High-speed large-scale automated isolation of SARS-CoV-2 from clinical samples using miniaturized co-culture coupled to high-content screening

Abstract

Objectives A novel coronavirus, SARS-CoV-2, is responsible for the current COVID-19 global pandemic. Only a few laboratories routinely isolate the virus, which is because the current co-culture strategy is highly time-consuming and requires working in a biosafety level 3 laboratory. This work aimed to develop a new high-throughput isolation strategy using novel technologies for rapid and automated isolation of SARS-CoV-2. Methods We used an automated microscope based on high-content screening (HCS), and we applied specific image analysis algorithms targeting cytopathic effects of SARS-CoV-2 on Vero E6 cells. A randomized panel of 104 samples, including 72 that tested positive by RT-PCR and 32 that tested negative, were processed with our HCS strategy and were compared to the classical isolation procedure. Results The isolation rate was 43% (31/72) with both strategies on RT-PCR-positive samples and was correlated with the initial RNA viral load in the samples, in which we obtained a positivity threshold of 27 Ct. Co-culture delays were shorter with the HCS strategy, where 80% (25/31) of the positive samples were recovered by the third day of co-culture, compared to only 26% (8/30) with the classic strategy. Moreover, only the HCS strategy allowed us to recover all the positive samples (31 with HCS versus 27 with classic strategy) after 1 week of co-culture. Conclusions This system allows the rapid and automated screening of clinical samples with minimal operator workload, which reduces the risk of contamination, thus paving the way for future applications in clinical microbiology, such as large-scale drug susceptibility testing.

Assessing severe acute respiratory syndrome coronavirus 2 infectivity by reverse‐transcription polymerase chain reaction: A systematic review and meta‐analysis

“…After deduplication, 6219 records remained, of which 6074 were excluded during title and abstract screening (Figure 1 ). One hundred and fourty five full texts were screened for eligibility and 55 studies were eligible, 53 peer‐reviewed articles 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , ...…”

COVID-19 Management at IHU Méditerranée Infection: A One-Year Experience

“…More than 600,000 RT-PCR custom-made tests have been fabricated in the IHU. In parallel, the activity of the Biosafety Safety Level 3 laboratory was redirected towards the high throughput isolation and culture of SARS-CoV-2 strains, the majority from nasopharyngeal swabs used in parallel for RT-PCR diagnosis [15]. This activity was rapidly crucial in determining a cut-off value for the accurate interpretation of RT-PCR cycle threshold (CT) after we showed that a CT value of >34 allowed only 1% viable viruses [16].…”

Heterogeneity in susceptibility to hydroxychloroquine of SARS-CoV-2 isolates

“…Imaging and cell analyses was performed by high-content-screening using the CX7 automated cell- insight optical microscope (ThermoFisher Scientific, USA). The proof of concept used by Francis et al [28] was developed to automatically detect infections in cells. Briefly, at time points H0 and 72 hours post-infection, wells were stained by NucBlue™ Live ReadyProbes™ reagent (Molecular Probes, Life Technologies, USA), at a final concentration of 2 ng/mL (5 µL per well directly from stock solution).…”

“…Indeed, most in vitro studies evaluating susceptibility to antiviral drugs have used a unique strain or clone of SARS-CoV-2, and have considered that this clone is representative of all strains, despite the fact that the variability of antiviral activities on an enlarged panel of strains is unknown. In this study, we decided to use an automated model of Vero E6 to screen a single combination of azithromycin and hydroxychloroquine on multiple strains isolated from patients with persistent and non-persistent infections and who were randomly chosen to detect reduced susceptibility to this combination [28]. After this preliminary screening, a dose-response study to hydroxychloroquine was performed on suspect isolates and controls.…”