High-Content Screening, a Reliable System for Coxiella burnetii Isolation from Clinical Samples

Francis, Rania; Mioulane, Maxime; Bideau, Marion Le; Mati, Marie-Charlotte; Fournier, Pierre‐Edouard; Raoult, Didier; Khalil, Jacques Bou; Scola, Bernard La

doi:10.1128/jcm.02081-19

Cited by 7 publications

(13 citation statements)

References 28 publications

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“…For the diagnosis, there are many laboratory tools that available for detecting C. burnetii using many biological samples (12). Even though the direct detection of Coxiella is more useful in veterinary pathology, cultivating of this fastidious microorganism is difficult and restricts to reference centers as it requires biosafety-level 3 laboratories and relies on cell culture which is performed by expert technicians (13). Serological screening of humans and animals for the detection of the organism specific antibodies by enzyme-linked immunosorbent assay (ELISA) has been found to be more sensitive and easier to be performed (14).…”

Section: Gharban H and Yousif Amentioning

confidence: 99%

Serological and Molecular Phylogenetic Detection of Coxiella burnetii in Lactating Cows, Iraq

Gharban

Yousif

2020

Iraqi J. Vet. Med.

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This study is carried out to investigate the prevalence of Coxiella burnetii (C. burnetii) infections in cattle using an enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) assay targeting IS1111A transposase gene. A total of 130 lactating cows were randomly selected from different areas in Wasit province, Iraq and subjected to blood and milk sampling during the period extended between November 2018 and May 2019. ELISA and PCR tests revealed that 16.15% and 10% of the animals studied were respectively positive. Significant correlations (P<0.05) were detected between the positive results and clinical data. Two positive PCR products were analyzed phylogenetically, named as C. burnetii IQ-No.5 and C. burnetii IQ-No.6; and then recorded in the National Center for Biotechnology Information (NCBI) under an accession numbers of MN473204.1 and MN473205.1. Comparative identity of the local strains with NCBI-BLAST strains/isolates revealed 97% similarity and 0.1-0.6% of total genetic mutations/changes. NCBI-BLAST Homology Sequence reported high significant identity (P<0.05) between the local, C. burnetii IQ-No.5 and C. burnetii IQ-No.6; strains and C. burnetii 3345937 (CP014354.1) Netherlands isolate at 99.10% and 99.06%, respectively. The current study concluded that the percentage of infected cows with coxiellosis is relatively high, and Coxiella should be listed as abortive pathogen. Therefore, additional studies should be performed including different animals, samples, and regions

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Section: Gharban H and Yousif Amentioning

confidence: 99%

Serological and Molecular Phylogenetic Detection of Coxiella burnetii in Lactating Cows, Iraq

Gharban

Yousif

2020

Iraqi J. Vet. Med.

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“…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 photo-bleaching and possible cytotoxicity, as previously described (21).…”

Section: Detection Process Optimizationmentioning

confidence: 99%

“…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 (21). We optimized this application for the detection of cytopathic effects caused by Covid-19.…”

Section: Detection Process Optimizationmentioning

confidence: 99%

“…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 2 clusters using Kmeans clustering algorithm and then the percentage of injured cells per well was calculated, as previously described (21). We then compared the percentage of injured cells obtained to the total cell count in each well in order to detect cell lysis (ratio = % injured cells / cell count).…”

Section: Detection Process Optimizationmentioning

confidence: 99%

“…The best would be to have an automated system allowing the rapid screening and monitoring of co-cultures at large scale. 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 (20) , (21). 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 1 week and omit blind sub-cultures.…”

Section: Introductionmentioning

confidence: 99%

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

Francis

Bideau

Jardot

et al. 2020

Preprint

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19SARS-CoV-2, a novel coronavirus infecting humans, is responsible for the current COVID-19 20 global pandemic. If several strains could be isolated worldwide, especially for in-vitro drug 21 susceptibility testing and vaccine development, few laboratories routinely isolate SARS- This is due to the fact that the current co-culture strategy is highly time consuming and requires 23 working in a biosafety level 3 laboratory. In this work, we present a new strategy based on high 24 content screening automated microscopy (HCS) allowing large scale isolation of SARS-CoV-2 25 from clinical samples in 1 week. A randomized panel of 104 samples, including 72 tested 26 positive by RT-PCR and 32 tested negative, were processed with our HCS procedure and were 27 compared to the classical isolation procedure. Isolation rate was 43 % with both strategies on 28 RT-PCR positive samples, and was correlated with the initial RNA viral load in the samples, 29 where we obtained a positivity threshold of 27 Ct. Co-culture delays were shorter with HCS 30 strategy, where 80 % of the positive samples were recovered by the third day of co-culture, as 31 compared to only 25 % with the classic strategy. Moreover, only the HCS strategy allowed us to 32 recover all the positive elements after 1 week of co-culture. This system allows rapid and 33 automated screening of clinical samples with minimal operator work load, thus reducing the risks 34 of contamination. 35 36 109 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).111 3.Large scale co-culture of clinical samples 112 We applied this strategy for the detection of SARS-CoV-2 in randomly chosen 104 anonymized 113 nasopharyngeal swab samples. Initial RT-PCR ranged from 12 Ct to 34 Ct in 72 samples, and 32 114 samples with negative initial PCR were used as negative controls. Sample preparation and co-115 culture were performed as previously described(6). Briefly, 500 µl of the sample were processed 116 7 into 0.22 µm pore sized centrifugal filter (Merck millipore, Darmstadt, Germany) and 117 centrifuged at 12 000 g for 5 minutes. 50 µl were then inoculated on a monolayer of Vero E6 118 cells cultured in 96-well microplates. A negative control consisting of uninfected cells and a 119 positive control consisting of cells infected with a 10 -4 dilution of the IHUMI-3 strain were 120 considered. A centrifugation step (700 x g for 1 h) was performed to enhance the entrance of the 121 virus inside the cells. Plates were then incubated at 37°C and monitored for 7 days to search for 122 cytopathic effects. In parallel, the same samples were processed using the classical isolation 123 strategy based on the manual observation of cytopathic effects under an inverted microscope, in 124 order to validate our strategy(6) , (19) , (22). For this strategy, co-cultures showing no cythopatic 125 effects after 1 week were sub-cultured at day 7 and day 14 onto a fresh...

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

Francis

Bideau

Jardot

et al. 2021

Clinical Microbiology and Infection

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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.

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High-Content Screening, a Reliable System for Coxiella burnetii Isolation from Clinical Samples

Cited by 7 publications

References 28 publications

Serological and Molecular Phylogenetic Detection of Coxiella burnetii in Lactating Cows, Iraq

Serological and Molecular Phylogenetic Detection of Coxiella burnetii in Lactating Cows, Iraq

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

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

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