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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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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I10-R327-article-35096 |
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ojs |
| institution |
Universidad Nacional de Córdoba |
| institution_str |
I-10 |
| repository_str |
R-327 |
| container_title_str |
Revista de la Facultad de Ciencias Médicas de Córdoba |
| format |
Artículo revista |
| topic |
SARS CoV-2 Covid 19 direct immunofluorescence Epidemiology Public Health Argentine SARSCoV-2/Covid-19 Inmunofluorescencia directa epidemiologia salud pública Argentina . |
| spellingShingle |
SARS CoV-2 Covid 19 direct immunofluorescence Epidemiology Public Health Argentine SARSCoV-2/Covid-19 Inmunofluorescencia directa epidemiologia salud pública Argentina . Bravo, MM Frutos , MC Garay , MP Lopez , MA Kassar, MA Arrúa, SM Pedranti , MS Alonso, RG Cajal, SP Colazo Salbetti, MB Herrera Simó, C Olivera , NL Pérez , IN Zalazar, JA Moreno , LB Raskovsky, VI Adamo , MP Cámara, A Development of an immunofluorescence-based SARS-CoV-2 detection technique in respiratory samples from patients with COVID-19 |
| topic_facet |
SARS CoV-2 Covid 19 direct immunofluorescence Epidemiology Public Health Argentine SARSCoV-2/Covid-19 Inmunofluorescencia directa epidemiologia salud pública Argentina . |
| author |
Bravo, MM Frutos , MC Garay , MP Lopez , MA Kassar, MA Arrúa, SM Pedranti , MS Alonso, RG Cajal, SP Colazo Salbetti, MB Herrera Simó, C Olivera , NL Pérez , IN Zalazar, JA Moreno , LB Raskovsky, VI Adamo , MP Cámara, A |
| author_facet |
Bravo, MM Frutos , MC Garay , MP Lopez , MA Kassar, MA Arrúa, SM Pedranti , MS Alonso, RG Cajal, SP Colazo Salbetti, MB Herrera Simó, C Olivera , NL Pérez , IN Zalazar, JA Moreno , LB Raskovsky, VI Adamo , MP Cámara, A |
| author_sort |
Bravo, MM |
| title |
Development of an immunofluorescence-based SARS-CoV-2 detection technique in respiratory samples from patients with COVID-19 |
| title_short |
Development of an immunofluorescence-based SARS-CoV-2 detection technique in respiratory samples from patients with COVID-19 |
| title_full |
Development of an immunofluorescence-based SARS-CoV-2 detection technique in respiratory samples from patients with COVID-19 |
| title_fullStr |
Development of an immunofluorescence-based SARS-CoV-2 detection technique in respiratory samples from patients with COVID-19 |
| title_full_unstemmed |
Development of an immunofluorescence-based SARS-CoV-2 detection technique in respiratory samples from patients with COVID-19 |
| title_sort |
development of an immunofluorescence-based sars-cov-2 detection technique in respiratory samples from patients with covid-19 |
| description |
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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| publisher |
Universidad Nacional Córdoba. Facultad de Ciencias Médicas. Secretaria de Ciencia y Tecnología |
| publishDate |
2021 |
| url |
https://revistas.unc.edu.ar/index.php/med/article/view/35096 |
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2024-09-03T21:03:12Z |
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2024-09-03T21:03:12Z |
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I10-R327-article-350962024-04-15T16:19:09Z Development of an immunofluorescence-based SARS-CoV-2 detection technique in respiratory samples from patients with COVID-19 Desarrollo de una técnica de detección de SARS-CoV-2 basada en inmunofluorescencia en muestras respiratorias de pacientes con COVID-19 A Bravo, MM Frutos , MC Garay , MP Lopez , MA Kassar, MA Arrúa, SM Pedranti , MS Alonso, RG Cajal, SP Colazo Salbetti, MB Herrera Simó, C Olivera , NL Pérez , IN Zalazar, JA Moreno , LB Raskovsky, VI Adamo , MP Cámara, A SARS CoV-2 Covid 19 direct immunofluorescence Epidemiology Public Health Argentine SARSCoV-2/Covid-19 Inmunofluorescencia directa epidemiologia salud pública Argentina . 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. Resumen: El SARS-CoV-2 es causante de COVID-19, un nuevo virus respiratorio de Wuhan, China, desde diciembre de 2019 y devino en la pandemia actual. La circulación e infección viral se volverían endémicas, por lo que la Inmunofluorescencia (IF) directa sería una metodología diagnóstica económica para la vigilancia centinela sostenida. Se propuso desarrollar una IF para SARS-CoV-2 en improntas con muestras respiratorias de pacientes enfrentadas con anticuerpos del suero de pacientes convalecientes como reactivo intermediario para el posterior marcaje con conjugado con fluorocromo. Con aprobaciones del Comités de Ética, se obtuvieron hisopados nasofaríngeos y sueros de pacientes con COVID-19 confirmado por PCR. Las improntas se elaboraron siguiendo el método estándar en cabina de bioseguridad biológica. Las condiciones para la realización de la técnica de IF fueron: 1-Se ensayó con sueros problemas titulados por CovidAR, CMIA Arquitect-ABBOTT y Neutralización. 2-IgG anti-FC de IgG humana obtenidas en ratón y cabra conjugada con fluoresceína. 3-Bloqueo con albúmina y tween 20 en diferentes etapas de la técnica. Los resultados obtenidos hasta ahora indican que la mejor combinación de condiciones implica el uso de bloqueo con albúmina al 1% + tween 20 al 0,05% sobre la impronta durante 20 minutos y una capa del suero humano con título de anticuerpos específicos de 1/80 NT. Además, funcionó mejor el conjugado de cabra. Permanece a la espera la validación y estandarización con anticuerpos monoclonales. Se realizarán análisis descriptivos mediante tablas y gráficos, estableciendo frecuencias absolutas y relativas (%). Se aplicarán análisis Chi cuadrado estimando sensibilidad, especificidad, valores predictivos positivos y negativos. Se harán ensayos de validación interespecíficos e intraespecíficos y análisis estadísticos de IFD con respecto a biología molecular. Se utilizará el soft R-Medic y en todos los casos el nivel de significación será del 5%. Si bien la IF posee menor sensibilidad que los métodos moleculares, ofrece practicidad en la tarea inicial de tamizaje. La relevancia del trabajo radica en poder implementar a futuro, una vez estandarizada y validada la técnica, la detección de SARS-Cov-2 como diagnóstico diferencial por IF, para distinguirlo entre los otros agentes virales del panel respiratorio, aportando al diagnóstico en la práctica clínica y a la vigilancia epidemiológica en Salud Pública. . Universidad Nacional Córdoba. Facultad de Ciencias Médicas. Secretaria de Ciencia y Tecnología 2021-10-12 info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion texto texto texto https://revistas.unc.edu.ar/index.php/med/article/view/35096 Revista de la Facultad de Ciencias Médicas de Córdoba.; Vol. 78 No. Suplemento (2021): Suplemento JIC XXII Revista de la Facultad de Ciencias Médicas de Córdoba; Vol. 78 Núm. Suplemento (2021): Suplemento JIC XXII Revista da Faculdade de Ciências Médicas de Córdoba; v. 78 n. Suplemento (2021): Suplemento JIC XXII 1853-0605 0014-6722 http://creativecommons.org/licenses/by-nc/4.0 |