key: cord-282576-mcx0xq0w
authors: Boutin, Catherine-Audrey; Grandjean-Lapierre, Simon; Gagnon, Simon; Labbé, Annie-Claude; Charest, Hugues; Roger, Michel; Coutlée, François
title: Comparison of SARS-CoV-2 detection from combined nasopharyngeal/oropharyngeal swab samples by a laboratory-developed real-time RT-PCR test and the Roche SARS-CoV-2 assay on a cobas 8800 instrument
date: 2020-09-04
journal: J Clin Virol
DOI: 10.1016/j.jcv.2020.104615
sha: 
doc_id: 282576
cord_uid: mcx0xq0w

OBJECTIVE: Although several assays have been developed to detect SARS-CoV-2 RNA in clinical specimens, their relative performance is unknown. METHODS: The concordance between the cobas 8800 SARS-CoV-2 and a laboratory developed (LD) reverse transcriptase-polymerase chain reaction (RT-PCR) assay was assessed on 377 combined nasopharyngeal/oropharyngeal swabs in Hanks medium. RESULTS: The positive and negative agreement between these assays were 99.3 % (95 % CI, 97.3–99.9) and 77.1 % (95 % CI, 67.7–84.4), respectively, for an overall agreement of 93.6 % (95 % CI, 90.7–95.7) beyond random chance (kappa of 0.82, 95 % CI, 0.75−0.85). Of the 22 samples positive by cobas SARS-CoV-2 only, 9 were positive only for ORF-1 gene and had Cycle thresholds (Ct) > 35.1, 8 were positive only for the E gene with Ct > 35.5 and 5 were positive for both targets with Ct > 33.9. Samples positive only with the cobas assay were more often positive with only one gene target (77.3 %) than samples positive in both assays (16.9 %, p < 0.0001). Ct values in the cobas SARS-CoV-2 assay were significantly higher in the 279 samples testing positive in both assays (32.9 %, 95 % CI 32.3–33.6) compared to the 22 samples with discordant results (36.6 %, 95 % CI 36.2–37.1; p = 0.0009). An excellent correlation (r(2) = 0.98) was obtained between Ct values of the ORF-1 and E targets in the cobas assays and a good correlation was obtained between LD RT-PCR test and cobas SARS CoV-2 ORF-1 target (r(2) = 0.82). CONCLUSION: Our study demonstrated an excellent concordance between a LD RT-PCR and the cobas SARS-CoV-2 tests on the 8800 platform.

Since December 2019, a novel coronavirus, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged as the cause of a severe respiratory disease and is causing a worldwide pandemic. Tests based on reverse transcription-polymerase chain reaction (RT-PCR) are the most suitable mode of rapid diagnosis of acute SARS-CoV-2 infections which is essential for patient management and contact tracing (1) . Several commercial and laboratory developed (LD) assays are available but few manufacture-independent evaluations and few comparisons between assays have been published up to now (1) . Moreover, the comparison between assays is hampered by the absence of accepted gold standard test as well as our incomplete knowledge of the natural history of SARS-CoV-2 infection. Studies evaluating the concordance between assays are thus needed at this point in the pandemic.

We evaluated the concordance between the two-target cobas SARS-CoV-2 test (Roche Molecular Diagnostics, Laval, Canada) on the fully automated cobas 8800 platform authorized by Health Canada and a laboratory-developed (LD) standardized RT-PCR test using widely used primer set and probe (2, 3) in samples submitted at the diagnostic laboratory for patient care at the Centre Hospitalier de l'Université de Montréal.

A subset of combined nasopharyngeal/oropharyngeal swabs routinely collected in Hanks media from 377 individuals between 21 April 2020 and 11 June 2020 were transported to the laboratory the same day and tested in parallel as part of routine clinical care using the cobas SARS- Prior to testing on the cobas 8800, each specimen was treated to inactivate potential infectious viruses: 400 µl of specimen in Hanks was added to 200 µL of cobas 8800 lysis buffer, followed by an incubation at room temperature for 10 minutes. The detection of SARS-CoV-2 RNA was then performed on the inactivated 600 µl-aliquot with the cobas 8800 instrument, as suggested by the manufacturer. A two-target RT-PCR was used on this instrument: one targeting Orf1, a nonstructural region that is unique to SARS-CoV-2 coding for the RdRp activity of the virus, and the second targeting a conserved region in the structural protein envelope E gene for pan-sabercovirus detection. An internal RNA control was detected in each sample to control for amplification efficiency and RNA extraction from sample. Uracil-N-glycosylase is also included in the master mix to catalyze contaminating amplicons. A data management software in the automated apparatus assigns test results to each sample. A sample was considered positive if a positive result was obtained for the Orf1 gene only or for both Orf1 and E genes, as suggested by the manufacturer's instructions. Samples testing positive only for the E gene were considered as SARS-CoV-2 presumptive positive. Testing was performed in 96 samples batches, including one positive control and one negative control. The Limit of detection of the assay was 20 viral RNA copies per mL of medium (data not shown). Samples with discrepant results between the cobas and LDT assays were tested, when enough sample left was available, in the Abbott RealTime SARS-CoV-2 test on the Abbott m2000 system (Abbott Molecular Inc., Des Plaines, IL), as suggested by the manufacturer (4).

In the absence of gold standard for SARS-CoV-2 RNA detection, data was analyzed using a contingency table to assess the overall, positive and negative agreement with 95% confidence intervals (95% CI) calculated. The level of agreement was also assessed using kappa statistics. By 

The results of the comparison of the cobas SARS-CoV-2 and LD RT-PCR on 377 routinely collected nasopharyngeal/oropharyngeal swabs are summarized in Table 1 one showed inhibition, 8 were 'detected' with low cycle number (CN) values between 26.7 and 31.5, and 11 were 'not detected'. Since these latter samples were weakly reactive initially in the cobas assay, had been frozen for 6 to 9 weeks and thawed, the 11 negative samples in the Abbott

RealTime test yet initially positive in the cobas assay were retested in the cobas assay : only one out of 10 then scored positive in the cobas test.

An excellent correlation (r 2 = 0.98, p<0.0001) was obtained between Ct values for SARS-CoV-2 ORF-1 target and E target in the cobas SARS-CoV-2 assays (Figure 1 ). The correlation between Ct values obtained in the LD RT-PCR test and cobas SARS CoV-2 ORF-1 target for positive samples in both assays was good (r 2 = 0.82, data not shown). RT-PCR test. Only discordant samples were tested in a third assay, we could thus not calculate the performance of the cobas test with an expanded gold standard.

In conclusion, this study demonstrated an excellent agreement between the cobas SARS-CoV-2 test in the 8800 platform and a LD RT-PCR test, although the cobas assay detected a greater number of samples with low viral load infections. Further studies comparing different platforms in parallel, as well as using clinical information will allow establishing a gold standard and determine the true performance of automated high throughput platforms. 

Laboratory diagnosis of emerging human coronavirus infections -the state of the art

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

Clinical Evaluation of the cobas SARS-CoV-2 Test and a Diagnostic Platform Switch during 48 Hours in the Midst of the COVID-19 Pandemic

Comparison of Abbott ID Now and Abbott m2000 Methods for the Detection of SARS-CoV-2 from Nasopharyngeal and Nasal Swabs from Symptomatic Patients

Statistical methods for rates and proportions

US CDC Real-Time Reverse Transcription PCR Panel for Detection of Severe Acute Respiratory Syndrome Coronavirus 2

Comparative Performance of SARS-CoV-2 Detection Assays Using Seven Different Primer-Probe Sets and One Assay Kit

Table 1. Detection of SARS-CoV-2 RNA in 377 nasopharyngeal-oropharyngeal samples in Hanks with cobas 8800 SARS-CoV-2 and LD RT-PCR assays Overall agreement : 93

8 samples were positive only with the E gene and were presumptive positive samples. The other 14 samples were positive with for at least the SARS-CoV-2 Orf1 gene

We would like to thank all the laboratory technologists who performed the real time PCR assays for this study.J o u r n a l P r e -p r o o f