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, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is responsible for the current coronavirus disease 2019 global pandemic. Only a few laboratories routinely isolate the virus, which is because the current co-culture strategy is highly time-consuming and requires 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 with 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 with 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 and paves the way for future applications in clinical microbiology, such as large-scale drug susceptibility testing.

Keywords: Co-culture; Coronavirus disease 2019; High-content screening; Isolation; Severe acute respiratory syndrome coronavirus 2.

Fig. 1

Automated detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in co-culture. (a) Ratio of the percentage of injured cells on the total cell count of SARS-CoV-2-infected cells at different concentrations compared with the negative control over a period of 6 days. (b) Ratio of the percentage of injured cells on the total cell count of ten clinical samples with different initial viral loads over a period of 6 days. Initial viral loads were negative in S1 and S2, 32 Ct in S3, 30 Ct in S4, 29 Ct in S5, 28 Ct in S6, 23 Ct in S7, 22 Ct in S8, 16 Ct in S9 and 15 Ct in S10.

Fig. 2

Cumulative percentage of isolated strains per day using the classic and the new high-content screening isolation strategies for samples detected as positive in co-culture.

Fig. 3

Scanning electron microscopy images obtained with the SU5000 microscope showing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles isolated from clinical samples (white arrows). Acquisition settings and scale bars are generated on the original micrographs.

Fig. 4

Isolation rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from nasopharyngeal samples according to initial Ct values in samples using the classic and the new high-content screening isolation strategies (40 Ct represents the samples with a negative initial PCR). Note that the curves are overlapping before 28 Ct, showing similar isolation rates for both strategies.

Fig. 5

Receiver operating characteristics curves and positivity threshold analysis for positive samples detected by the classic (a) and the high-content screening (b) isolation strategies.