Cell culture differentiation and proliferation conditions influence the in vitro regeneration of the human airway epithelium (preprint)

The human airway mucociliary epithelium can be recapitulated in vitro using primary cells cultured in an Air-Liquid Interface (ALI), a reliable surrogate to perform pathophysiological studies. As tremendous variations exist between media used for ALI-cultured human airway epithelial cells, our study aimed to evaluate the impact of several media (BEGM, PneumaCult, "Half&Half" and "Clancy") on cell type distribution using single-cell RNA sequencing and imaging. Our work revealed the impact of these media on cell composition, gene expression profile, cell signaling or epithelial morphology. We found higher proportions of multiciliated cells in PneumaCult-ALI and Half&Half, stronger EGF signaling from basal cells in BEGM-ALI, differential expression of the SARS-CoV-2 entry factor ACE2, and distinct secretome transcripts depending on media used. We also established that proliferation in PneumaCult-Ex Plus favored secretory cell fate, showing the key influence of proliferation media on late differentiation epithelial characteristics. Altogether, our data offer a comprehensive repertoire for evaluating the effects of culture conditions on airway epithelial differentiation and will help to choose the most relevant medium according to the processes to be investigated such as cilia, mucus biology or viral infection. We detail useful parameters that should be explored to document airway epithelial cell fate and morphology.

Monitoring SARS-CoV-2 variants alterations in Nice neighborhoods by wastewater nanopore sequencing (preprint)

Background: Wastewater surveillance has been proposed as an epidemiological tool to define the prevalence and evolution of the SARS-CoV-2 epidemics. However, most implemented SARS-CoV-2 wastewater surveillance projects were relying on qPCR measurement of virus titers and did not address the mutational spectrum of SARS-CoV-2 circulating in the population. Methods: We have implemented a nanopore RNA sequencing monitoring system in the city of Nice (France, 550,000 inhabitants). Between October 2020 and March 2021, we monthly analyzed the SARS-CoV-2 variants in 113 wastewater samples collected in the main wastewater treatment plant and 20 neighborhoods. Findings: We initially detected the lineages predominant in Europe at the end of 2020 (B.1.160, B.1.177, B.1.367, B.1.474, and B.1.221). In January, a localized emergence of a variant (Spike:A522S) of the B.1.1.7 lineage occurred in one neighborhood. It rapidly spread and became dominant all over the city. Other variants of concern (B.1.351, P.1) were also detected in some neighborhoods, but at low frequency. Comparison with individual clinical samples collected during the same week showed that wastewater sequencing correctly identified the same lineages as those found in COVID-19 patients. Interpretation: Wastewater sequencing allowed to document the diversity of SARS-CoV-2 sequences within the different neighborhoods of the city of Nice. Our results illustrate how sequencing of sewage samples can be used to track pathogen sequence diversity in the current pandemics and in future infectious disease outbreaks.

Spread of the SARS-CoV-2 UK-variant in the South East of France: impact on COVID-19 patients age, comorbidity profiles and clinical presentation, week 50 2020 to week 8 2021 (preprint)

Abstract Objectives: The variant 20I/501Y.V1, associated to a higher risk of transmissibility, emerged in Nice city (South East of France, French Riviera) during January 2021. The pandemic has resumed late December 2020 in this aera. A high incidence rate together with a fast turn-over of the main circulating variants, provided us the opportunity to analyze modifications in clinical profile and outcome traits. Methods: Observational study in the University hospital of Nice from December 2020 to February 2021. We analyzed data of sequencing of SARS-CoV-2 from the sewage collector and PCR screening from all positive samples at the hospital. Then, we described the characteristics of all COVID-19 patients admitted in the emergency department (ED) (n=1247) and those hospitalized in the infectious diseases ward or ICU (n=232). Demographic data, clinical signs and severity were documented by the NEWS-2, SAPS-2 and SOFA scores were recorded and analyzed. Results: the UK-variant was absent in the area in December, then increasingly spread in January representing 59% of the PCR screening performed mid-February. The rate of patients over 65 years admitted to the ED decreased from 63% to 50% (p=0.001). The mean age of hospitalized patients in the infectious diseases ward decreased from 70.7 to 59.2 (p<0.001) while the proportion of patients without comorbidity increased from 16% to 42% (p=0.007). Neither the NEWS-2 score nor the main signs of clinical severity have changed over time. Conclusion: Spread of the UK-variant in the South East of France affects younger and healthier patients.

Spread of the SARS-CoV-2 UK-variant in the South East of France: impact on COVID-19 patients age, comorbidity profiles and clinical presentation (preprint)

Objectives: The variant 20I/501Y.V1, associated to a higher risk of transmissibility, emerged in Nice city (South East of France, French Riviera) during January 2021. The pandemic has resumed late December 2020 in this aera. A high incidence rate together with a fast turn-over of the main circulating variants, provided us the opportunity to analyze modifications in clinical profile and outcome traits. Methods: : Observational study in the University hospital of Nice from December 2020 to February 2021. We analyzed data of sequencing of SARS-CoV-2 from the sewage collector and PCR screening from all positive samples at the hospital. Then, we described the characteristics of all COVID-19 patients admitted in the emergency department (ED) (n=1247) and those hospitalized in the infectious diseases ward or ICU (n=232). Results: : the UK-variant was absent in this area in December, then increasingly spread in January representing 59% of the PCR screening performed mid-February. The rate of patients over 65 years admitted to the ED decreased from 63% to 50% (p=0.001). The mean age of hospitalized patients in the infectious diseases ward decreased from 70.7 to 59.2 (p<0.001) while the proportion of patients without comorbidity increased from 16% to 42% (p=0.007). Conclusion: Spread of the UK-variant in the South East of France affects younger and healthier patients.

Versatile and flexible microfluidic qPCR test for high-throughput SARS-CoV-2 and cellular response detection in nasopharyngeal swab samples (preprint)

The emergence and quick spread of SARS-CoV-2 has pointed at a low capacity response for testing large populations in many countries, in line of material, technical and staff limitations. The traditional RT-qPCR diagnostic test remains the reference method and is by far the most widely used test. These assays are limited to a couple of probe sets, require large sample PCR reaction volumes, along with an expensive and time-consuming RNA extraction steps. Here we describe a quantitative nanofluidic assay that overcomes some of these shortcomings, based on the Biomark instrument from Fluidigm. This system offers the possibility of performing 4608 qPCR end-points in a single run, equivalent to 192 clinical samples combined with 12 pairs of primers/probe sets in duplicate, thus allowing the monitoring in addition to SARS-CoV-2 probes of other pathogens and/or host cellular responses (virus receptors, response markers, microRNAs). Its 10 nL range volume is compatible with sensitive and reproducible reactions that can be easily and cost-effectively adapted to various RT-qPCR configurations and sets of primers/probe. Finally, we also evaluated the use of inactivating lysis buffers composed of various detergents in the presence or absence of proteinase K to assess the compatibility of these buffers with a direct reverse transcription enzymatic step and we propose several procedures, bypassing the need for RNA purification. We advocate that the combined utilization of an optimized processing buffer and a high-throughput real-time PCR device would contribute to improve the turn-around-time to deliver the test results to patients and increase the SARS-CoV-2 testing capacities.