Clinical validation of quantitative SARS-CoV-2 antigen assays to estimate SARS-CoV-2 viral loads in nasopharyngeal swabs

Abstract

Background: Expansion of the testing capacity for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important issue to mitigate the pandemic of coronavirus disease-2019 (COVID-19) caused by this virus. Recently, a sensitive quantitative antigen test (SQT), Lumipulse® SARS-CoV-2 Ag, was developed. It is a fully automated chemiluminescent enzyme immunoassay system for SARS-CoV-2.

Methods: In this study, the analytical performance of SQT was examined using clinical specimens from nasopharyngeal swabs using reverse transcription polymerase chain reaction (RT-PCR) as a control.

Results: Receiver operating characteristic analysis of 24 SARS-CoV-2-positive and 524 -negative patients showed an area under the curve of 0.957 ± 0.063. Using a cut-off value of 1.34 pg/ml, the sensitivity was 91.7%, the specificity was 98.5%, and the overall rate of agreement was 98.2%. In the distribution of negative cases, the 99.5 percentile value was 1.03 pg/ml. There was a high correlation between the viral load calculated using the cycle threshold value of RT-PCR and the concentration of antigen. The tendency for the antigen concentration to decrease with time after disease onset correlated with that of the viral load.

Conclusions: Presented results indicate that SQT is highly concordant with RT-PCR and should be useful for the diagnosis of COVID-19 in any clinical setting. Therefore, this fully automated kit will contribute to the expansion of the testing capability for SARS-CoV-2.

Keywords: Chemiluminescent enzyme immunoassay; Covid-19; Nasopharyngeal swab; Nasopharynx; SARS-CoV-2; Saliva; Sensitive quantitative antigen test.

Fig. 1

ROC curve and negative distribution of measuremented SARS-CoV-2 Ag value by Lumipulse®. (A) Receiver operating characteristic (ROC) curve for sensitive quantitative antigen test (SQT). ROC was performed by SQT (n = 548; n = 30 SQT-positive patients and n = 518 SQT-negative patients). The area under the curve (AUC) was 0.957 ± 0.063. (B) Distribution of SARS-CoV-2 antigen concentration in Lumipulse®-negative cases. This figure shows SQT-negative (−) cases without a history of COVID-19 (+) (n = 510). The 99.5 percentile was 1.03 pg/mL.

Fig. 2

Analysis of viral load and antigen concentration. (A) Correlation analysis between viral load and SARS-CoV-2 antigen concentration. This figure shows cases with a positive test for either RT-PCR or SQT. Open circles indicate RT-PCR-positive and SQT-positive cases (n = 22). Cross marks indicate RT-PCR-negative and SQT-positive cases (n = 8). Triangles indicate RT-PCR-positive and SQT-negative cases (n = 2). (B) Relationship between days after onset and viral load or SARS-CoV-2 antigen concentration. The viral load in RT-PCR-positive cases and the antigen concentrations in SQT-positive cases are shown. Black circles indicate viral load (n = 24) and gray squares indicate SARS-CoV-2 antigen concentrations (n = 30). Black arrowheads indicate PCR-negative and SQT-positive 8 cases, and gray indicates PCR-positive and SQT-negative 2 cases. The dotted line represents the approximate line of viral load, and the dashed-and-dotted line represents the approximate line of SARS-CoV-2 antigen.