Background
Wastewater surveillance was 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 based 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.
Translation
For the French translation of the abstract see Supplementary Materials section.
BackgroundWastewater 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.MethodsWe 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.FindingsWe 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.InterpretationWastewater 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.
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 few probe sets, require large sample PCR reaction volumes, along with an expensive and time-consuming RNA extraction step. Here we describe a quantitative nanofluidic assay that overcomes some of these shortcomings, based on the BiomarkTM 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 of SARS-CoV-2 including the detection of specific SARS-CoV-2 variants, as well as the detection other pathogens and/or host cellular responses (virus receptors, response markers, microRNAs). The 10 nL-range volume of BiomarkTM reactions 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 protocols, 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.
Reliable measurement of cellular cytotoxicity is essential for the characterization of immune responses and for the monitoring of antibody treatment efficacy. Until now, the standard Cr-release assay has remained the sole sensitive assay that measures cellular cytotoxicity. Alternative non-radioactive assays have been developed but they do not provide accurate measurement of target cell cytotoxicity. The cost and hazard of handling radioactivity are strong incentives to find alternative solutions to Cr. We took advantage of the recent development of cell-imaging multimode readers to develop a novel non-radioactive and real-time cytotoxic assay that demonstrates good reproducibility and sensitivity. The extent of target-cell cytotoxicity is monitored over time by imaging and quantifying live fluorescent target cells in 96-well plates. We have developed classical natural killer cell assays in the presence or absence of blocking antibodies and antibody-dependent cell-mediated cytotoxicity. We show that in these assays, cell killing occurs within the first 2 hr with half maximum killing reached after 30 min. This technology has numerous applications such as natural killer and T-cell cytotoxicity assays and can be extended to cell survival and apoptosis measurement assays.
Lineage dedifferentiation toward a mesenchymal‐like state displaying myofibroblast and fibrotic features is a common mechanism of adaptive and acquired resistance to targeted therapy in melanoma. Here, we show that the anti‐fibrotic drug nintedanib is active to normalize the fibrous ECM network, enhance the efficacy of MAPK‐targeted therapy, and delay tumor relapse in a preclinical model of melanoma. Acquisition of this resistant phenotype and its reversion by nintedanib pointed to miR‐143/‐145 pro‐fibrotic cluster as a driver of this mesenchymal‐like phenotype. Upregulation of the miR‐143/‐145 cluster under BRAFi/MAPKi therapy was observed in melanoma cells in vitro and in vivo and was associated with an invasive/undifferentiated profile. The 2 mature miRNAs generated from this cluster, miR‐143‐3p and miR‐145‐5p, collaborated to mediate transition toward a drug‐resistant undifferentiated mesenchymal‐like state by targeting Fascin actin‐bundling protein 1 (FSCN1), modulating the dynamic crosstalk between the actin cytoskeleton and the ECM through the regulation of focal adhesion dynamics and mechanotransduction pathways. Our study brings insights into a novel miRNA‐mediated regulatory network that contributes to non‐genetic adaptive drug resistance and provides proof of principle that preventing MAPKi‐induced pro‐fibrotic stromal response is a viable therapeutic opportunity for patients on targeted therapy.
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