Improved recovery of SARS-CoV-2 from wastewater through application of RNA and DNA stabilising agents.

Wastewater Based Epidemiology (WBE) has become an integral part of the public health effort to track the levels of SARS-CoV-2 within communities. Detection of SARS-CoV-2 in wastewater can be challenging due to relatively low levels of virus within the sample. The wastewater matrix is also comprised of commercial and domestically derived contaminants, as well as RNases, all of which can adversely affect RT-qPCR analysis. To improve SARS-CoV-2 detection within wastewater samples we investigated both the effect of template dilution (as a means to reduce RT-qPCR inhibition) and sample stabilisation via addition of DNA/RNA Shield™ and/or RNA Later™ (to prevent RNA degradation via RNases) as a means to improve viral fragment detection. Using both methodologies, a significant improvement in SARS-CoV-2 detection from wastewater samples was observed. No adverse effects of stabilising agent addition on downstream Next-Generation Sequencing workflows were detected.

Single-cell transcriptome analyses reveal distinct gene expression signatures of severe COVID-19 in the presence of clonal hematopoiesis.

Clonal hematopoiesis of indeterminate potential (CHIP), a common aging-related process that predisposes individuals to various inflammatory responses, has been reported to be associated with COVID-19 severity. However, the immunological signature and the exact gene expression program by which the presence of CHIP exerts its clinical impact on COVID-19 remain to be elucidated. In this study, we generated a single-cell transcriptome landscape of severe COVID-19 according to the presence of CHIP using peripheral blood mononuclear cells. Patients with CHIP exhibited a potent IFN-γ response in exacerbating inflammation, particularly in classical monocytes, compared to patients without CHIP. To dissect the regulatory mechanism of CHIP (+)-specific IFN-γ response gene expression in severe COVID-19, we identified DNMT3A CHIP mutation-dependent differentially methylated regions (DMRs) and annotated their putative target genes based on long-range chromatin interactions. We revealed that CHIP mutant-driven hypo-DMRs at poised cis-regulatory elements appear to facilitate the CHIP (+)-specific IFN-γ-mediated inflammatory immune response. Our results highlight that the presence of CHIP may increase the susceptibility to hyperinflammation through the reorganization of chromatin architecture, establishing a novel subgroup of severe COVID-19 patients.