Secretory Phospholipase A2 and Interleukin-6 Levels as Predictive Markers of the Severity and Outcome of Patients with COVID-19 Infections

Coronavirus disease (COVID-19) has become a global pandemic. COVID-19 patients need immediate diagnosis and rehabilitation, which makes it urgent to identify new protein markers for a prognosis of the severity and outcome of the disease. The aim of this study was to analyze the levels of interleukin-6 (IL-6) and secretory phospholipase (sPLA2) in the blood of patients regarding the severity and outcome of COVID-19 infection. The study included clinical and biochemical data obtained from 158 patients with COVID-19 treated at St. Petersburg City Hospital No. 40. A detailed clinical blood test was performed on all patients, as well as an assessment of IL-6, sPLA2, aspartate aminotransferase (AST), total protein, albumin, lactate dehydrogenase (LDH), APTT, fibrinogen, procalcitonin, D-dimer, C-reactive protein (CRB), ferritin, and glomerular filtration rate (GFR) levels. It was found that the levels of PLA2, IL-6, APTV, AST, CRP, LDH, IL-6, D-dimer, and ferritin, as well as the number of neutrophils, significantly increased in patients with mild to severe COVID-19 infections. The levels of IL-6 were positively correlated with APTT; the levels of AST, LDH, CRP, D-dimer, and ferritin; and the number of neutrophils. The increase in the level of sPLA2 was positively correlated with the levels of CRP, LDH, D-dimer, and ferritin, the number of neutrophils, and APTT, and negatively correlated with the levels of GFR and lymphocytes. High levels of IL-6 and PLA2 significantly increase the risk of a severe course by 13.7 and 2.24 times, and increase the risk of death from COVID-19 infection by 14.82 and 5.32 times, respectively. We have shown that the blood levels of sPLA2 and IL-6 increase in cases which eventually result in death and when patients are transferred to the ICU (as the severity of COVID-19 infection increases), showing that IL-6 and sPLA2 can be considered as early predictors of aggravation of COVID-19 infections.


Introduction
Coronaviruses (CoV) are the causative agents of acute severe respiratory syndrome (SARS-CoV), which first caused a global epidemic in 2002 [1]. In December 2019, a new form of coronavirus (SARS-CoV-2) was recorded in China, which caused a global pandemic of coronavirus disease , which spread to 228 countries in 2020-2023 [2]. To date, the pandemic has caused more than 669 million infections and more than 6.7 million deaths worldwide [3]. outcome of the disease, which will allow personalizing medical rehabilitation and therapy programs. The definition of such new biomarkers should be accessible for clinical diagnosis, as cheaply and informatively as possible. The availability of biomarkers is determined by the possibility of their rapid detection in tissue samples or biological fluids of the patient using methods used in laboratory diagnostics.
The aim of this work was to analyze the levels of interleukin-6 and secretory phospholipase in the blood of patients depending on the severity and outcome of the COVID-19 infection.

Results
Initially, we studied the severity and outcome in 158 patients with COVID-19 infections. Depending on the severity of the COVID-19 infection, patients were divided into groups with mild 38.6% (n = 61), average 23.4% (n = 37), and severe 38.0% (n = 60) severity. The survival rate (discharge) of patients was 80.4% (127/158) and the mortality was 19.6% (31/158). Survival among men was 81.6%, while among women it was 78.3% (Table 1). Statistically significant differences depending on age among discharged and deceased patients and among men and women were not found. The results are presented as the arithmetic means ± standard deviations.
Then, the influence of the cardiological history and some clinical indicators, such as transfer to the ICU, BMI, number of days from the onset of the disease from biobanking, and maximum CT on the severity of COVID-19 infection, was determined ( Table 2). The results presented in Table 2 show that the transfer to the ICU, the number of days from the onset of the disease, and the CT index statistically significantly increased with an increased severity of the disease. The severity of COVID-19 infection was affected by pulmonary circulatory disorders, the incidence of patients with hypertension, coagulopathy, and iron deficiency anemia.
The results shown in Figure 1 show that the levels of APTV, AST, CRP, LTK, IL-6, Ddimer, and ferritin, as well as the number of neutrophils, significantly increased in patients with mild and severe COVID-19 infections ( Figure 1). On the contrary, hematocrit, GFR, lymphocyte, and eosinophil levels were statistically significantly reduced in these groups of patients compared to the values in patients with a mild infection. The PLA2 level also significantly increased in mild to moderate COVID-19 infections, but it did not change in patients with severe infections. These data on changes in the level of PLA2 in the blood show that this enzyme can be considered as an early marker of exacerbation of COVID-19 infection.
IL-6 and PLA2 levels were also analyzed depending on the severity of the disease according to computed tomography ( The results are presented as the arithmetic means ± standard deviation. The significance levels evaluated as * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001. The results shown in Figure 1 show that the levels of APTV, AST, CRP, LTK, IL-6, Ddimer, and ferritin, as well as the number of neutrophils, significantly increased in patients with mild and severe COVID-19 infections ( Figure 1). On the contrary, hematocrit, GFR, lymphocyte, and eosinophil levels were statistically significantly reduced in these groups of patients compared to the values in patients with a mild infection. The PLA2 level also significantly increased in mild to moderate COVID-19 infections, but it did not change in patients with severe infections. These data on changes in the level of PLA2 in the blood show that this enzyme can be considered as an early marker of exacerbation of COVID-19 infection.
IL-6 and PLA2 levels were also analyzed depending on the severity of the disease according to computed tomography ( Figure 2). The results shown in Figure 2 show that the levels of IL-6 and PLA2 increase statistically significantly with an increase in the CT score. Moreover, the increase in PLA2 is almost 2 times higher than the increase in IL-6 at the beginning of the disease, when the CT score changes from 0 to 1. On the contrary, the level of IL-6 increases two-fold at the height of the disease, when the CT score increases from 2 to 3. In addition, the dependence of the PLA2 level on the severity of infection is also confirmed by the presence of a sufficiently high correlation coefficient between the two (r = 0.311171, p = 0.00069).
An analysis of laboratory tests was carried out depending on the levels of IL-6 and sPLA-2.
Patient samples were also divided into groups with high and low levels of IL-6 and PLA2. The criteria for dividing IL-6 and PLA2 into low-and high-level groups were the values of their first quartile (Q1); values below Q1 were assigned to the low-level group The results are presented as medians and confidence intervals. The significance levels evaluated as * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001.
The results shown in Figure 2 show that the levels of IL-6 and PLA2 increase statistically significantly with an increase in the CT score. Moreover, the increase in PLA2 is almost 2 times higher than the increase in IL-6 at the beginning of the disease, when the CT score changes from 0 to 1. On the contrary, the level of IL-6 increases two-fold at the height of the disease, when the CT score increases from 2 to 3. In addition, the dependence of the PLA2 level on the severity of infection is also confirmed by the presence of a sufficiently high correlation coefficient between the two (r = 0.311171, p = 0.00069).
An analysis of laboratory tests was carried out depending on the levels of IL-6 and sPLA-2. Patient samples were also divided into groups with high and low levels of IL-6 and PLA2. The criteria for dividing IL-6 and PLA2 into low-and high-level groups were the values of their first quartile (Q1); values below Q1 were assigned to the low-level group and all values above Q1 were assigned to the high-level group of analytes (Table 3 and Figure 3).  The results shown in Figure 2 show that the levels of IL-6 and PLA2 increase statistically significantly with an increase in the CT score. Moreover, the increase in PLA2 is almost 2 times higher than the increase in IL-6 at the beginning of the disease, when the CT score changes from 0 to 1. On the contrary, the level of IL-6 increases two-fold at the height of the disease, when the CT score increases from 2 to 3. In addition, the dependence of the PLA2 level on the severity of infection is also confirmed by the presence of a sufficiently high correlation coefficient between the two (r = 0.311171, p = 0.00069).
An analysis of laboratory tests was carried out depending on the levels of IL-6 and sPLA-2.
Patient samples were also divided into groups with high and low levels of IL-6 and PLA2. The criteria for dividing IL-6 and PLA2 into low-and high-level groups were the values of their first quartile (Q1); values below Q1 were assigned to the low-level group and all values above Q1 were assigned to the high-level group of analytes (Table 3 and Figure 3).  The results shown in Table 3 show that the increased in the levels of APTT, AST, LDH, CRP, D-dimer, ferritin, and the number of neutrophils are statistically significantly different in groups with low and high concentrations of IL-6. On the contrary, hematocrit, GFR, lymphocyte, and eosinophil levels significantly decrease in the group of patients with medium and high concentrations of IL-6. The results shown in Table 3 show that the increase in the level of CRP and the decrease in lymphocytes are statistically significantly different in groups with low, medium, and high concentrations of PLA2. A statistically significant increase in LDH and D-dimer levels was observed between groups of patients with low and medium, and low and high concentrations of PLA2. An increase in APTT, ferritin, and the number of neutrophils and a decrease in GFR were statistically significantly observed between the groups of patients with low and high concentrations of PLA2. A statistically significant increase in AST levels was observed only between groups of patients with low and medium concentrations of PLA2. The number of eosinophils and hematocrit decreased between the groups of patients with low and high and medium and high severity of COVID-19 infection.
Correlation coefficients were calculated between the levels of IL-6, sPLA2 concentrations, demographics, clinical parameters, and history of concomitant diseases (Table 4). The results in Table 4 show that the levels of IL-6 and PLA2 are statistically significantly correlated with the transfer of patients to the ICU, CT score, the presence in the anamnesis of uncomplicated diabetes mellitus, and iron deficiency anemia in patients. In addition, IL-6 showed a weak but significant correlation with weight. PLA2 also showed a weak but significant correlation with obesity, and the level of IL-6 correlates with the presence of pulmonary circulatory disorders, pulmonary artery pressure, peripheral vascular disorders, coagulopathy, renal insufficiency, rheumatoid arthritis, collagen, and vascular diseases.
Correlations between the levels of PLA2 and IL-6 and the studied laboratory parameters were also analyzed ( Table 5 and Figure 3). Note: Statistically significant correlations at p < 0.05 are highlighted in bold. * IL-6 group (1: up to 100 pg/mL; 2: 100-200 pg/mL; 3: more than 200 pg/mL); ** group IL-6 (1: up to 40 and remained up to 40 pg/mL; 2: up to 40 and rose to more than 40 pg/mL; 3: more than 40 and remained more than 40 pg/mL).
The results in Table 6 and Figure 3 show that the level of IL-6 is statistically significantly positively correlated with the levels of PLA2, ASIA, AST, DV, SKI, and the laboratory score, and is negatively correlated with the number of lymphocytes and the PAC index. The level of PLA2 also correlates positively with the level of IL-6, SKI, and the laboratory score, and negatively correlates with the number of lymphocytes and the PAC index. Moreover, the correlation values for IL-6 were higher than for sPLA2. At the final stage, the endpoint (outcome of COVID-19 infection) was analyzed depending on the severity, the levels of IL-6, PLA2, ARTT, AST, LDH, CRB, GFR, procalcitonin, D-dimer, ferritin, and hematocrit, the number of lymphocytes, leukocytes, neutrophils, and eosinophils, and the laboratory score (Tables 6 and 7).
The results presented in Table 7 show that the probability of death from a COVID-19 infection is greatly increased in patients with a severe course (p < 0.0001); high levels of IL-6, PLA2, ARTT, AST, LDH, CRB, d-dimer, ferritin, and neutrophil counts; and low levels of GFR, lymphocytes, and hematocrit. The dependence of the outcome of COVID-19 infections on the level of PLA2 is also confirmed by the presence of a sufficiently high correlation coefficient between them (r = 0.310402, p = 0.0000721).
Finally, we calculated the odd ratio (OR) for the severity and the outcome of COVID-19 infections using our analytes (Figure 4).    The results presented in Figure 4 show that increased levels of LDH, IL-6, and procalcitonin are the factors most predictive of the development of a severe or fatal COVID-19 infection in patients. Elevated levels of PLA2, ACT, D-dimer, APTT, and hemocrit also predicted a severe or fatal COVID-19 infection in patients, with odd ratios of 2.24, 5.46, 5.68, 4.71, 6.39 and 5.32, 4.14, 2.85, 4.51, 12.81, respectively. A combination of elevated levels of PLA2 and IL-6 increased the risk of a severe infection and death of the patients from COVID-19 by 20.0 and 9.68 times.
In this study, it was found that sPLA2 levels increase in seriously ill patients, especially in the eventual case of death, and positively correlate with the severity and outcome of COVID-19 infections (Tables 2 and 5-7, Figures 1-4). These results are consistent with the data of a single study, which also showed that high levels of sPLA2-IIA in blood plasma correlate with its activity (r 2 = 0.84, p = 1.2 × 10 −13 ) and the severity of COVID-19 infection in 127 patients. In the group of patients who died from COVID-19, sPLA2-IIA levels could reach 1020 ng/mL, and were higher (89.3 ng/mL) than in those with severe (17.9 ng/mL) and mild (9.3 ng/mL) courses of the disease and those without coronavirus infection (8.9 ng/mL). Additionally, using a regression analysis model, sPLA2-IIA and urea nitrogen (BUN), at levels of 10 ng/mL and 16 mg/dL, respectively, were determined as the main clinical parameters for predicting mortality from COVID-19 infections with high accuracy (AUROC 0.93-1.0) and a sensitivity of 75.4% [31]. In another study, sPLA2 levels were elevated (269 ± 137.3 ng/mL, p = 0.01) in the blood plasma of 14 children with severe COVID-19 infections compared with those with asymptomatic (2.0 ± 3 ng/mL) and mild (23.0 ng/mL) cases. At the same time, the level of sPLA2 was increased (p = 0.04) in patients in the acute phase of the disease (540 ± 510 ng/mL) compared with the recovery period (2 ± 1 ng/mL). No correlations were found between sPLA2 and CRB and D-dimer levels and the leukocyte count [34]. At the same time, positive correlations between sPLA2-IIA levels and NEWS2 indicators and glucose levels, and negative correlations between urea creatinine, glomerular filtration rate, hematocrit, and hemoglobin saturation were determined, which also confirms the dependence of sPLA2-IIA on the severity of COVID-19 infection [14,35].
In addition, the severity of COVID-19 infection is positively correlated with the level of viremia and the number of apoptotic cells expressing PS. Consequently, such cells will be destroyed by sPLA2, which further increases systemic inflammation [36]. Inhibition of sPLA2-induced cell damage can be considered as a new approach against uncontrolled inflammation and cytokine storms.
This study also shows that the level of IL-6 increases in seriously ill patients and in the cases of unfavorable outcomes and it is correlated with the severity and death in patients (Tables 2 and 5-7, Figures 1-3). Our results are consistent with the research of other authors evaluating the prognostic significance of IL-6 in COVID-19 infections [37]. For example, in a retrospective single-center study conducted on 728 patients with COVID-19, the prognostic significance of elevated IL-6 levels for assessing mortality and a severe disease course was studied [38]. Using the logistic regression analysis of Cox in this study, the adjusted ratio of mortality risks and the severity of the disease in patients was evaluated. The authors concluded that elevated IL-6 levels may serve as an independent risk factor for severity and mortality in patients with COVID-19 [38]. A meta-analysis deserves attention, in which prognostic factors and the significance of elevated IL-6 levels in this pathology were determined in 1426 patients infected with COVID-19 [39]. It was shown that the best predictor of mortality or severe COVID-19 infection in patients is IL-6 (OR = 11.6460, 95%CI = 2.8123-48.2277), which predicts the onset of endpoints (outcomes) with an accuracy of 80.8%. Consequently, the level of IL-6 can serve as a prognostic marker of severe course, and especially where the outcome could be death, in patients with COVID-19 infections [39].
In an earlier study, we developed a model for assessing the risk of cytokine storms (CS) in 458 patients with COVID-19 infections [40]. The patients were divided into two groups, comparable in age. The first group consisted of 100 (21.8%) patients with clinical and radiological features characterizing a stable course of the disease of moderate severity and the second group consisted of 358 (78.2%) people with progressive moderate, severe, and extremely severe cases of the disease. When conducting a comparative analysis of clinical, instrumental, and laboratory data from the selected groups of patients, we found significant differences in the dynamics of the index on the NEWS scale, the absolute number of lymphocytes, and the levels of CRP, ferritin, D-dimer, and IL-6 between the groups, which can serve as the most important indicators characterizing the development of a CS. Using the method of constructing classification trees, the threshold levels for risk factors for the development of a CS were identified. We performed a comprehensive assessment of the risk of CSH by ranking the indicators, which, in accordance with the rank of prognostic significance obtained by the method of constructing "classification trees", at the beginning of CSH therapy was as follows: dynamics of the index on the NEWS scale; blood IL-6 level above 23 pg/mL; blood CRP level equal to or above 50 mg/L; absolute number of lymphocytes less than 0.72 × 10 9 /L; positive test result for coronavirus RNA (SARS-CoV-2); and an age of 40 years or older. These biomarkers can be used as criteria for assessing the risk of a CS. An increase in the frequency of CS cases correlates with an increase in the number of risk factors (correlation coefficient Rg = 0.91, p < 0.001). The following risk categories are identified for the practical application of our predictive model: category 1 (0-1 factor): the risk of CSH is practically absent; category 2 (2-3 factors): the risk of CSH increases sharply to 55%, a 35.5-fold increase compared to category 1; category 3 (4 or more factors): the risk of CSH reaches 96%, an 718-fold increase 718 compared to category 1. The results obtained are consistent with the assessment of risk factors for CS in COVID-19 by other authors [41,42] and allowed us to justify the choice of therapeutic tactics with early prescriptions of proactive anti-inflammatory therapy and anticoid plasma convalescents for patients with a high risk of CS.

Patients
The retrospective cohort study included clinical and biochemical data obtained from 158 patients (98 men and 60 women aged 51.2 ± 11.6 years), who showed a positive test results for the presence of SARS-CoV-2 RNA by nucleic acid amplification in polymerase chain reaction (PCR), treated at the budget healthcare institution "City Hospital No. 40 of St. Petersburg Resort administrative district", the boarding house "Zarya", and "City Hospital We determined the severity of COVID-19 infection in our patients based on assessment of their clinical status using the NEWS-2 scale, the degree of lung involvement on CT, the blood level of serum and plasma biomarkers, and their laboratory score.
The design of the study is presented in Figure 5.

Clinical Methods and Treatment of Patients with COVID-19
All patients were admitted for inpatient treatment in the emergency room to the infectious diseases department. It was mandatory to provide the entire volume of medical services in accordance with medical and economic standards and in accordance with the version of clinical recommendations in force at the time [4]. Data were collected from the patients, e.g., their epidemiological history, and the presence of clinical symptoms (cough, shortness of breath, fever, fever, weakness, loss of sense of smell and taste, and heaviness in the chest). We also conducted an objective examination of patients with an assessment of hemodynamic parameters, an assessment of the respiratory system (HR, HR, BP, and SpO2), and an assessment of the NEWS scale recommended for use for patients with COVID-19 [43]. On the day of admission or the next day, biomaterial was collected for laboratory tests and an electrocardiogram (ECG) was performed. Computed tomography (CT) of the chest organs was performed with an assessment of the form of the disease on a 4-digit scale without intravenous contrast enhancement (CT-1, CT-2, CT-3, and CT-4). The bilateral lower lobe; the peripheral, perivascular, multilobular character; numerous peripheral seals in the form of "frosted glass" with a rounded shape of various lengths; flattening of the interlobular interstitium in the type of a "cobblestone pavement"; areas

Clinical Methods and Treatment of Patients with COVID-19
All patients were admitted for inpatient treatment in the emergency room to the infectious diseases department. It was mandatory to provide the entire volume of medical services in accordance with medical and economic standards and in accordance with the version of clinical recommendations in force at the time [4]. Data were collected from the patients, e.g., their epidemiological history, and the presence of clinical symptoms (cough, shortness of breath, fever, fever, weakness, loss of sense of smell and taste, and heaviness in the chest). We also conducted an objective examination of patients with an assessment of hemodynamic parameters, an assessment of the respiratory system (HR, HR, BP, and SpO 2 ), and an assessment of the NEWS scale recommended for use for patients with COVID-19 [43]. On the day of admission or the next day, biomaterial was collected for laboratory tests and an electrocardiogram (ECG) was performed. Computed tomography (CT) of the chest organs was performed with an assessment of the form of the disease on a 4-digit scale without intravenous contrast enhancement (CT-1, CT-2, CT-3, and CT-4). The bilateral lower lobe; the peripheral, perivascular, multilobular character; numerous peripheral seals in the form of "frosted glass" with a rounded shape of various lengths; flattening of the interlobular interstitium in the type of a "cobblestone pavement"; areas of consolidation; symptoms of an air bronchogram, etc. [44], were mainly assessed and, if necessary, additional instrumental methods were used.
According to the national recommendations for the diagnosis and treatment of COVID-19 [4], we used the following classification of COVID-19 according to severity:

•
Mild course: body temperature < 38 • C, cough, weakness, and a sore throat. Absence of criteria for moderate and severe courses. • Moderate course: body temperature > 38 • C, respiratory rate > 22/min, shortness of breath during physical exertion, changes in CT (radiography) typical of a viral lesion, SpO 2 < 95%, and serum CRP > 10 mg/L. • Severe course: respiratory rate > 30/min; SpO 2 ≤ 93%; PaO 2 /FiO 2 ≤ 300 mmHg; decreased level of consciousness; agitation; unstable hemodynamics (systolic blood pressure less than 90 mmHg or diastolic blood pressure less than 60 mmHg, diuresis less than 20 mL/h); changes in the lungs in CT (radiography) typical of a viral lesion; arterial blood lactate > 2 mmol/L; and qSOFA > 2 points. • Extremely severe course: persistent febrile fever; ARDS; acute respiratory failure (ARF) with the need for respiratory support (invasive ventilation); septic shock; multiple organ failure; changes in the lungs on CT (X-ray) typical of a critical viral lesion or ARDS.
Treatment of COVID-19 infections and its complications included antiviral drugs, prevention of hypercoagulation and DIC syndrome, symptomatic treatment, and oxygen therapy. In patients with a progressive course of the disease, for the prevention or treatment of a cytokine storm (CS), standard therapy was supplemented with the appointment of pathogeninduced plasma convalescents, anti-cytokine drugs, interleukin-6 receptor inhibitors (IL-6) (tocilizumab, olokizumab, and levilimab), IL-1 (kanakinumab and RH104), JAK kinases (tofacitinib, ruxolitinib, and baricitinib), tyrosine kinase Bcr-Abl (radotinib), and, in some cases, glucocorticosteroids [40]. According to the indications, staged respiratory therapy, antibacterial therapy, treatment of sepsis and septic shock, extracorporeal detoxification and hemocorrection, and extracorporeal membrane oxygenation were performed.
A statement of biological death was made by the ICU doctor. The transfer of the patient in accordance with the indications specified in the clinical recommendations of the ICU was carried out and registered at the conclusion of the examination by the on-duty resuscitator. A decrease in blood saturation during dynamic observation was recorded by the medical staff and the attending physician: below 95% in the air and the moment of supply of moistened oxygen through a mask or nasal cannulas in a volume of 5 L per minute, or until saturation of more than 95% with a constant flow of oxygen. Desaturation was reported to the duty officer and attending physicians and noted in the observation sheet. The doctor gave the command to start oxygen insufflation.

Biochemical Blood Analysis
A detailed clinical blood test was performed on all patients, which included an assessment of 24 laboratory parameters: dynamic analysis of the indicators of the acid-base state of the blood (concentrations of calcium ions, ionized calcium (Ca 2+ ), sodium ions (Na + ), potassium (K + ), BE (Ecf) excess bases outside the cell fluid, pH, partial pressure of carbon dioxide (pCO 2 ), and bicarbonate in plasma (HCO 3 − )), lactate, blood oxygen saturation, and partial pressure of oxygen (pO 2 ). Biochemical blood analyses included the determination of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein, albumin, total bilirubin, direct (bound), bilirubin, glucose, lactate dehydrogenase (LDH), creatinine, and urea by an automatic hemoanalyzer XN-1000 (Sysmex Corporation, Kobe, Japan) according to the operating instructions [45]. Coagulogram parameters were also measured, e.g., APTT, fibrinogen, prothrombin time, and D-dimer. For example, APTT was evaluated using a set of reagents, STA ® Cephascreen ® (Diagnostica Stago S.A.S, Asnières-sur-Seine, France), on an STA ® analyzer according to the instructions [46]. The amounts of immunoglobulins (IgA, IgM, and IgG), CRP, ferritin, IL-6, and procalcitonin (PCT) were also assessed in the blood. Blood serum and plasma samples were collected in the morning on an empty stomach using vacutenirs containing Li-heparin separation gel, K2-EDTA, or K3-EDTA plasma. The blood was sent to the clinical diagnostic laboratory for examination within 1 h. The biochemical and clinical parameters and a history of comorbidities are presented in Table 8. The IL-6 levels in blood sera were determined using an Elecsys ® IL-6 electrochemiluminescent immunoassay on a cobas e602 analyzer (Roche Diagnostics Corporation, Indianapolis, IN, USA) according to the method in [47]. This test takes 18 min to measure the concentration of IL-6 in the range of 1.5-5000 pg/mL in a sample volume of 30 µL. The immunoassay is based on the sandwich principle, when a blood sample is added to immobilized microparticles coated with streptavidin. After incubation, imaging is performed using biotinylated mouse monoclonal antibodies to IL-6 (0.9 mcg/mL) diluted in a phosphate buffer of 95 mmol/L, at pH 7.3 [48].

Evaluation of Procalcitonin Using an Immunochromatographic Test
Procalcitonin in serum and plasma was evaluated using the BRAHMS PCT-Q immunochromatographic test (BRAHMS, Hennigsdorf, Germany) according to the instructions [49]. Six drops of a blood sample were dropped into a round hole on a BRAHMS PCT-Q tablet using a pipette and incubated for 30 min at room temperature. After the specified time (maximum 45 min), the concentration range of the PCT sample was determined. At the beginning, a distinct appearance of the control band was visually recorded. Tests in which only the control band is observed were negative. In these tests, the concentration of PCT was less than 0.5 ng/mL. The tests in which the control and test strips were visualized were positive. The PCT concentration was determined by comparing the intensity of staining of the test strip with the colored stripes on the control card included in the kit. The BRAHMS PCT-Q express test obtain results with a 90-92% diagnostic sensitivity and a 92-98% specificity. Phospholipase A2 in blood samples was determined using the sPLA2 kit (Cayman Chemical, Ann Arbor, MI, USA) for enzyme immunoassay based on the "sandwich" method of double antibodies, according to the instructions [56]. Each well of a 96-well microplate was coated with a monoclonal antibody of human sPLA2 type IIA. Such an antibody will bind to human sPLA2 type IIA located in the well. One hundred milliliters of control and biological samples were added to each well and incubated for 2 h at room temperature on a shaker. Then, the wells of the tablet were washed four times with a washing buffer and 100 µL of secondary HRP-conjugated mouse monoclonal antibodies specific to sPLA2 were added. These antibodies were used to visualize the captured sPLA2 type IIA. The samples were incubated for 1 h at room temperature on a shaker. Excess antibodies were washed 4 times with a washing buffer. The human sPLA2 type IIA concentration was determined by measuring the enzymatic activity of HRP with the addition of 100 µL of the chromogenic substrate 3,3 ,5,5 -tetramethylbenzidine (TMB). The samples were then incubated for 30 min at room temperature in the dark. The formation of a blue color was monitored. The reaction was stopped by adding 100 µL of acid, and a bright yellow product was formed, which was measured on an ELISA reader at 450 nm. The intensity of this color is directly proportional to the amount of bound HRP-streptavidin conjugate and the concentration of human sPLA2 type IIA.

Genetic Methods
In smears from the nasopharyngeal mucosa, the presence of RNA of the SARScoronavirus-2 virus, as well as concomitant factors of mixed infection, i.e., RNA of influenza A and B, parainfluenza virus, respiratory syncytial virus, rhinoviruses, DNA of bokavirus and adenovirus, and metapneumovirus, was evaluated by RT-PCR. Sampling of smears was carried out with sterile swabs from both nasal entrances and the nasopharynx. Smears were transported in saline solution in sterile Eppendorf-type microprobes.

RNA Isolation
The isolation of SARS-CoV-2 virus RNA from nasopharyngeal smears was carried out according to the instructions for the extraction reagent kit, GeneJET (Thermo Scientific, Waltham, MA, USA) RNA Purification kit, at the Magna Pure System station (Roche, Indianapolis, IN, USA) and KingFisher™ [57]. The RNA concentration was measured on a Quantus fluorimeter using a Quantifluorine RNA System (Promega, Madison, WI, USA) [58]. The RNA quality was assessed using the TapeStation 4200 system device and a Highly Sensitive RNA Video Recording Analysis kit [59]. The selection of positive samples (with Cq < 25) for further investigation was carried out using real-time PCR with one of the kits for the diagnosis of the SARS-CoV-2 virus. The excavation of samples into tablets was carried out with the use of the automated stations Xiril AG and Eppendorf epMotion 5075tc. In cases of poor RNA quality, its post-purification was carried out using the GeneJET kit (Thermofisher, Waltham, MA, USA). cDNA was made using a set of SuperScpipt, Mint-2 (Eurogene, Moscow, Russia), or a set for the synthesis of double-stranded cDNA Maxima H Minus (ThermoFisher, Waltham, MA, USA) [60,61].

Polymerase Chain Reaction
A RT-PCR analysis was performed using reagent kits for detecting coronavirus RNA SARS-coronavirus-2 in clinical material (produced by the Pasteur Research Institute of EM), GeneFinderTM, and COVID-19 plus RealAmp (OSANG Medicine Co. Ltd., Anyang-si, Korea) on CFX96 PCR devices in a real-time detection system (Biorad, Hercules, CA, USA) [62].

Statistical Methods
The results are presented as the arithmetic means ± standard deviation for the sample volume n (M ± m) and in terms of the median and the first quartile. The significance level was evaluated as * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001. Statistical data processing (descriptive statistics and graphical analysis of data relationships from different tables) was performed using the GraphPad application on the Prism 8.01 platform. The frequency characteristics of qualitative indicators (gender, degree of form and pathological processes, and complaints) were evaluated using nonparametric methods, χ 2 . A Fisher's exact test was used to compare mortality and disease severity in groups. Differences between groups were identified using the Kruskal-Wallis test and the Mann-Whitney test as a post hoc analysis [63].

Conclusions
We have shown that the blood levels of sPLA2 and IL-6 in 158 patients increase statistically significantly in cases which eventually result in death and when patients are transferred to the ICU (as the severity of the COVID-19 infection increases), showing that IL-6 and sPLA2 can be considered as early predictors of aggravation of COVID-19 infections.  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Written informed consent was obtained from the patient(s) to publish this paper.