High Content Screening, a reliable system forCoxiella burnetiiisolation from clinical samples

Abstract

24Q fever, caused by Coxiella burnetii, is a worldwide zoonotic disease that may cause severe 25 forms in humans and requires a specific and prolonged antibiotic treatment. Although the current 26 serological and molecular detection tools enable a reliable diagnosis of the disease, culture of C. 27 burnetii strains is mandatory to evaluate their antibiotic susceptibility and sequence their genome 28 in order to optimize patient management and epidemiological studies. However, cultivating this 29 fastidious microorganism is difficult and restricted to reference centers as it requires biosafety-30 level 3 laboratories and relies on cell culture performed by experienced technicians. In addition, 31 the culture yield is low, which results in a small number of isolates being available. In this work, 32 we developed a novel high content screening (HCS) isolation strategy based on optimized high-33 throughput cell culture and automated microscopic detection of infected cells with specifically-34 designed algorithms targeting cytopathic effects. This method was more efficient than the shell-35 vial assay when applied to both frozen specimens (7 isolates recovered by HCS only, sensitivity 36 91% vs 78% for shell-vial) and fresh samples (1 additional isolate using HCS, sensitivity 7% vs 37 5% for shell-vial). In addition, detecting positive cultures by an automated microscope reduced 38 the need for expertise and saved 24% of technician working time. Application of HCS to 39 antibiotic susceptibility testing of 12 strains demonstrated that it was as efficient as the standard 40 procedure that combines shell-vial culture and quantitative PCR. Overall, this high-throughput 41 HCS system paves the way to the development of improved cell culture isolation of human 42 viruses.43

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

“…DNA staining was performed with NucBlue™ Live ReadyProbes™ reagent (Molecular Probes, Life Technologies, USA). A concentration of 4 ng/ml was used (equivalent to 10 μl per well directly from stock solution), and a different well was stained each day to avoid photobleaching and possible cytotoxicity, as previously described [ 10 ].…”

“…Cell debris were removed using area cutoffs. The entire well (80 fields per well) was screened on a daily basis, and data were extracted and analyzed in a dedicated application that we recently developed in R Studio® for the detection of the intracellular bacteria, Coxiella burnetii [ 10 ]. We optimized this application for the detection of cytopathic effects caused by COVID-19.…”

“…The following features were selected: the average, total and variation of the nuclear fluorescence intensity per cell, the nuclear area, the skewness of the brightfield intensity distribution, the kurtosis of the brightfield intensity distribution and the total intensity of the brightfield within the regions of interest (ObjectAvgIntenCh1, ObjectTotalIntenCh1, ObjectVarIntenCh1, ObjectAreaCh1, ROI_SkewIntenCh3, ROI_KurtIntenCh3 and ROI_TotalIntenCh3 respectively). These parameters were used to generate two clusters using the K-means clustering algorithm, and the percentage of injured cells per well was calculated as previously described [ 10 ]. We then compared the percentage of injured cells obtained to the total cell count in each well to detect cell lysis.…”

“…In previous works, we developed a screening strategy based on high-content screening microscopy (HCS) for the isolation of environmental giant viruses in amoeba and the strict intracellular bacterium Coxiella burnetii [ 9 ],[ 10 ]. In this work, we used the same automated high-throughput method and adapted it for SARS-CoV-2 isolation from clinical samples with the objective to discard the negative co-cultures after one week and omit blind subcultures.…”