key: cord-1004386-6f1pqx54
authors: van der Moeren, N.; Zwart, V.F.; Goderski, G.; Rijkers, G.T.; Bijllaardt, W.; Veenemans, J.; Kluytmans, J.A.J.W.; Pas, S.D.; Meijer, A.; Verweij, J.J.; Murk, J.L.A.N.; Stohr, J.J.J.M.
title: Performance of the Diasorin SARS-CoV-2 antigen detection assay on the LIAISON XL
date: 2021-07-08
journal: J Clin Virol
DOI: 10.1016/j.jcv.2021.104909
sha: 9c6484cb8e7ca3fd16042a5e4da87671af21b40f
doc_id: 1004386
cord_uid: 6f1pqx54

BACKGROUND: The current reference standard to diagnose a SARS-CoV-2 infection is real-time reverse transcriptase polymerase chain reaction (RT-PCR). This test poses substantial challenges for large-scale community testing, especially with respect to the long turnaround times. SARS-CoV-2 antigen tests are an alternative, but typically use a lateral flow assay format rendering them less suitable for analysis of large numbers of samples. METHODS: We conducted an evaluation of the Diasorin SARS-CoV-2 antigen detection assay (DAA) compared to real-time RT-PCR (Abbott). The study was performed on 248 (74 qRT-PCR positive, 174 qRT-PCR negative) clinical combined oro-nasopharyngeal samples of individuals with COVID-19-like symptoms obtained at a Municipal Health Service test centre. In addition, we evaluated the analytical performance of DAA with a 10-fold dilution series of SARS-CoV-2 containing culture supernatant and compared it with the lateral flow assay SARS-CoV-2 Roche/SD Biosensor Rapid Antigen test (RRA). RESULTS: The DAA had an overall specificity of 100% (95%CI 97.9%-100%) and sensitivity of 73% (95%CI 61.3% -82.7%) for the clinical samples. Sensitivity was 86% (CI95% 74.6%-93.3%) for samples with Ct-value below 30. Both the DAA and RRA detected SARS-CoV-2 up to a dilution containing 5.2 × 10(2) fifty-percent-tissue-culture-infective-dose (TCID50)/ml. DISCUSSION: The DAA performed adequately for clinical samples with a Ct-value below 30. Test performance may be further optimised by lowering the relative light unit (RLU) threshold for positivity assuming the in this study used pre-analytical protocol . The test has potential for use as a diagnostic assay for symptomatic community-dwelling individuals early after disease onset in the context of disease control.

Accurate and sustainable test strategies are key in the control of SARS-CoV-2 community spread. 1, 2, 3 The current reference standard to diagnose a SARS-CoV-2 infection is real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR). Real-time RT-PCR is a highly sensitive and specific test, but raises substantial challenges when applied for large-scale community testing due to the long turnaround time (6-8 hours after arrival of the specimen in the lab) and the need for a highly specialised laboratory environment. Furthermore, the testing capacity is limited by availability of extraction and PCR reagents and disposables. SARS-CoV-2 antigen lateral flow assays (LFA) have been proposed as an alternative for large-scale community testing of symptomatic individuals in the context of disease control. 2, 3, 4 Multiple LFA platforms have been evaluated in this context and showed satisfactory clinical performance for application as a diagnostic test in symptomatic community dwelling individuals within a limited number of days after symptom onset. 5, 6, 7 The performance of the LFA on large numbers of samples, however, is labour intensive and creates specific logistic challenges due to the strict time intervals to be respected when performing the test and the absence of automatic processing and registration. SARS-CoV-2 antigen assays that can be performed on existing automated analysers could potentially form an alternative as they are less dependent on manual labour and allow large numbers of samples to be processed in a shorter period of time. Moreover, some of these SARS-CoV-2 antigen assays can be performed on oro-/nasopharyngeal swabs suspended in virus transport medium (VTM) which can also be used to perform a confirmatory real-time RT-PCR for SARS-CoV-2 or in-depth genetic typing when needed. 8, 9 The objective of this study was to evaluate the test performance of the 'Diasorin SARS-CoV-2 Antigen detection assay' (DAA), a 96-well microtiterplate based two-step sandwich chemiluminescence immunoassay for the quantitative determination of SARS-CoV-2 nucleocapsid antigen performed on high-throughput platform, compared to the real-time RT-PCR performed on the Alinity M (Abbott) as the reference method.

Analytical performance evaluation with virus culture supernatant Analytical sensitivity and repeatability were evaluated by diluting a cell cultured SARS-CoV-2 strain (SARS hCoV-19/Netherlands/NoordBrabant_10003/2020 SARS-CoV-2; heat inactivated for 2 hours at 60 ºC in a biosafety level 3 laboratory before use for the current experiments at BSL-2 level) with 50% 

A positive result in the DAA was obtained from dilution 10 -1 until dilution 10 -5 of the cell cultured SARS-CoV-2 strain in all three series ( Table 1) . The RLU progressively decreased up to a dilution of 10 -5 after which the signal became indistinguishable from the background of the blank sample ( Table 1) .

The average decrease in Ct-value of the real-time RT-PCR was 3.6 for every step of sample dilution, starting at a mean Ct-value of 10.23 in step 10 -1 until a Ct-value of 35.32 in step 10 -8 . The highest Ctvalue at which the DAA in every dilution series still had a positive result was dilution step 10 -4 (Ctvalue 20.20) ( Table 1) . Table 2 ). In all real-time RT-PCR negative samples the RLU value of DAA was 60 or less. (Table 3) .

Sensitivity and specificity stratified by CT-value category are presented in Table 2 . Table 2 . Table 2 The performance of a number of SARS-CoV-2 LFA for use amongst symptomatic community-dwelling individuals has been evaluated and is in line with the results found in this study. 5, 6, 7 Clinical specificity of the 'BD Veritor System for Rapid Detection of SARS-CoV-2', the RRA and 'Abbott PanbioTM COVID-19 Ag Rapid Test' varied from 99.5% to 100% and the observed sensitivity on clinical samples ranged between 73% and 85% overall. All LFA had a higher sensitivity for samples with lower real-time RT-PCR Ct-values, varying from 94.3% for samples with a Ct-value below 30 for the Roche/SD Biosensor test to 98.0% for samples with a Ct-value below 32 for the Abbott Panbio test. 5, 6, 7 Studies evaluating the performance of currently available automated SARS-CoV-2 Antigen assays widely vary in study design (composition of the clinical cohort, pre-analytical protocols) and gain very different results. The LUMIPULSE SARS-CoV-2 Ag kit (Fujirebio, Tokyo, Japan) and VITROS Immunodiagnostic Products SARS-CoV-2 Antigen test (Ortho Clinical Diagnostics, Raritan, USA) were found to have respectively a 55.2% and 83.3% sensitivity and 99.6% and 100% specificity compared to the used real-time RT-PCR when performed on clinical samples. Analytical sensitivity was not determined. 10, 11 The mariPOC SARS-CoV-2 Antigen Test (ArcDia International, Turku, Finland) gained a sensitivity of 100.0% when directly performed on clinical swab specimens (84.4% in undefined transport mediums) and a specificity 100.0%. The observed limit of detection was 2.7 TCID50/test. 12 The First, information about the number of days between symptom onset and sampling was not available. As a number of performance evaluation studies of SARS-CoV-2 LFA have shown a substantial difference in test performance correlated with the time since symptom onset, further research on the DAA performance in relation to the timing of sampling is needed. 5, 6 Furthermore, the swabs were not directly preserved in DAA inactivation buffer: they were kept into 3 ml of GLYmedium, of which 1 ml was added to 1 ml of DAA inactivation buffer before testing. This dilution could theoretically reduce the assay signal by more than 50%. Differences between types of swabs and quantity and quality of absorbed clinical material could also account for differences in sensitivity.

One of the strengths of the study is the correlation of real-time RT-PCR Ct-values and test resulted in an improvement of test performance when conducting the pre-analytical steps as described above, we also found that 'background' signal varied between different types of swabs (data not shown).

Thus, the distribution of the RLU values among patients without SARS-CoV-2 infection may be affected by changes in the pre-analytical procedures, or by changes in the population tested. We therefore emphasize that any modification in the test cut-off should be validated in each specific test-setting.

In conclusion, our quantitative data confirmed the close relation between Ct-values and RLU and the DAA showed an adequate sensitivity compared with real-time RT-PCR among clinical samples with Ct-value below 30. The DAA has potential for use as a diagnostic test for symptomatic communitydwelling individuals in the context of transmission control.

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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.