Culturing of SARS-CoV-2 from patient samples: Protocol for optimal virus recovery and assessment of infectious viral load

IF 2.2 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS Journal of virological methods Pub Date : 2024-03-05 DOI:10.1016/j.jviromet.2024.114912

Line L. Bang , Ditte R. Tornby , Stephanie T.D. Pham , Kristian Assing , Sören Möller , Yaseelan Palarasah , Lone W. Madsen , Karina G. Thomsen , Isik S. Johansen , Rune M. Pedersen , Thomas E. Andersen

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Abstract

Optimal sampling, preservation, and culturing of SARS-CoV-2 from COVID-19 patients are critical for successful recovery of virus isolates and to accurately estimate contagiousness of the patient. In this study, we investigated the influence of the type of sampling media, storage time, freezing conditions, sterile filtration, and combinations of these to determine the optimal pre-analytic conditions for virus recovery and estimation of infectious viral load in COVID-19 patients. Further, we investigated the viral shedding kinetics and mucosal antibody response in 38 COVID-19 hospitalized patients. We found Universal Transport Medium (Copan) to be the most optimal medium for preservation of SARS-CoV-2 infectivity. Our data showed that the probability of a positive viral culture was strongly correlated to Ct values, however some samples did not follow the general trend. We found a significant correlation between plaque forming units and levels of mucosal antibodies and found that high levels of mucosal antibodies correlated with reduced chance of isolating the virus. Our data reveals essential parameters to consider from specimen collection over storage to culturing technique for optimal chance of isolating SARS-CoV-2 and accurately estimating patient contagiousness.

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从患者样本中培养 SARS-CoV-2 ：最佳病毒回收和传染性病毒载量评估方案。

从 COVID-19 患者身上进行 SARS-CoV-2 的最佳采样、保存和培养是成功回收病毒分离物和准确估计患者传染性的关键。在本研究中，我们研究了采样培养基类型、储存时间、冷冻条件、无菌过滤以及这些因素组合的影响，以评估 COVID-19 患者病毒回收和传染性病毒载量估计的最佳分析前条件。此外，我们还研究了 38 名 COVID-19 住院患者的病毒脱落动力学和粘膜抗体反应。我们发现通用运输培养基（Copan）是保存 SARS-CoV-2 感染性的最佳培养基。我们的数据显示，病毒培养呈阳性的概率与 Ct 值密切相关，但有些样本并不遵循总体趋势。我们发现斑块形成单位与粘膜抗体水平之间存在明显的相关性，并发现高水平的粘膜抗体与分离病毒的几率降低有关。我们的数据揭示了从标本的采集、储存到培养技术都需要考虑的重要参数，以获得分离出 SARS-CoV-2 的最佳机会并准确估计病人的传染性。

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来源期刊

Journal of virological methods 医学-病毒学

CiteScore

5.80

自引率

0.00%

发文量

209

审稿时长

41 days

期刊介绍： The Journal of Virological Methods focuses on original, high quality research papers that describe novel and comprehensively tested methods which enhance human, animal, plant, bacterial or environmental virology and prions research and discovery. The methods may include, but not limited to, the study of: Viral components and morphology- Virus isolation, propagation and development of viral vectors- Viral pathogenesis, oncogenesis, vaccines and antivirals- Virus replication, host-pathogen interactions and responses- Virus transmission, prevention, control and treatment- Viral metagenomics and virome- Virus ecology, adaption and evolution- Applied virology such as nanotechnology- Viral diagnosis with novelty and comprehensive evaluation. We seek articles, systematic reviews, meta-analyses and laboratory protocols that include comprehensive technical details with statistical confirmations that provide validations against current best practice, international standards or quality assurance programs and which advance knowledge in virology leading to improved medical, veterinary or agricultural practices and management.