Department of Pharmaceutical Microbiology and Immunology, Faculty of Pharmacy, Beni-Suef University, Beni-Suef, 625 11, Egypt.
General Administration of Biological Products, Central Administration of Biological and Innovative Products and Clinical Trials, Egyptian Drug Authority (EDA), Giza, Egypt.
Sci Rep. 2023 Jul 18;13(1):11606. doi: 10.1038/s41598-023-38657-x.
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.
在一个充满快速增长的大流行病的不稳定世界中，疫苗生产领域取得了相当大的发展。卡介苗（BCG）是一种减毒活疫苗，是 157 个国家免疫计划的一部分。质量控制基于通过活细胞计数进行效力测试。集落形成单位（CFU）测定法是官方方法，但它经常产生波动的结果，易受培养基破裂的影响，并且需要冗长的分析（~28 天）。流式细胞术分析早些时候已经提出，但它与库尔特计数器结合使用，用于测量整个细菌群体（活/死）。在本研究中，噻唑橙/碘化丙啶染料补充了荧光参考珠，用于活菌计数，从而无需库尔特计数器。流式细胞术和使用四唑盐的比色技术都经过了验证，并与 CFU 测定法进行了比较。比色测定法显示出高精度、准确性以及与 CFU 测定法的强正相关性。流式细胞术测定法显示出高精度和区分不同形式的卡介苗细胞（活细胞、受损细胞和死细胞）的显著能力。它还与 CFU 测定法呈完美正相关。这两种方法都将分析时间缩短了>26 天，并通过自动化测试消除了对人工干预的需求。
