ABSTRACT
The coronavirus infectious disease (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses. The pandemic has emerged as a global public health crisis, and the threat of fast-spreading of the latest variants of the coronavirus (such as omicron, delta) is rampant. Therefore, a fast and reliable diagnostic assay is needed to make the clinical decision for further treatment. The study aims to develop a Centers for Disease and Prevention (CDC)-modified qualitative real-time reverse transcriptase PCR (RT-qPCR) assay and parallel assessment of commercially available RT-qPCR assay (Altona, Seegene, BD, and GBC) to detect SARS-CoV-2. Two hundred nine samples were chosen randomly out of around two hundred thousand samples. The panel consisted of SARS-CoV-2-positive (n = 156) and SARS-CoV-2-negative (n = 52) nasopharyngeal swab specimens for a primary clinical evaluation. Furthermore, 29 positive samples were sequenced using Oxford Nanopore Minion technology. Two hundred nine patient sample data of the cycle threshold (Ct) readings for target genes of five assays are 100% sensitive for Ct values. Mean Ct values for N1, N2, RdRp, S, and E of the positive controls in CDC assay, RealStar®, Allplex, GBC, and SD Biosensor were 17.5 ± 0.49, 16.9 ± 0.51, 20 ± 0.49, 21.7 ± 0.38, and 23.1 ± 0.43, respectively. F test value shows ≥ 1, which was statistically significant. All assays showed an efficiency of < 120% and R squares were < 0.99, which is well above the required threshold value. Thus, when taking the CDC-modified assay as a gold standard, the other four assays demonstrated a p value of 0.0000, concordance at 100%, and a Kappa at 1.000. A maximum-likelihood (ML) tree was constructed and compared based on full-length SARS-CoV-2 with Wuhan isolate. These isolates are closely related to the B.1.617 lineage and reference sequences. Therefore, we conclude that all RT-PCR kits assessed in this study shall be used for routine diagnostics of COVID-19 in patients. Supplementary information: The online version contains supplementary material available at 10.1007/s00580-022-03356-y.
ABSTRACT
Although a defective vitamin D pathway has been widely suspected to be associated in SARS-CoV-2 pathobiology, the status of the vitamin D pathway and vitamin D-modulated genes in lung cells of patients infected with SARS-CoV-2 remains unknown. To understand the significance of the vitamin D pathway in SARS-CoV-2 pathobiology, computational approaches were applied to transcriptomic datasets from bronchoalveolar lavage fluid (BALF) cells of such patients or healthy individuals. Levels of vitamin D receptor, retinoid X receptor, and CYP27A1 in BALF cells of patients infected with SARS-CoV-2 were found to be reduced. Additionally, 107 differentially expressed, predominantly downregulated genes modulated by vitamin D were identified in transcriptomic datasets from patient's cells. Further analysis of differentially expressed genes provided eight novel genes with a conserved motif with vitamin D-responsive elements, implying the role of both direct and indirect mechanisms of gene expression by the dysregulated vitamin D pathway in SARS-CoV-2-infected cells. Network analysis of differentially expressed vitamin D-modulated genes identified pathways in the immune system, NF-KB;cytokine signaling, and cell cycle regulation as top predicted pathways that might be affected in the cells of such patients. In brief, the results provided computational evidence to implicate a dysregulated vitamin D pathway in the pathobiology of SARS-CoV-2 infection.