Severe COVID-19 patient characteristics in stabilized stage
A total of 28 patients with confirmed severe COVID-19 and 25 healthy volunteers participated in this study. As shown in Table 1, there was no statistical difference in age between the two groups (64.93±1.63 vs 64.56±1.55, p=0.872) and gender (male/female, 16 /12 vs 14/11, p=0.935). In the parameters of blood routine, white blood cell (WBC) count (6.98±0.53 vs 5.81±0.34, p=0.067), lymphocyte count (2.09±0.30 vs 1.66±0.13, p=0.172), neutrophil count (3.91±0.34 vs 3.41±0.25, p=0.244), monocyte count (0.65±0.06 vs 0.56±0.06, p=0.352), neutrophil/lymphocyte ratio (1.82±0.34 vs 2.34±0.24, p=0.255), monocyte/lymphocyte ratio (0.28±0.05 vs 0.35±0.025, p=0.157) had no statistical difference between two group. Among the cytokines, there were no significant statistical difference in IL-1β (9.10±3.28 vs 4.65±4.10, p=0.411), IL-2 (2.96±1.04 vs 1.18±1.11, p=0.279), IL-4 (4.97±1.60 vs 1.69±1.51, p=0.184), IL-12p70 (5.04±2.22 vs 0.13±0.11, p=0.110) and TNF-α (9.83±3.81 vs 3.08±2.89, p=0.162). But, there were significant statistical difference in IL-5 (1.72±0.40 vs 0.13±0.10, p<0.01), IL-6 (23.93±4.89 vs 3.78±1.49, p<0.01), IL-10 (9.74±1.75 vs 1.50±0.19, p<0.01), IL-17A (24.94±4.39 vs 2.90±1.03, p<0.01), IFN-γ (6.65±1.76 vs 0.55±0.22, p<0.01) and IFN-α (17.71±4.92 vs 1.70±1.55, p<0.01). At the same time, we detected the indicators of type II alveolar epithelial injury and found that SP-A (247.04±22.45 vs 17.83±8.01, p<0.01) and KL-6 (977.39±136.93 vs 251.96±20.63, p<0.01) were significant higher in COVID-19 patients.
ROC analysis of significant indicators in stabilized stage of severe COVID-19 patients
ROC analysis was performed to test the prediction power of the signature of severe COVID-19 in stabilized stage. ROC analysis distinguished these two groups with an area under the curve of IL-5 (AUC Signature=0.938, Figure 1A), IL-6(AUC Signature=0.934, Figure 1B), IL-10 (AUC Signature=0.967, Figure 1C), IL-17A (AUC Signature=0.791, Figure 1D), IFN-γ (AUC Signature=0.917, Figure 1E), IFN-α (AUC Signature=0.947, Figure 1F), SP-A (AUC Signature=0.989, Figure 1G), KL-6 (AUC Signature=0.931, Figure 1H).
The blood indexes variation of severe COVID-19 patients from stabilized stage toward convalescence
From the above results, we know that during the stabilized stage of severe COVID-19 patients, the parameters of blood routine and some cytokines (IL-1β, IL-2, IL-4, IL-12p70 and TNF-α) returned to normal, but some cytokines (IL-5, IL-6, IL-10, IL-17A, IFN-γ and IFN-α) and type II alveolar epithelial injury indicators (SP-A and KL-6) were still higher than healthy control group. Therefore, we continuously monitored the blood indexes of severe COVID-19 patients from stabilized stage to convalescence (day 1, 30, 60 and 90 of experiment enrollment). The variation of WBC count (F=0.789, p=0.453) (Figure 2A), lymphocyte count (F=0.599, p=0.618) (Figure 2B), neutrophil count (F=0.974, p=0.389) (Figure 2C), neutrophil/lymphocyte ratio (F=3.015, p=0.055) (Figure 2E), IL-1β (F=2.729, p=0.109) (Figure 2G), IL-2 (F=3.568, p=0.071) (Figure 2H), IL-4 (F=2.004, p=0.172) (Figure 2I), IL-6 (F=0.594, p=0.477) (Figure 2K) and TNF-α (F=1.627, p=0.217) (Figure 2Q) were not statistically significant. The monocyte count (F=51.081, p<0.01) (Figure 2D), monocyte/lymphocyte ratio (F=40.715, p<0.01) (Figure 2F), IL-5 (F=12.571, p<0.01) (Figure 2J), IL-10 (F=16.943, p<0.01) (Figure 2L), IL-12p70 (F=9.015, p<0.01) (Figure 2M), IL-17A (F=4.402, p=0.029) (Figure 2N), IFN-γ (F=7.288, p=0.005) (Figure 2O), IFN-α (F=5.928, p=0.021) (Figure 2P), SP-A (F=13.710, p<0.01) (Figure 2R) and KL-6 (F=13.168, p<0.01) (Figure 2S) were downward trend and the variation were statistically significant.
Serum KL-6 level reflect the severity of lung injury in severe COVID-19 patients from stabilized stage toward convalescence
The COVID-19 patients group were divided into lung injury range>1000 mm3 group (n=10) and lung injury range<1000 mm3 group (n=18) based on the CT results of day 1 enrollment. Following, we continuously monitored the blood indexes from stabilized stage to convalescence (day 1, 30, 60 and 90 of experiment enrollment). Our data showed that the WBC count (F=0.301, p=0.589) (Figure 3A), lymphocyte count (F=4.054, p=0.055) (Figure 3B), neutrophil count (F=1.274, p= 0.270) (Figure 3C), monocytes count (F=0.021, p=0.887) (Figure 3D), neutrophil/lymphocyte ratio (F=3.440, p=0.076) (Figure 3E), monocyte/lymphocyte ratio (F=3.670, p=0.067 ) (Figure 3F), IL-β (F=0.221, p=0.644) (Figure 3G), IL-2 (F=0.299, p=0.638) (Figure 3H), IL-4 (F=0.383, p=0.543) (Figure 3I), IL-5 (F=0.827, p=0.374) (Figure 3J), IL-6 (F=0.159, p=0.694) (Figure 3K), IL-10 (F=0.449,p=0.511 ) (Figure 3L), IL-12p70 (0.809, p=0.380) (Figure 3M), IL-17A (F=1.828, p=0.192) (Figure 3N), IFN-γ (F=0.721, p=0.406) (Figure 3O), IFN-α (F=0.239, p=0.630) (Figure 3P), TNF-α (F=0.290,p=0.597) (Figure 3Q) and SP-A (F=0.005, p=0.942) (Figure 3R) were no statistically significant between two groups. Meanwhile, we found that the serum KL-6 (F=5.824, p=0.023) (Figure 3S) was statistically significant between the two groups, and KL-6 was higher in lung injury range>1000 mm3 group than lung injury range<1000 mm3 group. Above data implied that KL-6 maybe serve as a good indicator for monitoring lung injury repair in severe COVID-19 patient from stabilized stage toward convalescence.
The clinical characteristics of a severe COVID-19 patient case from stabilized stage toward convalescence
We selected a 57-year-old female patient confirmed severe COVID-19. Figures 4A to 4D showed the CT imaging results of this patient at different time points (day 1, 30, 60 and 90 of experiment enrollment). Figure 4E was the variation trend of CT results, it showed that the lung injury range decreased along time. Figure 4F and 4G respectively showed the variation of serum SP-A and KL-6 levels at the time points corresponding to the CT results. Combining the results of CT and serology, we found that as the extent of lung injury shrinks, the levels of SP-A and KL-6 gradually decrease. In addition, the variation of parameters of blood routine and cytokines did not match with the CT results, and the data not show.