Performance evaluation of an in-house immunofluorescence assay and a commercial rapid test kit for detection of SARS-CoV-2 antigens in clinical samples

Abstract

The coronavirus disease 19 (COVID-19) pandemic has prompted the development of various diagnostic tools, with antigen detection methods emerging as promising options due to their rapidity, simplicity and accuracy. This study focuses on two rapid antigen detection approaches, namely indirect immunofluorescence and a commercial rapid diagnostic test, to evaluate their potential in diagnosing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in clinical samples. As a traditional diagnostic method for respiratory viruses, indirect immunofluorescence was selected in this study as an in-house assay to identify SARS-CoV-2 antigens from clinical samples. Nasopharyngeal swab (NPS) from 29 COVID-19 patients and 20 non-infected individuals, in addition to cells from viral culture, were collected for assessing the performance of immunofluorescence. The tested primary antibodies target nucleocapsid (N) proteins of SARS-CoV and SARS-CoV-2, as well as receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein. Among the 3 primary antibodies, the highest sensitivity and specificity have been demonstrated by the polyclonal antibody against SARS-CoV-2 N protein, detecting 17 out of 29 laboratory-confirmed COVID-19 patients by reverse transcription-polymerase chain reaction (RT-PCR) without cross-reacting with other tested viruses except SARS-CoV. While indirect immunofluorescence can identify infected individuals by detecting intact virusinfected cells by targeting SARS-CoV-2 N protein, its accuracy is limited by quality of clinical samples especially the quantity of respiratory epithelial cells in each sample. The commercial rapid diagnostic test was evaluated using serially diluted SARS-CoV-2 isolates and 110 NPS from COVID-19 patients for its sensitivity, and other viral isolates and 20 NPS from non-infected individuals for its specificity. In addition, 10 clinical samples from COVID-19 patients infected with SARS-CoV-2 variants were tested to assess its potential application in identifying variants. The overall sensitivity was 92%, which could be stratified by viral loads of clinical samples represented by RT-PCR cycle threshold (Ct) values, reaching 100% for Ct < 25 samples including SARS-CoV-2 variants, but 11.11% for Ct ≥ 30 samples. The analytical sensitivity was determined to be log10 TCID50/mL 2.0 SARS-CoV-2, and the specificity of 97% with only cross-reactivity with SARS-CoV yielded a Youden index of 0.89. The benefits and potential applications of immunofluorescence and rapid diagnostic test in the diagnosis of COVID-19 were demonstrated. In particular, incorporation of immunofluorescence targeting SARS-CoV-2 into commonly used multiplex immunofluorescence panels for respiratory viruses could be considered to enable routine testing and surveillance of SARS-CoV-2 even in periods of low prevalence of COVID-19. Besides, the rapid diagnostic test could serve as a complementary tool to other tests including RT-PCR, or as a point-of-care solution for large-scale, particularly for pandemic areas, airport border infection control or patient screening at outpatient clinics and Accident and Emergency Departments.