Immune responses in SARS-CoV-2-infected patients at Moi Teaching and Referral Hospital, Eldoret, Kenya

Abstract

Background: Emerging and re-emerging infections pose significant global health challenge. Clinical spectrum of Coronavirus Disease 19 (COVID-19) ranges from asymptomatic to severe life-threatening conditions. Recent research suggests that immune responses may influence disease severity, but the dynamics between immune responses and COVID-19 progression remain unclear. Objective: To determine viral load, levels of anti-viral IgG, interferon gene expressions, C-reactive protein (CRP) and lactate dehydrogenase (LDH) in COVID-19 patients at Moi Teaching and Referral Hospital (MTRH) and Kenya Medical Research Institute (KEMRI) in Kenya Methodology: This was a retrospective cross-sectional study of 48 asymptomatic patients with moderate to severe COVID-19 and 48 SARS-CoV-2-negative individuals at MTRH. Real-time quantitative PCR (RT-qPCR) (Bio-Rad, Sweden) was used to quantify viral load in swabs and interferon mRNA gene expression in blood and mucosal compartments. Antibody levels were quantified using MSD-MULTISPOT® assay in blood and nasopharyngeal samples, while CRP and LDH in blood were determined using VITROS 5600/XT 7600 (Integrated Systems, USA). Data Analysis: Variables were tested for normality. Unpaired t-test, Mann-Whitney U-test, one-way ANOVA, Kruskal-Wallis were used to test for differences between the groups and regression analysis, using GraphPad Prism version 9.0, p<0.05 was considered statistically significant. Result: Severely ill patients demonstrated higher viral loads, copies/ml, mean±SD11.9±9.55x 106 compared to moderate 7.44±6.81 x 106 and asymptomatic 4.18±3.12 x 106 p=0.02. Severely ill patients recorded higher systemic anti-spike IgG, BAU/ml, 1219±124 and anti-nucleocapsid 872.3±388.7 as compared to asymptomatic 554.1±145.4 vs 403.8±464.3 p=0.0245 and p<0.0001 respectively. Asymptomatic individuals had higher mucosal anti-spike IgG, BAU/ml, 6.023±12.79 and receptor binding domain (RBD) 7.236±14.70 in comparison to severely ill, 0.5738±0.8877, p=0.0034 and 0.7275±1.103, p=0.007 respectively. Systemic IFN-α mRNA transcript (normalized values) was significantly higher in asymptomatic individuals (median [IQR], 95% CI 1.473 [0.5907-2.412]) compared to severely ill individuals (0.1542 [0.1157-0.4940]) and healthy individuals (1.0). In blood, severely ill patients had significantly lower mean IFN-γ mRNA transcript 0.1737(0.0426-1.045) in contrast to healthy, moderate 0.6803 (0.4982-1.271) and asymptomatic 1.032(0.2707-1.197) individuals (p=0.0311). In mucosa, IFN-γ mRNA levels of asymptomatic persons were significantly higher (1.123±0.8701) than healthy (p=0.0201), severely 0.1894(0.0365-0.7772) and moderately ill individuals 0.3602(0.0965-0.7789). Severely ill patients recorded significantly lower systemic IFI-16 mRNA transcript levels 0.1166(0.1009-0.1459) in comparison to moderate 0.2721(0.1693-0.4199), asymptomatic 0.2545(0.1291-0.5104) and healthy. Regression analysis showed a positive association between CRP (0.5433; p=0.0006) with a 95% CI: 9.361-110.9 and LDH (0.2484; p<0.0001) and a 95% CI between 0.6948-1.252 with COVID-19 severity. Discriminative accuracy was highest when asymptomatic patients were compared to severe COVID-19 for CRP (AUC: 0.8867, 95% CI: 0.7532-1.000) and LDH (AUC: 1.000, 95% CI: 1.000-1.000). Conclusion: SARS-CoV-2 viral load increased illness severity. Mucosal anti-spike and RBD IgG appeared protective against severe COVID-19, while systemic anti-spike, RBD and nucleocapsid correlated with disease progression. Both mucosal and systemic interferon responses are suppressed in severe disease. Increased CRP and LDH levels correlated with COVID-19 severity. Recommendation: Mucosal IFN mRNA expression, CRP, LDH and IgG levels may be used to predict the course of COVID-19 and targeted treatments for COVID-19 patients. A prospective study on interferon protein levels may provide insight into COVID-19 progression.