Clinical features of COVID-19 patients
All patients included in study met the criteria for COVID-19 (11) and according to disease severity classified into the four groups. Among them 160 (57.1%) were male. As the disease progresses, statistically significant decrement in percentage of female patients and increment in percentage of male patients was detected (p = 0.041; Table 1). The median age of the patients in the mild group (55.1± 1.6) was significantly different from moderate (58.5 ± 1.3), sever (63.5 ± 1.5) or critical one (69.2 ± 1.3) (Table 1). Patients in the moderate, severe and critical groups were more likely to have fever, fatigue, dyspnea, chest pain, auscultatory attenuated breathing sound with audible crackles diffusely or whistling compared with the mild one with statistical significance difference (p < 0.05) ( Table 1). The most frequent auscultatory finding in group I was sharpened respiratory sound in 45,7%, normal breathing in 41.4%, attenuated breathing in 40% of patients. Only 8.6% of patients had audible cracks diffusely and 5.7% whistling. CXR findings in group I described like CXR I in 80% and CXR II in 20% of patients, and no one had CXR III, IV, V. In group II, 41.4 % of patients had attenuated breathing sound, sharpened respiratory sound 32.9%, audible cracks diffusly 50%, whistling 2.8% of patients. CXR finding are described like CXR II in 60%, CXR III in 40% of patients. In group III the most frequent auscultatory finding was attenuated breathing sound in 44.3% of patients with audible cracks diffusely in 67.1% of patients. All of them had CXR IV. In group IV attenuated breathing sound had 88.6% of patients, audible cracks diffusly 91.4%, whistling 37.1% of patients. All of them had CXR V.
Significant differences in laboratory features noted between defined group. Patients in moderate, severe and critical group had an increase with statistically significant differences (p < 0.05) in white blood cell count, neutrophil count, levels of glycemia, urea, creatinine, TBIL, DBIL, AST, ALT, LDH, CK, D-dimer, CRP, PCT, Fe, Ferritin and reduced lymphocyte count, monocyte count, decreased levels of albumin, lower values of Sa02 and p02 (Table 2). There were no statistically significant differences in number of erythrocytes and platelets, levels of hemoglobin, K+, Na+, Fe, pH of blood, pC02 (Table 2).
Patients with severe COVID-19 had increased Gal-3, IL-10 and proinflammatory cytokines
Patients in stage IV of COVID-19 had significantly higher serum level of pro-inflammatory cytokines IL-1β (p=0.001), TNF-α (p=0.012) and IL-12 (p=0.001) as well as immunosuppressive IL-10 (p=0.001) compared to the patients in less severe stages of disease (Figure 1A, 1B, 1C, 1D). Value of Gal-3 (p=0.001) was significantly higher in sera of patients in stage IV in comparison to patients in other stages of disease (Figure 1E). Ratios of serum levels of IL-10 and Gal-3 with IL-1β, TNF-α and IL-12 in all stages of COVID-19 were made. No differences were obtained in ratios IL-10/IL-1β, IL-10/TNF-α, IL-10/IL-12 as well as Gal-3/IL-1β, Gal-3/TNF-α, Gal-3/IL-12 between defined groups.
Increased expression of Gal-3, TNF-α and CCR5 in PBMCs in patients with severe COVID-19
Flow cytometry analysis of lymphocytes in peripheral blood didn’t detect any statistically significant difference in the percentage of CD56-CD3+T cells between defined groups (Fig 2a). Significantly increased percentages of CD56-CD3+TNF-α+T cells (p=0.050) as well as CD56-CD3+ Gal-3+T cells (p=0.007) were detected in the peripheral blood of patients in stage IV compared to the patients in other stages (Fig 2b, 2c). Patients in stage IV had significantly higher percentage of CD56-CD3+T cells expressing CCR5 chemokine (p=0.005) (Figure 2d). Analyses of dendritic cells (DCs) and macrophages (Mf) revealed that there were no differences in the percentage of CD11b+ cells or CD11c+ cells in peripheral blood derived from patients in different stages of COVID-19 (Figure 3a, 3b). Higher percentage of CD11c+IL1-β+ (p=0.078), and significantly higher percentage CD11b+IL1-β+ (p=0.006) and CD11b+TNF-α+ (p=0.012) cells were measured in stage IV (Figure 3c, 3d, 3e).
Strong correlation between Gal-3 and proinflammatory cytokines
Next, analyses of correlation between Gal-3 and pro- and anti- inflammatory cytokines and their ratios in COVID-19 patients were performed. Strong positive correlation was detected between Gal-3 and IL-1β (p=0.001), moderate positive correlation between Gal-3 and TNF-α (p=0.001) and IL-12 (p=0.001) (Table 3). Moderate negative correlation noted between Gal-3, IL-10/IL-1β (p=0.001) and IL-10/TNF-α (p=0.001) (Table 3).
Moderate correlation between Gal-3 and clinical parameters of COVID-19
Further, correlation between Gal-3 and clinical parameters of COVID-19 has been done. Results revealed moderate positive correlation between Gal-3 and D dimer (p=0.001), CXR findings (p=0.001) and urea (p=0.001), weak positive correlation between Gal-3 and PCT (p=0.001), glycemia (p=0.004), granulocytes no. (p=0.001), monocytes no. (p=0.003), CRP (p=0.011), LDH (p=0.002), AST (p=0.020) and creatinine (p=0.028) (Table 4). Moderate negative correlation was measured between Gal-3 and p02 (p=0.001), Sa02 (p=0.001), lymphocyte percentage (p=0.001) and monocyte percentage (p=0.003) (Table 4).
The univariate logistic regression analysis showed that age (> 65 years), dyspnea, chest pain, attenuated breathing sound, audible cracks diffusely, platelet count, lymphocyte, albumin, urea, creatinin, d-dimer, CRP, PCT, IL-1 (> 120 pg/mL), IL-10 (> 300 pg/mL), TNF-α (> 65 pg/mL) and Gal-3 (> 5000 pg/mL) were associated with critical stage of COVID-19 (Table 5). Multivariate logistic regression analysis identify that Gal-3 (OR 2.97, 95% CI 1.006–8.764; p=0.049), IL-10 (OR 3.117, 95% CI 1.119–8.105; p=0.020), creatinin (> 100 umol/L) (OR 4.035, 95% CI 1.549–10.506; p=0.004), platelet count (< 100 × 109/L) (OR 9.608, 95% CI 1.255–73.535; p=0.029), chest pain (OR 4.964, 95% CI 1.158–21.278; p=0.031), attenuated breathing sound (OR 4.703, 95% CI 1.601–13.812; p=0.005) and audible cracks diffusely (OR 2.467, 95% CI 1.006–6.048; p=0.048) were independently significant factors for critical stage of COVID-19.