel with 1-10 replicates. The analytical sensitivities of the BioFire RP2.1, Liat SARS-CoV-2 and Influenza A/B, and Cepheid Xpert Xpress SARS-CoV-2/Flu/RSV tests were defined as the lowest dilution at which all replicates were identified as positive for SARS-CoV-2.

Comparison of Clinical Test Performance Using Clinical Samples
We included 325 retrospective nasopharyngeal swab specimens from patients hospitalized at the Tri-Service General Hospital (Taipei City, Taiwan). Clinical testing was performed for all 325 clinical specimens, and the results were compared to those of our developed RT-PCR, which was used as the reference method. 15

Analytic Sensitivities of Three Sample-to-Answer Platforms for Detecting SARS-CoV-2
We assessed the empirical sensitivity of the BioFire RP2.1, Liat SARS-CoV-2 and Influenza A/B, and Cepheid Xpert Xpress SARS-CoV-2/Flu/RSV for detecting SARS-CoV-2. For consistency, we used the unit copies/mL to compare the results obtained from all three platforms when determining the LoD of SARS-CoV-2. Accordingly, we defined the LoD as the minimum concentration at which a detection rate of 100% could be achieved for 3-10 replicates. The LoD established as per this criterion ranged from 2000 to 25 copies/mL in the three platforms (Table 1).  Figure 1). This sample had a Ct value of 26.5 on the cobas Liat System. This result indicates that a novel mutation caused mismatches with the primer for S-gene assays in the VOC of SARS-CoV-2.

Workflow Evaluation
We next evaluated the total turnaround time (TAT) for each specimen, including the sample preparation, hand-  on time, assay detection time, and data interpretation steps. Table 3 shows the overall workflow assessment and a comparison of the three POCT platforms. Analysis of the overall TAT, from sample collection to results, showed that the cobas Liat System had the lowest TAT (approximately 20 min), followed by the Cepheid Xpert Xpress system (42 min) and BioFire RP2.1 (45 min). All three platforms evaluated in this study decreased the hands-on time and reduced the risk of exposure compared to conventional RT-PCR methods, which is particularly important when handling samples from patients suspected of having COVID-19. These POCT platforms

Discussion
In vitro diagnostic nucleic acid-amplification tests assays for SARS-CoV-2 employ RT-PCR, which takes less than 1 h or a few hours depending on the number of samples processed or if a specific facility is well-equipped, which facilitates reliable and easy-to-implement in vitro diagnostic assays for the molecular diagnosis of COVID-19. 17,18 In this study, we determined the analytic sensitivity of three POCT platforms. Similar LoD values were obtained for the BioFire RP2. When relying on fully automated platforms from samples to answers, all of the three evaluated platforms have shortened the time required to obtain results and expanded the number of laboratories capable of testing for SARS-CoV-2, while also testing for co-infecting pathogens (such as other upper respiratory pathogens) as alternative diagnoses, which is key for determining the most effective treatment regimen. Various co-factors influence SARS-CoV-2-detection results, including the input volume, extraction methods, and workflow requirements. 19 Our study provides a perspective for deciding which molecular diagnostic test should be implemented in clinical laboratories. To date, several reports of concurrent infections with other pathogens, such as influenza virus and other seasonal coronaviruses, have suggested that coinfection influences the morbidity and mortality of patients with COVID-19. 20-22 Therefore, it is crucial for clinicians to rule out SARS-CoV-2 or other upper respiratory viral infections. Notably, BioFire RP2.1 provided more than 20 detection results, including SARS-CoV-2, providing important data for laboratories to rapidly detect and differentiate cocirculating respiratory pathogens and make these tests accessible in remote areas where higher-complexity assays are not feasible. 23

Conclusions
The three molecular-diagnostics platforms examined simultaneously test for SARS-CoV-2, influenza A/B, and/ or RSV, which can greatly benefit hospitals by enabling the management and control of infectious diseases.

Institutional Review Board Statement Funding
This study was supported by the Tri-Service General Hospital, Taipei, Taiwan, ROC, (grant number TSGH-D-110100). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.