key: cord-1012896-370fqkat
authors: Gong, Jing; Dong, Hui; Xia, Song Qing; Huang, Yi Zhao; Wang, Dingkun; Zhao, Yan; Liu, Wenhua; Tu, Shenghao; Zhang, Mingmin; Wang, Qi; Lu, Fuer
title: Correlation Analysis Between Disease Severity and Inflammation-related Parameters in Patients with COVID-19 Pneumonia
date: 2020-02-27
journal: nan
DOI: 10.1101/2020.02.25.20025643
sha: c8d206a4f9af0709b6e9ee90c4d854d482cb0784
doc_id: 1012896
cord_uid: 370fqkat

Aim: The new coronavirus pneumonia (COVID-19) outbreaking at the end of 2019 is highly contagious. Crude mortality rate reached 49% in critical patients. Inflammation matters on disease progression. This study analyzed blood inflammation indicators among mild, severe and critical patients, helping to identify severe or critical patients early. Methods: In this cross-sectional study, 100 patients were included and divided to mild, severe or critical groups. Correlation of peripheral blood inflammation-related indicators with disease criticality was analyzed. Cut-off values for critically ill patients were speculated through the ROC curve. Results：Significantly, disease severity were associated with age (R=-0.564, P<0.001), interleukin-2 receptor (IL2R) (R=-0.534, P<0.001), interleukin-6 (IL-6) (R=-0.535, P<0.001), interleukin-8 (IL-8) (R=-0.308, P<0.001), interleukin-10 (IL-10) (R=-0.422, P<0.001), tumor necrosis factor α (TNFα) (R=-0.322, P<0.001), C-reactive protein (CRP) (R=-0.604, P<0.001), ferroprotein (R=-0.508, P<0.001), procalcitonin (R=-0.650, P<0.001), white cell counts (WBC) (R=-0.54, P<0.001), lymphocyte counts (LC) (R=-0.56, P<0.001), neutrophil count (NC) (R=-0.585, P<0.001) and eosinophil counts (EC) (R=-0.299, P=0.01). Conclusion：With following parameters such as age >67.5 years, IL2R >793.5U/mL, CRP >30.7ng/mL, ferroprotein >2252μg/L, WBC>9.5*10^9/L or NC >7.305*10^9/L, the progress of COVID-19 to critical stage should be closely observed and possibly prevented. Inflammation is closely related to severity of COVID-19, and IL-6, TNFα and IL-8 might be promising therapeutic targets.

Critical patients should have any of the following conditions:

1. Respiratory failure occurs and mechanical ventilation required; 2. Shock occurs;

3. Combining other organ failure and requiring treatment in ICU.

All tests were completed in the clinical laboratory in Tongji Hospital. Interleukin-1β (IL-1β), interleukin-2 receptor (IL2R), interleukin-8 (IL-8), IL-10 and TNFα were detected by Siemens chemiluminescence method and IL-6 were detected by Roche electrochemiluminescence method according to the manufacturer's instruction. The ultrasensitive CRP regent was provided by Nippon Denkasei Co., Ltd, and CRP was detected by immunoturbidimetry method. Ferroprotein was detected by Roche granule-enhanced immune turbidimetry. Procalcitonin (PCT) was detected by Roche electrochemiluminescence method. ESR was measured by Westergren's international standard method. Peripheral blood cell was detected by fluorescence staining flow cytometry, and we analyzed the differences of white blood cell (WBC), NC, LC and eosinophils (EC) among three groups.

For data of normal distribution (IL2R, ESR, ferroprotein, WBC and NC), comparisons among critical, severe and mild groups were analyzed by ANOVA analysis. With different homogeneity of variance, the pairwise comparison between groups was performed using the Bonferroni test or Dunnett's T3 test. For non-normal distribution (CRP and LC), the data were conversed using square root, followed by ANOVA analysis and pairwise comparison. With data below the detectable limit, including IL-1β, IL-6, IL-8, IL-10, TNFα, PCT, and EC, the data were ranked referring to the reference range and value rank (Table 1) ; then non-parametric Kruskal-Wallis test were performed. In correlation analysis, Spearman correlation coefficient was used for the variables of normal distribution, Pearson correlation coefficient for those of skewed distribution, and Kendall's tau-b correlation coefficient for ranked data.

. CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.02.25.20025643 doi: medRxiv preprint patients were <31 pg/mL, three ranked grades were set, namely, <31 pg/mL, 31-62 pg/mL and >62 pg/mL. Figure 1D and Table 2 showed that all IL-8 levels in mild and severe patients were within the reference range (<62 pg/mL), and there were significant differences between critical and severe patients or critical and mild group.

With regard to IL-10 and TNFα, there was significant difference between mild and severe or mild and critical patients, and no significant difference was found between severe and critical group ( Figure 1E -F). With regard to cytokine IL-1β, no significant difference was found among three groups, and the data were not shown.

In addition, levels of CRP, ferroprotein and PCT were statistically different among mild, severe and critical patients ( Figure Figure 2D ) indicated the best cut-off point was 2252μg/L with a specificity of 96.6% and a sensitivity of 52.2%. There was no statistical difference on ESR among three groups ( Figure 3D ).

In peripheral blood cell analysis, there were significant differences in WBC count between the critical and severe groups or critical and mild groups, and no significance was found between the mild and severe groups ( Figure 4A ). ROC curve of WBC (AUC =0.838, p =0.000, Figure 2E ) suggested the best cut-off point was 7.92*10^9/L with 77% specificity and 78.1% sensitivity. If the detected values of WBC are above the upper reference limit, it often indicates infection. Because WBC count is influenced by therapy, clinically the upper limit of reference range of WBC, namely 9.5*10^9/L, seems more meaningful.

NC was significantly different among the three groups ( Figure 4B ), and the average was 10.80*10^9/L in critical group, 5.47*10^9/L in severe group and 3.53 *10^9/L in mild group. ROC curve of NC (AUC=0.814, P=0.000, Figure 2F ) indicated the best cut-off point was 7.305*10^9/L with 85.2% specificity and 75% sensitivity. There was . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.02.25.20025643 doi: medRxiv preprint significantly difference about LC between critical and severe group or critical and mild group ( Figure 4C ). Many patients in each group had a decreased level of EC, and 0 EC was detected in 65.63% patients in critical group, 55.88% in severe group, and 25.93% in mild group. There were significant differences between severe and mild or critical and mild patients (Table 2, Figure 4D ). Table 3 (Table 3 ). There was no significant correlation on IL-1β, gender and ESR.

The new coronavirus (COVID-19) pneumonia outbreaking at the end of 2019 is highly contagious, with a crude mortality rate of about 2.3% [1] . Currently, more than 74677 patients have been reported in the Mainland of China. About 80.9% patients are with mild to moderate severity [1] , and with a better prognosis. However, for patients developing into severe or critical levels, the mortality rate was significantly increased, and crude mortality rate reached 49% in critical patients [1] . It plays key roles to identify severe and critical patients even earlier, aiming to improve the recovery rate and reduce mortality.

Clinically, many patients appeared short-term progressive aggravation, scholars speculated that "inflammatory storms" occurred, namely, overreaction of cytokine. In a preprint article in medrxiv, the lymphocyte subsets and cytokines of 123 patients (102 mild and 21 severe patients) were analyzed and the researchers found the numbers of CD4+ T cells and CD8+ T cells decreased, and the levels of IL-6 and IL-10 increased in severe cases [7] . In this study, a retrospective analysis was conducted about cytokines and other inflammatory indicators of 100 patients (including 66 severe or critical patients), and more indicators were expected to better identify critical patients and help clinical decision-making.

. CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.02. 25.20025643 doi: medRxiv preprint In accordance with Wan S's [7] and Liu J's [8] study, this study also found that the levels of IL-6 and IL-10 were associated with the severity of COVID-19 pneumonia; similar to Chaolin Huang's study [4] , TNFα concentration, NC count and LC count were also found correlated with disease severity. In addition, IL2R levels, ferroprotein levels, PCT levels, and EC counts were also related to the disease severity. Besides, we also found the IL-6 levels in mild patients were lower than 100pg/mL. For eight patients who were deceased in critical group, IL-6 level was >100pg/mL in three persons.

IL-6 >100pg/mL might represent the emergence of "inflammatory storm". IL-8 levels in mild and severe patients were normal, and for patients with IL-8 >62 pg/mL, more attention need to avoid the disease progression. Based on clinical practice and ROC analysis between critical and non-critical patients, some cut-off values of the test items were obtained. With age >67.5 years, IL2R >793.5U/mL, CRP >30.7ng/mL, ferroprotein >2252μg/L, WBC >9.5*10^9/L or NC>7.305*10^9/L, progress to critical illness should be closely observed and prevented.

Preventing recognition or blocking the occurrence of inflammatory action, new drugs development on immune regulation, might be new breakthroughs in the control of COVID-19 pneumonia. Pathological examination found lymphocyte-dominated mononuclear cell infiltration in interstitial pulmonary tissue, CD4+ and CD8+ T cells were significantly reduced in peripheral blood cells, but a high ratio of CD38+ (CD4 3.47%) and HLA-DR+ (CD8 39.4%) T cells [9] , which suggested excessive activation of pro-inflammatory cells. Presumably, lymphocyte deposition in lung tissue might contribute to lymphocyte reduction in peripheral blood. In addition, researchers found that CCR4+ CCR6+ Th17 cells were increased, and the IL-17 inhibitor (Secukinumab) against activated Th17 cells is promising for disease control [9] . In this study, IL-6, TNFα and IL-8 may be potential targets for immunotherapy of COVID-19. With IL-6 >100pg/mL, the patient's condition was extremely critical. According to the news, a research team from First Affiliated Hospital of University of Science and Technology of China has used IL-6 receptor recombinant monoclonal antibody, Tozhu monoclonal antibody, in 14 critically or severely ill COVID-19 patients [10] .

The results were reported to be encouraging. Respiratory oxygenation indices of 14 Table 3 Correlation coefficient and P value between items and disease severity. . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.02.25.20025643 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.02.25.20025643 doi: medRxiv preprint

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Thanks to all people who work so hard to fight against the COVID-19 pneumonia.