Study design and participants

This case–control study was conducted on 150 adult COVID-19-assured cases recruited from the Departments of Tropical Medicine, Chest, and Intensive Care Unit of Menoufia University Hospital in collaboration with the National Liver Institute Hospital from September 2022 to February 2023. Patients 18 years of age or older who had COVID-19 confirmed by polymerase chain reaction (PCR) from nasopharyngeal and oropharyngeal samples were included. Exclusion criteria included patients with pre-existing cardiac, hepatic, renal, chest, or coagulation disorders. In addition to COVID-19-vaccinated participants and those with a previous history of COVID-19, all patients who declined to participate in the study, skipped the study, or lacked the necessary data were not included. Furthermore, 74 healthy participants were included in the healthy control (HC) group. HC were selected from healthcare providers who appeared healthy and showed no signs of COVID-19 infection based on common clinical criteria and laboratory testing (scheduled screening for health care workers by detection for both S and A antigens). The Epi-Info website was utilized for calculating the sample size (https://www.cdc.gov/Epi-Info/unmatched-case-control).

Clinical assessment of studied patients

Throughout the research period, patients who presented to the COVID-19 isolation unit at the Faculty of Medicine, Menoufia University Hospital, with a clinical suspicion of the virus were evaluated. Confirmed cases were categorized as non-severe, severe, or critical after evaluations by clinical, laboratory, and radiological methods. In cases deemed non-severe, a prescription for outpatient treatment was given, and additional follow-up was done via phone or at the COVID-19 outpatient clinic. COVID-19 patients exhibiting severe or critical symptoms were admitted to the COVID-19 isolation ward or intensive care unit (ICU), where initial clinical, laboratory, and radiological data were documented. In addition, a daily assessment of the illness’s progression and the patient’s response to treatment were noted and assessed. Patients were monitored until their death or discharge from the hospital.

COVID-19 patients were classified based on CT presentations using the COVID-19 Reporting and Data System (CO-RAD) radiological score. The findings of CO-RADS 1 are either normal or non-infectious; CO-RADS 2 is idealistic for other infectious causes but not COVID-19 pneumonia; and CO-RADS 3 is comparable to COVID-19 pneumonia in addition to other illnesses. CO-RADS 4 and 5 are highly compatible with COVID-19 pneumonia, but with atypical features as well in CO-RADS 4 [15]. According to the World Health Organization (WHO) guidelines [16], patients were divided into three categories as follows: The non-severe COVID-19 group included 78 patients not meeting either critical or severe COVID-19 criteria. The severe COVID-19 group included 39 patients with any of the following criteria: on room air, oxygen saturation < 90%, or evidence of severe respiratory distress (respiratory rate > 30 breaths per minute, difficulty finishing the entire sentence, or using accessory muscles of respiration), in addition to signs of pneumonia. The critically ill COVID-19 group included 33 patients who met the criteria for acute respiratory distress syndrome, sepsis, septic shock, or conditions requiring life-sustaining therapeutic strategies.

Laboratory investigations

10 mL of blood samples were collected from all participants after overnight fasting and aliquoted into ethylenediaminetetraacetic acid (EDTA)-containing tubes, sodium citrate-containing tubes, and plain tubes for SNPs, CBC analysis, coagulation parameters, and biochemical investigations, respectively. After centrifugation at high speed for 10 min, sera were analyzed for blood chemistry (LDH, CRP, blood sugar, liver and kidney function tests, and lipid profile) using a fully automated chemistry analyzer, SYNCHRON CX9ALX Beckman Coulter (CA, USA), which was also utilized to estimate D-dimer. CBC was assayed by a semi-automated Sysmex analyzer (Siemens, Germany), INR/PT (The Sysmex CA-600 Systems, Siemens, Germany), and serum ferritin (Abbott chemiluminescence instrument, Architect, USA).

IL-6 assay

The IL-6 Human ELISA Kit (Invitrogen, USA) was utilized and then measured by a Multiskan Sky Microplate Spectrophotometer (Thermo Fisher Scientific, USA).

For health care workers (control group)

During scheduled screening, five mL of the blood were delivered to a vacutainer plain test tube. Serum was separated by centrifugation at 3000 rpm for 10 min and was used for detection of the specific anti-COVID-19 antibody by chemiluminescence immunoassay seronegative for IgG for S or N using Cobas 6000 (Roche, Germany).

COVID-19 RNA analysis

RNA extraction of nasopharyngeal swab specimens was performed using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Then, the cDNA first strand was prepared using the cDNA synthesis kit (Invitrogen) and specific primers using the Proflex cycler (USA). The diagnosis of COVID-19 was confirmed by a positive fluorescent signal and RT-PCR detection of cDNA. The signal reflects a positive test result.

SNP analyses of FURIN, IFNL4, and TLR2 genes

Genotyping of FURIN (rs6226), IFN4 (rs12979860), and TLR2 (rs3804099) genes was performed using allelic discrimination techniques that detect the variants of the gene. Unknown samples were classified as homozygotes (samples with only allele 1 or allele 2) and heterozygotes (samples with both allele 1 and allele 2). Genomic DNA extraction was conducted using a spin column method (Thermo Scientific, Lithuania, GeneJET whole blood genomic DNA purification mini kit). DNA concentration was measured using NanoDrop spectrophotometer (Thermofisher Scientific, USA). PCR was performed on a real-time PCR system (7500 Fast, Applied Biosystems, USA) using the TaqMan SNP genotyping assay: primers and probe (40  ×) (Thermo Fisher Scientific, MA, USA) and genotyping qPCR Master Mix (2 ×). For the amplification reaction, the master mix (total volume: 20 µL) consisted of 10 µL of genotyping qPCR master mix, 0.5 µL of genotyping assay, and 3.5 µL of DNAse-free water; then, 6 µL of extracted genomic DNA template was added. 6 µL of DNAse-free water was then added as a negative control reaction. The cycling parameters were set as follows: holding stage (pre-PCR): 60 °C for 1 min, initial denaturation step: 95 °C for 10 min, cycling stage: denaturation step: 95 °C for 1 min repeated for 35 cycles, annealing and extension: 60 °C for 1 min, and post-PCR (holding stage): 60 °C for 1 min. The TaqMan assays were predesigned (for FURIN rs6226: C_11947693_10, for INF4 rs12979860: C_7820462_10, and for TLR2 rs3804099: C_22274563_10).

Statistical analysis

Data were analyzed using IBM SPSS package version 20.0 (Armonk, NY: IBM Corp.). Categorical information was delineated as numbers and percentages. The Chi-square (χ2) test was used for comparing between two groups. Alternatively, the Fisher Exact correction test was applied when > 20% of the cells had an expected count < 5, and the Monte Carlo correction test was applied when > 20% of the cells had an anticipated count of < 5. The Kolmogorov–Smirnov test was applied to test for normality for continuous data. Quantitative data were expressed as range (minimum and maximum), mean, standard deviation, and median. For non-normally distributed quantitative variables, the Mann–Whitney test was applied for comparing between two groups, whereas the Kruskal–Wallis test was used to compare between more than two groups, and then the Post-Hoc test (Dunn’s multiple comparisons test) was utilized for pairwise comparison. The population of the studied sample was investigated in order to determine its equilibrium using the Hardy–Weinberg equation. The 95% confidence interval (CI) and odds ratio (OR) were calculated to assess the effects of alleles and genotypes. For additional analysis, OR was done in various genetic models (dominant, recessive, and additive). Regression analysis was performed to detect the independent factor (s) for COVID-19 susceptibility, severity, and mortality. A P value of < 0.05 was considered statistically significant.