SARS-CoV-2 Detection by Extraction-free qRT-PCR for Massive and Rapid COVID-19 Diagnosis During a Pandemic

Introduction/Objective COVID-19 pandemic severely impacted the healthcare and economy on a global scale. It is widely recognized that mass testing is an efficient way to contain the spread of SARS-CoV-2 infection as well as aid in the development of informed policies for disease management. Here we optimized two different protocols for qRT- PCR with direct samples and systematically compared them with the laboratory standard qRT-PCR detection assay. Methods/Case Report RNA samples from 270 subjects collected in two phases at 2020-2021. The groups consisted from positive (n = 240) and negative (n = 30) samples. We compared the performance of qRT-PCR in direct heat- inactivated (95 °C for 5 min, H), heat-inactivated and pelleted (95 °C for 5 min and centrifuged for 10 min at 12,000 g, HC) against standard laboratory protocol for SARS-CoV-2 qRT-PCR (targeting ORF1ab and N genes). Accuracy, sensitivity, and specificity for PCR assays were calculated using caret and epiR packages available in the R software environment for statistical computing. The Wilcoxon matched rank test was used to compare differences in Ct values. Results (if a Case Study enter NA) Our study suggests that HC samples show higher accuracy for SARS-CoV-2 detection PCR assay compared to direct H (89 % (95 % CI: 80–95 %) vs 83 % (95 % CI: 74–91 %) of the detection in RNA). The median ΔCt was lower by 1.55 and 2.29 cycles (Wilcoxon signed-rank test p = 0.0018 and < 0.0001 for ORF1ab and N genes, accordingly) in HC samples compared to H samples. Conclusion Our results suggest that purified RNA provides more accurate results;heat-inactivated and pelleted sample testing with qRT-PCR showed a slight drop in accuracy. However, the latter could also help to significantly increase testing capacity. Switching to the direct sample testing is justified if the number of tests is doubled at least.

Possible Biological Mechanisms Underlying the Association between COVID-19 Severity and HLA-C*04:01

A strong link between COVID-19 severity and HLAC* 04:01 allele has been replicated in several Caucasian populations including Armenians. The results have led to an idea that HLA-C*04:01 may affect the immune response via three biological mechanisms: (i) disruption of the HLA-C mediated protection harnessing natural killer cells (NK);(ii) causing NK hypo-responsiveness through KIR2DL1;or (iii) over-activation and exhaustion of CTL and NK cells by stimulating functional KIR2DS4. To test those hypotheses, we re-analyzed HLA-genotypes and RNA-sequencing data of Overmyer et al. [Cell Systems 2021;12:23-40]. An ordinal regression of patients' status (i.e., non-COVID vs. COVIDnon- ICU vs. COVID-ICU) against HLA-C has corroborated the increase in the disease severity with increasing HLA-C*04:01 dosage (p< 0.003). DESeq2 analyses of the transcriptome (16444 loci) within COVID subset mapped 3586 down-regulated and 4031 up-regulated loci to the disease severity at FDR p<0.05. The results of enrichment analyses of those 7617 genes indicated aberrations in processes, such as T cell activation, inflammatory response, positive regulation of both NK-mediated cytotoxicity and interferon-gamma production. However, only 563 down- and 341 up-regulated loci had nominally associated with the HLA-C*04:01 carriage, reflecting its genetic association with severe symptoms. Using GTEx data and rs5010528 as proxy for HLAC* 04:01 (R2 = 0.97, 1kG EUR cohort), we found that HLA-C*04:01 was associated with multiple tissue (e.g., lung, heart and blood) RNA expressional and splicing changes in >10 protein-coding loci situated close to HLA-C. The ontology analysis of the loci implicated HLA-C*04:01 in altering antigen processing and presentation of endogenous peptide antigen via HLA class I via ER pathway (FDR p<0.0001), protection from NKmediated cytotoxicity (p<0.004), and innate immune response to other organisms (p<0.009). The work was supported by the Science Committee of RA (grant E17).

Basic principles for the management of neurological patients during the COVID-19 pandemic

In a COVID-19 pandemic, a neurologist needs to be able to assess the risks of virus infection in patients with individual neurological diseases. The review presents categories of risk groups from the Association of British Neurologists for neuromuscular diseases, multiple sclerosis and other autoimmune diseases of the central nervous system, stroke, epilepsy and Parkinson's disease. The risk of infection and the management of patients with neuromuscular diseases are analyzed in detail. The use of multiple sclerosis disease modifying drugs, the treatment of stroke patients are discussed. The data from the international guidelines for the management of patients with epilepsy and Parkinson's disease are presented.