Abstract
Immunofluorescence is a traditional diagnostic method for respiratory viruses, allowing rapid, simple and accurate diagnosis, with specific benefits of direct visualization of antigens-of-interest and quality assessment. This study aims to evaluate the potential of indirect immunofluorescence as an in-house diagnostic method for SARS-CoV-2 antigens from nasopharyngeal swabs (NPS). Three primary antibodies raised from mice were used for immunofluorescence staining, including monoclonal antibody against SARS-CoV nucleocapsid protein, and polyclonal antibodies against SARS-CoV-2 nucleocapsid protein and receptor-binding domain of SARS-CoV-2 spike protein. Smears of cells from NPS of 29 COVID-19 patients and 20 non-infected individuals, and cells from viral culture were stained by the three antibodies. Immunofluorescence microscopy was used to identify respiratory epithelial cells with positive signals. Polyclonal antibody against SARS-CoV-2 N protein had the highest sensitivity and specificity among the three antibodies tested, detecting 17 out of 29 RT-PCR-confirmed COVID-19 cases and demonstrating no cross-reactivity with other tested viruses except SARS-CoV. Detection of virus-infected cells targeting SARS-CoV-2 N protein allow identification of infected individuals, although accuracy is limited by sample quality and number of respiratory epithelial cells. The potential of immunofluorescence as a simple diagnostic method was demonstrated, which could be applied by incorporating antibodies targeting SARS-CoV-2 into multiplex immunofluorescence panels used clinically, such as for respiratory viruses, thus allowing additional routine testing for diagnosis and surveillance of SARS-CoV-2 even after the epidemic has ended with low prevalence of COVID-19.
Highlights
Introduction distributed under the terms andAn integrated approach with considerations of clinical, radiological, epidemiological and molecular evidence is employed for diagnosis of coronavirus disease 19 (COVID-19)caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), [1]
Immunofluorescence using polyclonal antibody targeting SARS-CoV-2 N protein demonstrated higher sensitivity than monoclonal antibody against SARS-CoV N protein, and higher specificity than polyclonal antibody against SARS-CoV-2 receptorbinding domain (RBD), revealing that it would be the best option for staining of SARS-CoV-2-infected cells, with the sensitivity of
Index of 0.56, as compared to Youden (J) indices of 0.46 and 0.39 in immunofluorescence targeting SARS-CoV N protein and SARS-CoV-2 RBD, respectively, which is lower than the acceptable threshold of 0.50
Summary
Introduction distributed under the terms andAn integrated approach with considerations of clinical, radiological, epidemiological and molecular evidence is employed for diagnosis of coronavirus disease 19 (COVID-19)caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), [1]. Is the current gold standard for SARS-CoV-2 detection [3], which is highly sensitive but requires designated thermocyclers and specific expertise in molecular techniques, high expenses and generally long turnaround time due to complicated procedures, sample-to-result PCR-based methods have been developed for use in non-specialized laboratories to simplify and shorten the process with the cost per reaction being high. Direct viral isolation by electron microscopy or culture in cell lines, such as Vero E6 monkey kidney cells, could be used as a diagnostic tool, revealing cytopathic effects and presence of live SARS-CoV-2 that indicates infectivity of clinical samples; its complicated procedures that involve handling of dangerous live viruses in biosafety level 3 laboratories for a long duration, and its insensitivity prevent its application as a routine testing method [5,6,7]
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