JMIR Res Protoc. 2020 Nov 10. doi: 10.2196/21242. Online ahead of print.
ABSTRACT
BACKGROUND: COVID-19 pandemic caused by SARS-CoV-2 continued to impact health systems throughout the world with serious medical challenges being imposed on many African countries such as Nigeria. Although emerging studies have identified lymphopenia as a driver of cytokine storm, disease progression and poor outcomes in infected patients, its immunopathogenesis, environmental and genetic determinants remain unclear. Understanding the interplay of these determinants in the context of lymphopenia and COVID-19 complications in African patients may help in risk stratification and appropriate deployment of targeted treatment regimens with repurposed drugs to improve prognosis.
OBJECTIVE: This study is designed to investigate the role of vitamin D status, vasculopathy, apoptotic pathway and vitamin D receptor gene polymorphisms in the immunopathogenesis of lymphopenia among SARS-CoV-2 infected Africans.
METHODS: This cross-sectional study will enrol 200 study participants categorized as SARS-CoV-2 negative (n=69), COVID-19 mild (n=32), hospitalized (n=72) and recovered (n=37) from two purposively selected health facilities in Lagos State. Sociodemographic, travel history and co-morbidity information will be obtained from case file and study pre-tested interviewer-based structured questionnaire. Venous blood samples (5ml) collected between 8.00-10.00 h and aliquoted into EDTA and plain tubes will be used for complete blood count and CD4 T cells assays to determine lymphopenia (Lymphocyte count < 1000 cells/uL) and CD4 T lymphocyte levels as well as measuring the concentrations of vitamin D, caspase 3, sVCAM-1 and sFasL using an autoanalyzer, flow cytometry and ELISA techniques. Genomic DNA will be extracted from buffy coat and used as template for the amplification of apoptosis related genes (Bax, Bcl-2, BCl-2LI2) by PCR and genotyping of vitamin D receptor (VDR: Apa1, Foh1 and Bsm1) gene polymorphisms by PCR-RFLP and capillary sequencing. Total RNA will also be extracted, reverse transcribed and subsequently quantitated by RT-PCR to monitor the expression of apoptosis genes in the four categories of COVID-19 study participants enrolled. Data analyses, which include test of association between VDR gene polymorphisms and study outcomes (Lymphopenia and hypovitaminosis D prevalence, mild/moderate and severe infections) will be performed using the R Statistical Software. Hardy-Weinberg Equilibrium (HWE) and linkage disequilibrium analyses for alleles, genotype and haplotypes of the genotyped VDR gene will also be carried out.
RESULTS: A total of 45 participants comprising 37 SARS-CoV-2 negative and 8 COVID-19 recovered participants have been enrolled so far with their complete blood counts and CD4 T lymphocyte counts determined as well as having all their serum samples and genomic DNA and RNA sample extracted and stored at -200C until further analyses. Other expected outcomes include prevalence and distribution of lymphopenia and hypovitaminosis D among the control (SARS-CoV-2 negative), confirmed, hospitalized and recovered SARS-CoV-2 positive study participants, association lymphopenia with CD4 T lymphocyte level, serum vitamin D, sVCAM-1, sFasL and caspase 3 levels in hospitalized COVID-19 patients, expression levels of apoptosis related genes among hospitalized COVID-19 study participants, those with lymphopenia compared with those without lymphopenia and Frequency distribution of alleles, genotypes and haplotypes of VDR gene polymorphisms in COVID-19 afflicted Nigerians studied.
CONCLUSIONS: This study will aid in the genotypic and phenotypic stratification of COVID-19 afflicted Nigerian patients with and without lymphopenia to enable biomarker discovery and pave way for appropriate and timely deployment of patient-centered treatments to improve prognosis.
PMID:33621190 | DOI:10.2196/21242
