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
Bibliographic record
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.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.021 | 0.012 |
| Meta-epidemiology (narrow) | 0.002 | 0.002 |
| Meta-epidemiology (broad) | 0.003 | 0.003 |
| Bibliometrics | 0.002 | 0.002 |
| Science and technology studies | 0.003 | 0.001 |
| Scholarly communication | 0.002 | 0.002 |
| Open science | 0.002 | 0.002 |
| Research integrity | 0.003 | 0.003 |
| Insufficient payload (model declined to judge) | 0.025 | 0.005 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".