Assessment of longitudinal changes in immune responses in critically ill adults with COVID-19 (preprint)

Clinically, COVID-19 is often a mild or asymptomatic illness. However, in a subset of patients, a more severe illness with one or more organ dysfunction requiring intensive care (ICU) admission occurs (stated as critical COVID-19). Most studies assessing the immune responses in COVID-19 focus on patients with non-critical COVID-19, often assessing single biological domain (such as cytokines, leukocytes, proteomics, or transcriptomics) at single time point in patient’s illness. In this context, our cohort study of patients with critical COVID-19 with demographically similar pre-pandemic controls, characterised the longitudinal changes in multiple biological domains (28 plasma cytokines, 30 immune cell subsets identified using mass cytometry and pan-leukocyte transcriptome) at four clinically relevant timepoints between ICU admission and discharge. When compared with controls, on ICU admission day, patients with critical COVID-19, had altered cytokine/chemokine profile (high interleukin-6 (IL-6), IL-10, IL-13, CXCL10, with low CCL17, and CXCL5)), raised histones (H3.1, H3R8), robust plasmablast response despite lymphopenia, with enrichment of immunoglobulin production and interferon pathways in the transcriptome. Analyses of longitudinal transcriptome data highlights three immunologically distinct clusters that were discordant to clinical time points, indicating that the clinical time points do capture immune response trajectory. Complete integration of this multi-domain longitudinal data indicated that ~ 70% of immunological heterogeneity is explained by the transcriptome.

Highly-Sensitive Lineage Discrimination of SARS-CoV-2 Variants through Allele-Specific Probe Polymerase Chain Reaction (preprint)

Introduction: Tools to detect SARS-Coronavirus-2 variants of concern and track the ongoing evolution of the virus are necessary to support ongoing public health efforts and the design and evaluation of novel COVID-19 therapeutics and vaccines. Although next-generation sequencing (NGS) has been adopted as the gold standard method for discriminating SARS-CoV-2 lineages, alternative methods may be required when processing samples with low viral loads or low RNA quality. Methods: An allele-specific probe polymerase chain reaction (ASP-PCR) targeting lineage-specific single nucleotide polymorphisms (SNPs) was developed and used to screen 1,082 samples from two clinical trials in the United Kingdom and Brazil. Probit regression models were developed to compare ASP-PCR performance against 1,771 NGS results for the same cohorts. Results: Individual SNPs were shown to readily identify specific variants of concern. ASP-PCR was shown to discriminate SARS-CoV-2 lineages with a higher likelihood than NGS over a wide range of viral loads. Comparative advantage for ASP-PCR over NGS was most pronounced in samples with Ct values between 26-30 and in samples that showed evidence of degradation. Results for samples screened by ASP-PCR and NGS showed 99% concordant results. Discussion: ASP-PCR is well-suited to augment but not replace NGS. The method can differentiate SARS-COV-2 lineages with high accuracy and would be best deployed to screen samples with lower viral loads or that may suffer from degradation. Future work should investigate further destabilization from primer:target base mismatch through altered oligonucleotide chemistry or chemical additives.