Optimized QRT-PCR Approach for the Measurable Impact of Adjuvant Cholecalciferol Therapy in Ameliorating Cytokine Gene Expression

The endemic Vitamin D deficiency in Pakistan and the current COVID-19 epidemic have converged into a double whammy scenario in Pakistan [1]. Nutritional epigenomic studies have highlighted Vitamin D as a master Vitamin influencing various genomic expressions through its active metabolite 1α,25-dihydroxyvitamin D3 [2]. The objective of this study was to evaluate the measurable impact of adjuvant Cholecalciferol therapy in the Cytokine gene expression of COVID-19 patients by quantitative Real-Time Polymerase Chain Reaction analysis. The trial was a randomized control prospective open label interventional trial done on moderate to severe COVID-19 patients with deranged inflammatory and coagulation biomarkers. SunnyD STAT (Vitamin D3 200000 IU) softgels were given at Day 1, Day 3 and Day 5 of the treatment. Optimized quantitative Real-Time Polymerase Chain Reaction analysis showed decreased genetic expressions of Interleukin 6 (IL-6), Interleukin 2RA (IL-2RA) and Tumor Necrosis Factor (TNF-a) in the interventional group against the age and co-morbidities matched controls, providing molecular and genetic level evidence for the purported mechanism of amelioration of Cytokines induced pathogenic inflammation. However, inherent limitations of the design restrict the generalizability of the results and warrants caution for extrapolation. We recommend randomized placebo-controlled trials with larger sampling and genome wide profiling to infer more definite interpretations.