Improving the Understanding of the Immunopathogenesis of Lymphopenia as a Correlate of SARS-CoV-2 Infection Risk and Disease Progression in African Patients: Protocol for a Cross-sectional Study

摘要

Background The COVID-19 pandemic,caused by SARS-CoV-2,continues to impact health systems throughout the world with serious medicalchallenges being imposed onmanyAfrican countries like Nigeria. Although emerging studies haveidentified lymphopeniaasa driver ofcytokinestorm, disease progression,and poor outcomes in infected patients, its immunopathogenesis,as wellasenvironmentaland genetic determinants, remain unclear. Understanding theinterplay ofthese determinants in thecontext oflymphopeniaand COVID-19 complications in patients inAfrica may help with risk stratification and appropriate deployment oftargeted treatment regimens with repurposed drugs to improve prognosis. Objective This study is designed to investigatetherole of vitaminDstatus, vasculopathy,apoptotic pathways,and vitaminDreceptor (VDR) gene polymorphisms in theimmunopathogenesis oflymphopeniaamongAfrican peopleinfected with SARS-CoV-2. Methods Thiscross-sectionalstudywillenroll 230 participants,categorized as“SARS-CoV-2 negative”(n=69),“COVID-19 mild”(n=32),“hospitalized” (n=92),and “recovered”(n=37), fromtwo health facilities inLagos, Nigeria. Sociodemographic data, travel history,and information on comorbidities will be obtained fromcasefilesand through a pretested, interview-based structured questionnaire. Venous blood samples (5 mL) collected between 8 AMand 10 AMand aliquoted into EDTA(ethylenediaminetetraaceticacid)and plain tubes will be used forcomplete blood countand CD4 T cellassays to determinelymphopenia(lymphocytecount 1000 cells/μL)and CD4 T lymphocytelevels,as wellas to measure theconcentrations of vitaminD,caspase 3, soluble vascularcelladhesionmolecule-1 (sVCAM-1),and soluble Fas ligand (sFasL) using an autoanalyzer, flowcytometry,and ELISA(enzyme-linked immunosorbentassay) techniques. Genomic DNAwill beextracted fromthe buffy coat and used asatemplatefor theamplification ofapoptosis-related genes (Bax, Bcl-2, BCL2L12) by polymerasechain reaction (PCR)and genotyping ofVDR(Apa1, Fok1,and Bsm1) gene polymorphisms by the PCRrestriction fragment length polymorphismmethod and capillary sequencing. TotalRNAwillalso beextracted, reversetranscribed,and subsequently quantitated by reversetranscription PCR(RT-PCR) to monitor theexpression ofapoptosis genes in thefour participantcategories. Dataanalyses, which includeatest ofassociation betweenVDRgene polymorphismsand study outcomes (lymphopeniaand hypovitaminosis Dprevalence, mild/moderateand severeinfections) will be performed using the Rstatisticalsoftware. Hardy-Weinberg equilibriumand linkage disequilibriumanalyses for thealleles, genotypes,and haplotypes ofthe genotyped VDRgene willalso becarried out. Results Atotal of 45 participantscomprising 37 SARS-CoV-2–negativeand 8 COVID-19–recovered individuals have been enrolled so far. Their complete blood countsand CD4 T lymphocytecounts have been determined,and their serumsamplesand genomic DNAand RNAsamples have been extracted and stored at –20 °C untilfurtheranalyses. Otherexpected outcomes includethe prevalenceand distribution oflymphopeniaand hypovitaminosis Din thecontrol(SARS-CoV-2 negative),confirmed, hospitalized,and recovered SARS-CoV-2–positive participants; association oflymphopenia withCD4 T lymphocytelevel, serumvitaminD, sVCAM-1, sFasL,and caspase 3 levels in hospitalized patients with COVID-19;expression levels ofapoptosis-related genesamong hospitalized participants withCOVID-19,and those with lymphopeniacompared to those without lymphopenia;and frequency distribution ofthealleles, genotypes,and haplotypes ofVDRgene polymorphisms inCOVID-19– infected participants. Conclusions This studywillaid in the genotypicand phenotypicstratification ofCOVID-19–infected patients inNigeria with and without lymphopeniato enable biomarker discovery and pavethe way for theappropriateand timely deployment of patient-centered treatments to improve prognosis.