Development of an immunofluorescence-based SARS-CoV-2 detection technique in respiratory samples from patients with COVID-19
Abstract: SARS-CoV-2 is causing COVID-19, a new respiratory virus from Wuhan, China, since December 2019 and devolved into the current pandemic. Viral circulation and infection would become endemic, making direct immunofluorescence (IF) a cost-effective diagnostic methodology for sust...
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| Autores principales: | , , , , , , , , , , , , , , , , , |
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| Formato: | Artículo revista |
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Universidad Nacional Córdoba. Facultad de Ciencias Médicas. Secretaria de Ciencia y Tecnología
2021
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| Acceso en línea: | https://revistas.unc.edu.ar/index.php/med/article/view/35096 |
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| Sumario: | Abstract:
SARS-CoV-2 is causing COVID-19, a new respiratory virus from Wuhan, China, since December 2019 and devolved into the current pandemic. Viral circulation and infection would become endemic, making direct immunofluorescence (IF) a cost-effective diagnostic methodology for sustained sentinel surveillance. We proposed to develop an IF for SARS-CoV-2 in slides with respiratory samples from patients challenged with antibodies from the serum of convalescent patients as an intermediate reagent for subsequent labeling with fluorochrome conjugate.
With Ethics Committee approvals, nasopharyngeal swabs and sera were obtained from patients with PCR-confirmed COVID-19. The slides were prepared following the standard method in a biological biosafety cabinet. The conditions for performing the IF technique were: 1-Sera were assayed with CovidAR, CMIA Arquitect-ABBOTT and Neutralization-titrated challenge sera. 2-Anti-FC IgG of human IgG obtained in mouse and goat conjugated with fluorescein. 3-Blocking with albumin and tween 20 at different stages of the technique.
The results obtained so far indicate that the best combination of conditions involves the use of blocking with 1% albumin + 0.05% tween 20 on the slides for 20 minutes and a layer of human serum with specific antibody titer of 1/80 NT. In addition, the goat conjugate performed better. Validation and standardization with monoclonal antibodies are still pending. Descriptive analyses will be performed by means of tables and graphs, establishing absolute and relative frequencies (%). Chi-square analysis will be applied estimating sensitivity, specificity, positive and negative predictive values. Interspecific and intraspecific validation assays and statistical analysis of DIF with respect to molecular biology will be performed. The R-Medic software will be used and in all cases the significance level will be 5%. Although IF has lower sensitivity than molecular methods, it offers practicality in the initial screening task.
The relevance of this work lies in being able to implement in the future, once the technique is standardized and validated, the detection of SARS-Cov-2 as differential diagnosis by IF, to distinguish it among the other viral agents of the respiratory panel, contributing to the diagnosis in clinical practice and epidemiological surveillance in Public Health.
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