Altered Plasma Cytokine Concentrations Are Associated with the Clinical Severity of Patients with Covid-19

Introduction: The novel Coronavirus (SARS-CoV-2), responsible for severe acute respiratory syndrome, has emerged as a threat to humans since December 2019, and the search for a better understanding of the pathophysiology of coronavirus disease 2019 (COVID-19) and its definitive treatment is still in progress. Objective(s): To evaluate the plasma pro- and anti-inflammatory cytokines in COVID-19 patients and their associations with the disease severity and outcome. Method(s): Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR)-confirmed COVID-19 unvaccinated patients at the Hospital de Clinicas (HC), UNICAMP, Campinas, SP, were enrolled. Clinical and laboratory data were extracted from the medical records, and the plasma cytokines levels were quantified using LUMINEX and ELISA. Result(s): There were 154 COVID-19 patients (99 survivors and 55 non-survivors) with male:female of 1.4:1, and a median age of 60 years. The non-survivors were older than survivors (65 vs. 55 years, p < 0.0001);and coronary artery disease and autoimmunity, disease severity, and oxygen therapy, intensive care, and intubation were associated with mortality. Non-survivors had higher leukocyte and neutrophil counts, and RDW and lower lymphocyte count at diagnosis. Non-survivors had higher levels of pro-inflammatory (TNF-alpha, IL-6, IFN-gamma, CCL3, IL-17/IL-17A, IL-8, G-CSF, CCL2/MCP-1) and anti-inflammatory (IL-1ra and IL-27) cytokines, but lower TGF-beta levels than the survivors. TNF-alpha levels were positively correlated with all studied cytokines except TGF-beta, while TGF-beta levels were negatively correlated with TNF-alpha, IL-6, CCL3, G-CSF, and IL-27. IL-27 levels were significantly correlated with all the cytokines except IL-37 and IL-17E. More than half (55.2%) of our patients had severe COVID-19, 18.8% had moderate, 16.2% had critical, 5.2% had mild, and 4.5% were asymptomatic. Majority of the patients (68.2%) required ICU care and had higher TNF-alpha, IL-6, IL-8, IL-17, CCL3, CCL2, IL-1ra, and IL-27 than others. 59.7% of the patients required endotracheal intubation and had higher TNF-alpha, IL-6, IL-8, CCL3, CCL2, and IL-1ra than those who did not have intubation. TNF-alpha, IL-6, and IL-8 had the highest Area Under the Receiver Operating Characteristics (AUROC) curve, sensitivity, and specificity for predicting mortality in these COVID-19 patients. Discussion and conclusion: The altered levels of pro- and anti-inflammatory cytokines support the role of SARS-CoV-2 in inducing cytokine storm, and higher concentrations seen in the deceased patients meant a more severe storm. Also, the increased leukocytes and neutrophils in our patients could have led to the release of reactive oxygen species, and end-organ damage, thus leading to poor outcomes. This study showed that the levels of these cytokines could be used as markers of mortality in COVID-19. It is possible to suggest that TNF, IL-6, and IL-8 levels at diagnosis could be efficient predictors of fatal outcomes in COVID-19 patients. If properly measured at diagnosis, these markers could be useful for triaging and predicting the outcome of COVID-19, thus guiding the treatment of the COVID-19. Funding(s): CNPq (#190374/2017-9), CAPES, FAPESP and FAEPEX (#338619). Copyright © 2022

ASSOCIATION OF LYMPHOCYTE COUNTS, NLR AND PLR WITH MORTALITY IN COVID-19 PATIENTS

Background/objective: The severity and outcome of COVID-19 are determined by the level of overstimulation of the immune response, age, and comorbidities in the patients infected by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). Lymphopenia is the most consistent finding that characterizes the hemogram in COVID-19 patients. We evaluated the hemogram and compared the lymphocyte count (LC),neutrophil-lymphocyte ratio (NLR), and platelet-lymphocyte ratio (PLR) at diagnosis in COVID-19 patients hospitalized at the Clinical Hospital of the State University of Campinas (UNICAMP), Campinas, São Paulo, Brazil. Methods: In this retrospective study, we reviewed the medical notes of 320 adult hospitalized patients with PCR-confirmed COVID-19 at the Clinical Hospital of UNICAMP, Campinas, from March 2020 to March 2021. The hemogram (performed using automated counter-XN 9000™, Sysmex, Japan) at COVID-19 diagnosis was analyzed, and NLR and PLR were calculated. The primary outcomes were discharge (n = 257 patients who recovered from the disease and were discharged from the hospital), and death (n = 63 those who died during treatment). Statistical analyses were performed using SPSS (version 22). Unpaired data of deceased and discharged COVID-19 patients were compared using Mann-Whitney tests. All results were significant if p < 0.05 or except otherwise stated. Results: Compared to the 257 discharged patients, the 63 deceased patients were older 56.0 vs 64.7 ys respectively, p = 0.000), the males are more in each group and the duration of hospitalization was not different (18.6 vs 19.7 days respectively, p = 0.12). The leukocyte (8.89 ± 4.50 vs 10.37 ± 7.03, p = 0.289) and platelet counts (227.00 ± 91.15 vs 197.79 ± 97.47, p = 0.119) were not significantly different in the two groups, the hematocrit was higher in the discharged than in the deceased patients (38.84 ± 6.86 vs 35.89 ± 8.57, p = 0.021). The LC was lower in the deceased (0.81 ± 0.59 × 103 vs 1.09 ± 0.80x103/μL, p = 0.002), and negatively correlated with the age of the patients(r = -0.145, p=0.009 at a significant level of 0.01). The deceased group had a higher NLR (17.52 ± 19.20 vs 10.06 ± 12.31, p < 0.001) and PLR (366.32 ± 275.03 vs 319.23 ± 331.54, p = 0.047) higher than the discharged group, and both parameters were strongly correlated (r = 0.734, p < 0.001 significant level of 0.01). One hundred and thirty-eight (53.7%) of the discharged patients and 45 (71.4%) of the deceased had LC of < 1.0 × 103/μL. The LC is associated with the disease outcome (χ2 = 6.498, df = 1, p = 0.011), and the odds for a deceased to have a lymphopenia is 1.9 times that for the discharged patients [OR = 1.87 (95% CI = 1.135-3.085). Discussion: Though lymphopenia is consistent in COVID-19, the cause is unclear. Acute recruitment of lymphocytes to the site of infection (mainly the lung) may explain this, thus the lymphopenia may worsen and the LRs will be elevated with the increasing severity of COVID-19. The negative correlation of LC with age and higher odds of lymphopenia in the deceased patients suggest that LC and the LRs at diagnosis could be easily accessible and useful predictors of severity and mortality in these patients. Conclusion: Our study supports that lymphopenia is negatively associated with mortality in COVID-19 patients and that the deceased patients have elevated NLR and PLR at diagnosis. These parameters are easily derived from the hemogram and could be utilized as affordable and accessible predictors of outcomes in patients with COVID-19.