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

Abstract

Background The COVID-19 pandemic, caused by SARS-CoV-2, continues to impact health systems throughout the world with serious medical challenges being imposed on many African countries like Nigeria. Although emerging studies have identified lymphopenia as a driver of cytokine storm, disease progression, and poor outcomes in infected patients, its immunopathogenesis, as well as environmental and genetic determinants, remain unclear. Understanding the interplay of these determinants in the context of lymphopenia and COVID-19 complications in patients in Africa may help with 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 pathways, and vitamin D receptor (VDR) gene polymorphisms in the immunopathogenesis of lymphopenia among African people infected with SARS-CoV-2. Methods This cross-sectional study will enroll 230 participants, categorized as “SARS-CoV-2 negative” (n=69), “COVID-19 mild” (n=32), “hospitalized” (n=92), and “recovered” (n=37), from two health facilities in Lagos, Nigeria. Sociodemographic data, travel history, and information on comorbidities will be obtained from case files and through a pretested, interview-based structured questionnaire. Venous blood samples (5 mL) collected between 8 AM and 10 AM and aliquoted into EDTA (ethylenediaminetetraacetic acid) and plain tubes will be used for complete blood count and CD4 T cell assays to determine lymphopenia (lymphocyte count <1000 cells/µL) and CD4 T lymphocyte levels, as well as to measure the concentrations of vitamin D, caspase 3, soluble vascular cell adhesion molecule-1 (sVCAM-1), and soluble Fas ligand (sFasL) using an autoanalyzer, flow cytometry, and ELISA (enzyme-linked immunosorbent assay) techniques. Genomic DNA will be extracted from the buffy coat and used as a template for the amplification of apoptosis-related genes (Bax, Bcl-2, BCL2L12) by polymerase chain reaction (PCR) and genotyping of VDR (Apa1, Fok1, and Bsm1) gene polymorphisms by the PCR restriction fragment length polymorphism method and capillary sequencing. Total RNA will also be extracted, reverse transcribed, and subsequently quantitated by reverse transcription PCR (RT-PCR) to monitor the expression of apoptosis genes in the four participant categories. Data analyses, which include a 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 and linkage disequilibrium analyses for the alleles, genotypes, 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 individuals have been enrolled so far. Their complete blood counts and CD4 T lymphocyte counts have been determined, and their serum samples and genomic DNA and RNA samples have been extracted and stored at –20 °C until further analyses. Other expected outcomes include the prevalence and distribution of lymphopenia and hypovitaminosis D in the control (SARS-CoV-2 negative), confirmed, hospitalized, and recovered SARS-CoV-2–positive participants; association of lymphopenia with CD4 T lymphocyte level, serum vitamin D, sVCAM-1, sFasL, and caspase 3 levels in hospitalized patients with COVID-19; expression levels of apoptosis-related genes among hospitalized participants with COVID-19, and those with lymphopenia compared to those without lymphopenia; and frequency distribution of the alleles, genotypes, and haplotypes of VDR gene polymorphisms in COVID-19–infected participants. Conclusions This study will aid in the genotypic and phenotypic stratification of COVID-19–infected patients in Nigeria with and without lymphopenia to enable biomarker discovery and pave the way for the appropriate and timely deployment of patient-centered treatments to improve prognosis. International Registered Report Identifier (IRRID) DERR1-10.2196/21242

Blood concentrations of proapoptotic sFas and antiapoptotic Bcl2 and COVID-19 patient mortality

“…We recorded demographic and clinical data at ICU admission as sex, age, body max index, chronic renal failure, diabetes mellitus, COPD, ischemic heart disease, smoking, arterial hypertension, steroid agents, human immunodeficiency virus (HIV), solid tumor, hematological tumor, ARDS [ 15 ], Acute Physiology and Chronic Health Evaluation (APACHE)-II score [ 16 ] Sepsis-related Organ Failure Assessment [SOFA] score [ 17 ], and chest radiography findings. Also, we recorded laboratory data at ICU admission as lactic acid, creatinine, sodium, protein, glucose, creatine kinase, albumin, bilirubin, alanine transaminase, alkaline phosphatase, aspartate transaminase, lactate dehydrogenase, gamma-glutamyl transpeptidase, interleukin-6, ferritin, procalcitonin, C-reactive protein, hemoglobin, hematocrit, white blood cell, lymphocytes, basophils, neutrophils, monocytes, eosinophils, d -dimer, platelets, fibrinogen, activated partial thromboplastin time (aPTT), international normalized ratio (INR), pressure of arterial oxygen (PaO 2 ), and fraction inspired of oxygen (FIO 2 ).…”

“…Higher apoptosis of lymphocytes [ 11 , 12 ] and higher expression of Fas in lymphocytes [ 13 , 14 ] of COVID-19 patients than in control subjects have been found, and this is associated with lymphopenia of COVID-19 patients. In addition, higher blood concentrations of caspase-8 [ 15 ], caspase-3 [ 16 ], sFasL [ 16 ] and caspase-cleaved cytokeratin 18 [ 17 ] in COVID-19 patients than in control subjects have been found. However, we have not found data about the association between apoptosis and mortality of COVID-19 patients.…”

Clinical applications of plasma proteomics and peptidomics: Towards precision medicine

“…COVID‐19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), is highly contagious and has spread rapidly around the world, posing an unprecedented threat to global public health and economy and attracting widespread attention in the academic and social communities [205–208]. SARS‐CoV‐2 affects not only the respiratory system, but also the digestive system, nervous system, and cardiovascular system, so its clinical manifestations are characterized by a variety of symptoms, including fever, pneumonia, sepsis, respiratory failure, acute respiratory distress syndrome (ARDS), and multiorgan injury [209, 210]. Statistically, approximately 80% of patients are commonly asymptomatic or minimally symptomatic and are classified as having mild or moderate COVID‐19.…”

“…COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is highly contagious and has spread rapidly around the world, posing an unprecedented threat to global public health and economy and attracting widespread attention in the academic and social communities [205][206][207][208]. SARS-CoV-2 affects not only the respiratory system, but also the digestive system, nervous system, and cardiovascular system, so its clinical manifestations are characterized by a variety of symptoms, including fever, pneumonia, sepsis, respiratory failure, acute respiratory distress syndrome (ARDS), and multiorgan injury [209,210] Using a fully automated robotic liquid handler 96 samples can be processed within 4 h. A TFC-MS system allows multiplexed analysis of four samples within 10 min, enabling the processing of more than 500 samples per day [221]. However, this per se would not appear to be compatible with the 100,000 s of tests required per day in countries with major outbreaks.…”

Introductory Chapter: Applications of RNA-Seq Diagnostics in Biology and Medicine

“…Bioinformatics characterization of pathological host immune gene expression allowed for discoveries of novel biomarkers of immune reconstitution inflammatory syndrome (IRIS), in the HIV-infected population [19][20][21]. Additionally, severe illnesses that begin with uncontrolled overexpression of proinflammatory cytokines (the so-called "cytokine storm") have been discovered through transcriptomics in viral (e.g., SARS-CoV) infections and many bacterial infections [22][23][24][25].…”