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Development of a fast and precise potency test for BCG vaccine viability using flow cytometry compared to MTT and colony-forming unit assays

In a precarious world of rapidly growing pandemics, the field of vaccine production has witnessed considerable growth. Bacillus Calmette-Guérin (BCG) is a live-attenuated vaccine and a part of the immunization program in 157 countries. The quality control is based on a potency test through viable ce...

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Autores principales: Moghawry, Hend M., Rashed, Mohamed E., Gomaa, Kareeman, AbdelGhani, Sameh, Dishisha, Tarek
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10354009/
https://www.ncbi.nlm.nih.gov/pubmed/37464014
http://dx.doi.org/10.1038/s41598-023-38657-x
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author Moghawry, Hend M.
Rashed, Mohamed E.
Gomaa, Kareeman
AbdelGhani, Sameh
Dishisha, Tarek
author_facet Moghawry, Hend M.
Rashed, Mohamed E.
Gomaa, Kareeman
AbdelGhani, Sameh
Dishisha, Tarek
author_sort Moghawry, Hend M.
collection PubMed
description In a precarious world of rapidly growing pandemics, the field of vaccine production has witnessed considerable growth. Bacillus Calmette-Guérin (BCG) is a live-attenuated vaccine and a part of the immunization program in 157 countries. The quality control is based on a potency test through viable cell enumeration. The colony-forming unit (CFU) assay is the official method, however, it often yields fluctuating results, suffers from medium cracking, and requires lengthy analysis (~ 28 days). Flow cytometric analysis was proposed earlier, but it was coupled with a Coulter counter for measuring the entire bacterial population (live/dead). In the present study, thiazole orange/propidium iodide dyes supplemented with fluorogenic reference beads were employed for viable counting, eliminating the need for a Coulter counter. Both the flow cytometry and the colorimetric technique employing tetrazolium salt were validated and compared to the CFU assay. The colorimetric assay displayed high precision, accuracy, and a strong positive correlation with the CFU assay. The flow cytometry assay demonstrated high precision and a notable ability to distinguish different forms of BCG cells (live, injured, and dead). It also exhibited a perfect positive correlation with the CFU assay. Both methods reduced the analysis time by > 26 days and eliminated the need for human intervention by automating the test.
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spelling pubmed-103540092023-07-20 Development of a fast and precise potency test for BCG vaccine viability using flow cytometry compared to MTT and colony-forming unit assays Moghawry, Hend M. Rashed, Mohamed E. Gomaa, Kareeman AbdelGhani, Sameh Dishisha, Tarek Sci Rep Article In a precarious world of rapidly growing pandemics, the field of vaccine production has witnessed considerable growth. Bacillus Calmette-Guérin (BCG) is a live-attenuated vaccine and a part of the immunization program in 157 countries. The quality control is based on a potency test through viable cell enumeration. The colony-forming unit (CFU) assay is the official method, however, it often yields fluctuating results, suffers from medium cracking, and requires lengthy analysis (~ 28 days). Flow cytometric analysis was proposed earlier, but it was coupled with a Coulter counter for measuring the entire bacterial population (live/dead). In the present study, thiazole orange/propidium iodide dyes supplemented with fluorogenic reference beads were employed for viable counting, eliminating the need for a Coulter counter. Both the flow cytometry and the colorimetric technique employing tetrazolium salt were validated and compared to the CFU assay. The colorimetric assay displayed high precision, accuracy, and a strong positive correlation with the CFU assay. The flow cytometry assay demonstrated high precision and a notable ability to distinguish different forms of BCG cells (live, injured, and dead). It also exhibited a perfect positive correlation with the CFU assay. Both methods reduced the analysis time by > 26 days and eliminated the need for human intervention by automating the test. Nature Publishing Group UK 2023-07-18 /pmc/articles/PMC10354009/ /pubmed/37464014 http://dx.doi.org/10.1038/s41598-023-38657-x Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Moghawry, Hend M.
Rashed, Mohamed E.
Gomaa, Kareeman
AbdelGhani, Sameh
Dishisha, Tarek
Development of a fast and precise potency test for BCG vaccine viability using flow cytometry compared to MTT and colony-forming unit assays
title Development of a fast and precise potency test for BCG vaccine viability using flow cytometry compared to MTT and colony-forming unit assays
title_full Development of a fast and precise potency test for BCG vaccine viability using flow cytometry compared to MTT and colony-forming unit assays
title_fullStr Development of a fast and precise potency test for BCG vaccine viability using flow cytometry compared to MTT and colony-forming unit assays
title_full_unstemmed Development of a fast and precise potency test for BCG vaccine viability using flow cytometry compared to MTT and colony-forming unit assays
title_short Development of a fast and precise potency test for BCG vaccine viability using flow cytometry compared to MTT and colony-forming unit assays
title_sort development of a fast and precise potency test for bcg vaccine viability using flow cytometry compared to mtt and colony-forming unit assays
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10354009/
https://www.ncbi.nlm.nih.gov/pubmed/37464014
http://dx.doi.org/10.1038/s41598-023-38657-x
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