key: cord-0939587-vs8dzliy authors: Francis, Rania; Bideau, Marion Le; Jardot, Priscilla; Grimaldier, Clio; Raoult, Didier; Bou Khalil, Jacques Yaacoub; Scola, Bernard La title: High speed large scale automated isolation of SARS-CoV-2 from clinical samples using miniaturized co-culture coupled with high content screening date: 2020-05-15 journal: bioRxiv DOI: 10.1101/2020.05.14.097295 sha: 79fe7179b5223cf041ff568e3a4b8de00b9f5c4e doc_id: 939587 cord_uid: vs8dzliy SARS-CoV-2, a novel coronavirus infecting humans, is responsible for the current COVID-19 global pandemic. If several strains could be isolated worldwide, especially for in-vitro drug susceptibility testing and vaccine development, few laboratories routinely isolate SARS-CoV-2. This is due to the fact that the current co-culture strategy is highly time consuming and requires working in a biosafety level 3 laboratory. In this work, we present a new strategy based on high content screening automated microscopy (HCS) allowing large scale isolation of SARS-CoV-2 from clinical samples in 1 week. A randomized panel of 104 samples, including 72 tested positive by RT-PCR and 32 tested negative, were processed with our HCS procedure and were compared to the classical isolation procedure. Isolation rate was 43 % with both strategies on RT-PCR positive samples, and was correlated with the initial RNA viral load in the samples, where we obtained a positivity threshold of 27 Ct. Co-culture delays were shorter with HCS strategy, where 80 % of the positive samples were recovered by the third day of co-culture, as compared to only 25 % with the classic strategy. Moreover, only the HCS strategy allowed us to recover all the positive elements after 1 week of co-culture. This system allows rapid and automated screening of clinical samples with minimal operator work load, thus reducing the risks of contamination. Plates were incubated for 24 hours at 37°C in a 5 % CO2 atmosphere to allow cell adhesion. Infection was then carried out with 50 µl of the viral stock suspension diluted up to 10 -10 . The 81 plates were centrifuged for 1 hour at 700 x g and the total volume per well was adjusted to 250 82 µl with culture medium. Uninfected cells were considered negative control. ROI_TotalIntenCh3 respectively). These parameters were used to generate 2 clusters using K-108 means clustering algorithm and then the percentage of injured cells per well was calculated, as 109 previously described(21). We then compared the percentage of injured cells obtained to the total 110 cell count in each well in order to detect cell lysis (ratio = % injured cells / cell count). confirm that this effect was due to SARS-CoV-2 and to negative wells to confirm that the lack of 135 cytopathic effect was not due to microscopically undetectable minimal viral growth. The data extracted from the images were analyzed in the dedicated application in R Studio. 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