key: cord-0892569-wy81ey2x
authors: Blairon, Laurent; Cupaiolo, Roberto; Thomas, Isabelle; Piteüs, Sébastien; Wilmet, Alain; Beukinga, Ingrid; Tré‐Hardy, Marie
title: Efficacy comparison of three rapid antigen tests for SARS‐CoV‐2 and how viral load impact their performance
date: 2021-06-04
journal: J Med Virol
DOI: 10.1002/jmv.27108
sha: fe1953e6c4966a7a149d944bc2fcda661787eb9f
doc_id: 892569
cord_uid: wy81ey2x

More and more rapid antigen tests for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) appear in the market with varying performance. The sensitivity of these tests heavily depends on the viral load, extrapolated by the threshold cycle (Ct). It is therefore essential to verify their performance before their inclusion in routine. The Coronavirus Ag Rapid Test Cassette Bio‐Rad, the GSD NovaGen SARS‐CoV‐2 (COVID‐19) Antigen Rapid Test, and the Aegle Coronavirus Ag Rapid Test Cassette were evaluated on 199 samples: 150 fresh samples from the routine and positive in quantitative reverse‐transcription polymerase chain reaction (RT‐qPCR), nine fresh samples negative in RT‐qPCR, and 40 frozen samples, taken before the discovery of SARS‐CoV‐2 but positive for other respiratory viruses. Positive RT‐qPCR samples were categorized according to their Ct: Ct < 20 (18.7%), ≥ 20–< 25 (27.3%), ≥ 25–< 30 (18.7%), ≥ 30–35 (17.3%), and > 35 (18.0%). Sensitivities (95% confidence interval) for Ct below 25 were 95.7% (92.4–98.9), 97.1% (94.4–99.8), and 97.1% (94.4–99.8) for GSD NovaGen, Bio‐Rad, and Aegle, respectively but drastically dropped when Ct exceeded 27. Among samples with previously diagnosed viruses, seven false‐positive results were found with GSD NovaGen only (specificity 85.7%). Equivalent, high sensitivities were observed with the highest viral load samples. The GSD NovaGen assay showed less specificity. Although the three kits tested in this study are inadequate for routine testing in a high throughput laboratory, they can help to quickly identify the most infectious patients and screen their close contacts in an environment where molecular tests are not readily available.

methods and often depends on the sample viral burden. 1, 2 Moreover, while the specificities are often higher than 97%, the sensitivities fluctuate widely depending on the tests used, the targeted population, or the study design. [2] [3] [4] [5] [6] [7] [8] [9] [10] It is therefore important to assess the performance of a rapid antigen test before its implementation in clinical routine practice. The Coronavirus Ag Rapid Test Cassette, the GSD NovaGen SARS-CoV-2 (coronavirus disease 2019 ) Antigen Rapid Test, and Aegle Coronavirus Ag Rapid Test Cassette are three new immunochromatographic assays recently made available in the market. In this evaluation, we compared these three tests to a quantitative reverse-transcription polymerase chain reaction (RT-qPCR) considered as the reference method. The sensitivity for each test was calculated globally and after sample stratification according to their viral load approximated by their cycle threshold value (Ct).

This study was conducted from February 2 to March 3, 2021 ≥ 20-< 25; ≥ 25-< 30; ≥30-35; > 35). A Kruskal-Wallis test was used to assess differences between the Ct of the different targets and the median Ct. A test with a Cohen's kappa score was used to evaluate the agreement between the three kits and the reference RT-qPCR. A kappa score from 0.61 to 0.80 signs a substantial level of agreement.

Above 0.80, the level of agreement is almost perfect. 11 3 | RESULTS This is often the case in the presymptomatic phase (1-3 days before the onset of symptoms) and in the early symptomatic phase (during the first 5-7 days of illness). [15] [16] [17] It was therefore crucial not to focus only on overall performance but also consider the viral load distribution of the samples tested, approximated by the number of cycles corresponding to the positivity of RT-qPCR.

In addition, the contagiousness seems to sharply decrease beyond 33 cycles and it is, therefore, necessary to validate the results by requiring a good sensitivity below 33 cycles. 18 To mimic the real-life setting and to obtain a representative range of samples with different Ct, this study was deliberately prospective for positive samples. Samples intended to calculate sensitivity were selected from the samples diagnosed positive in RT-qPCR and were tested within 24 h with antigen rapid tests.

No selection was then made among the positives, so the diversity of Ct observed during the evaluation naturally reflected our routine Ct distribution. In fact, given the low prevalence in our hospital at the time of the study (6.2%), we would have obtained too many PCR-negative samples using consecutive patients and therefore less accuracy in the comparison between the Ct and the antigen test results.

One limitation of our study is that rapid antigen testing was not performed directly on a bedside nasopharyngeal smear as expected in the instructions for use of each assay because two additional nasopharyngeal samplings would have been required for each Abbreviations: CI, confidence interval; Ct, cycle threshold value; qRT-PCR, RT-PCR quantitative reverse-transcription polymerase chain reaction.

patient. Instead, the swabs from the different kits were incubated in the viral transport medium previously analyzed in RT-qPCR. We cannot exclude the fact that some dilution by the transport medium did happen to result in a systematic underestimation of the sensitivities we calculated.

As our RT-qPCR detects four targets with three fluorochromes and therefore provides three potential Ct values for each reaction, we selected the average Ct of the positive targets as reference. We chose to do so as no significant difference between the single target Ct was observed. Also, when the detection of 1 or 2 targets failed, the available Ct was always very high, pleading for a low viral load.

The specificity study was conducted by testing frozen samples positive for other respiratory viruses and collected before the SARS-CoV-2 discovery. This allowed us to assess at the same time potential Some more automated antigen tests, allow to achieve better performance but can only be used within a laboratory. 1,2 These kits are therefore not intended for the same use.

The main advantage of antigen testing is the ability to diagnose early and stop transmission quickly through targeted isolation and clustering of the most infectious cases and their close contacts. Nevertheless, the rapid kits tested in this study are inadequate for routine testing in a high throughput setting. Indeed, if the handling is fast and the result appears within 15 min, performing more than 6 tests at a time makes it difficult to respect the incubation times. Although 

This study is the first to report the external validation of three antigen kits (GSD NovaGen, Bio-Rad, and Aegle 

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The authors thank all the members of the clinical laboratory staff for technical assistance. Ag Rapid Test Cassette (Bio-Rad), the GSD NovaGen SARS-CoV-2 (COVID-19) Antigen Rapid Test (NovaTec Immunodoagnostica GmbH), and the Aegle Coronavirus Ag Rapid Test Cassette (LumiraDx) were provided at no cost by the companies. The three companies had no role in study design, data collection, and analysis, decision to publish, or manuscript preparation.

The authors have no relevant competing interest to disclose in relation to this study. 

All the procedures were in accordance with the 1964 Helsinki Declaration and its later amendments.

According to French Health Public Law (CSP Article L1121-1), this type of study did not require specific informed consent or ethics committee approval.

http://orcid.org/0000-0003-0304-7624Marie Tré-Hardy http://orcid.org/0000-0003-4915-1339