Estimating the release of inflammatory factors and use of glucocorticoid therapy for COVID-19 patients with comorbidities

Patient baseline characteristics

A total of 79 eligible cases were enrolled in this study and retrospective analysis was performed. All of the cases met the criteria for severe COVID-19 according to the guidelines for diagnosis and treatment issued by the National Health Commission of the People’s Republic of China. Among them, 45 cases had comorbidities, including malignancy in 11 (24.5%), diabetes mellitus in 10 (22.2%) and hypertension in 24 (53.3%). The age of patients with comorbidities was significantly higher than those without comorbidities, and the median age in the two groups were 69 and 56 years, respectively (P < 0.01). The percentage of male subjects in the comorbidity and no comorbidities groups were 53.3% and 41.1%, respectively, but did not statistically differ. The clinical presentations of patients at admission were fever (74.6%), dry cough (64.5%), and dyspnea (56.9%), with no significant difference between the comorbidity and non-comorbidity groups. All enrolled patients received antiviral treatment. The patients received antibacterial treatment at a rate of 86.7% (comorbidities group) versus 73.5% (no comorbidities group, P = 0.14). 86.6% of patients in the comorbidities group received nutritional supports, which was significantly higher than that of the non-comorbidities group (61.7%, Table 1).

Routine laboratory tests and CT features analysis

In baseline laboratory tests, most of patients showed abnormal white cell count (normal or mildly low), while patients in comorbidities group showed obvious lymphopenia (57.7%) and thrombocytopenia (22.2%) and a relative increase in the number of granulocytes (26.6%). Additionally, obvious malnutrition and abnormal coagulation, such as hypokalemia accounting for 22.2%, hypoproteinemia (26.6%), and marked rise in D-dimer and Fibrinogen were observed in comorbidities group. Hepatic dysfunction was also common in severe COVID-19 patients, showed with increased serum Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) levels, but without differences between the groups. Additionally, patients with comorbidities showed prominent increased inflammatory parameters, such as CRP (56.8% vs. 26.4%), ESR (91.1% vs. 82.3%) compared with non-comorbidities. Radiographic findings in COVID-19 patients included clumped, ground-glass and interstitial lesions, involving bilateral lung or multilobar lung, but without differences between two groups (Table 2).

Inflammatory factors and lymphocyte subset analysis

The proportion of CD3+, CD4+, and CD8+ T cell subsets in peripheral blood was also determined (Figure 1A–1C). We found that there was a decrease in the proportion of CD4+ T cells in both groups, which was more pronounced in the comorbidities group (Figure 1A). There was a decrease in the proportion of CD4+ T cells in both groups, which was more pronounced in the underlying diseases group. Baseline serum levels of inflammatory factors IL-2R, IL-4, IL-6, IL-10, and IFN-γ were analyzed (Figure 2A–2F). The patients with comorbidities exhibited higher serum levels of IL-6 and IFN-γ when compared to patients without comorbidities (Figure 2C, 2F). Furthermore, elevated levels of IL-6 were positively correlated with an increase in CRP (r=0.41, P=0.008), ESR (r=0.37, P=0.022) and D-dimer (r=0.47, P=0.004) (Figure 3A, 3B, 3D); and negatively correlated with albumin (r=-0.34, P=0.028), lymphocyte counts (r=-0.38, P=0.002), and the proportion of CD4+T cells subsets (r=-0.62, P<0.0001) (Figure 3C, 3E, 3F). A negative correlation existed between IFN-γ and albumin (r=-0.35, P=0.032), lymphocyte counts (r=-0.41, P=0.007), and the proportion of CD4+T cells subsets (r=-0.33, P=0.032) (Figure 3I, 3K, 3L); while a positive correlation existed between CRP (r=0.45, P=0.003) and D-dimer (r=0.70, P<0.0001) as shown in Figure 3G, 3J.

Treatment outcome and prognosis

All patients enrolled in this study were regularly examined for chest CT, blood, and serum levels of inflammatory cytokines every 7-10 days following COVID-19 treatment to evaluate the changes in disease development. The signs of disease remission included: relief of symptoms, negative nucleic acid detection of COVID-19 (twice within a 24-h interval), and if the CT images showed a reduction in the size of pulmonary lesions > 20% (Figure 4A). In the comorbidities group, 30 cases showed progress during treatment as characterized by inflammation development in the CT images (Figure 4B), accompanied by elevated levels of IL-6 and IFN-γ, and decreased lymphocyte counts (Figure 4C–4E). In 23 cases of the comorbidities group, decreased levels of IL-6 and IFN-γ as well as improved lymphocyte counts were observed after glucocorticoid treatment (Figure 4F–4H).

The time for complete remission, negative coronavirus nucleic acid detection, and CT images remission (27 ± 6.7, 19 ± 6.1, 21 ± 6.9 days, respectively) in the comorbidities group was significantly longer than that in non-comorbidities group (20 ± 6.5, 16 ± 5.8, 16 ± 6.8 days) (Figure 5A–5C). Multivariate COX regression analysis showed that serum levels of IL-6 and glucocorticoid treatment were independent and protective factors, respectively, for the prognosis of severe COVID-19 patients with coexisting comorbidities (Table 3).

Patient and study design

This study was a single-center retrospective clinical study and was approved by the Ethics Committee of Huazhong University of Science and Technology. Verbal consent was obtained for urgent data collection. The study population included 79 subjects consecutively admitted to Cancer Center of Union Hospital in Wuhan from February 10 to March 22, 2020. All the patients had laboratory-confirmed cases of COVID-19. Patient characteristics, clinical history, physical examination, laboratory investigations, radiologic findings, and response to treatment were collected and reviewed retrospectively. Subjects were included in this study based on the following eligibility criteria: Age range was 19-80 years (male/female), the patient was a confirmed COVID-19 case based on the CT imaging features, respiratory samples (throat swab/ nasopharyngeal swab/ endotracheal aspirates/ bronchoalveolar lavage) were positive for the RNA virus nucleic acid detection by real-time RT-PCR, or viral genome sequencing revealed a strong homology to COVID-19. Additionally, the clinical manifestations presented with severe pneumonia: fast breathing (respiratory rate ≥ 30/min), dyspnea, lip cyanosis; finger pulse oxygen saturation < 93% at rest; partial pressure of arterial oxygen pressure (PaO₂, mmHg) / fraction of inspired oxygen (FIO₂) ratios ≤ 300mmHg (1 mmHg = 0.133 kPa); the CT images showed multiple lesions in bilateral lungs or pulmonary lesions progression > 50% within 24-48 h [7]. Complete CT imaging data before, during, and after antiviral therapy, as well as relevant laboratory examinations were available for all enrolled patients. Follow-up was completed in all enrolled patients.

Inflammatory factors and lymphocyte subsets analysis

Peripheral blood samples of all enrolled patients were collected and transported to the Immune Research Laboratories, Union Hospital, Huazhong University of Science and Technology. Serum levels of the following cytokines were determined: IL-2R, IL-4, IL-6, IL-10, IFN-γ and TNF-α. Levels of T lymphocyte subsets (CD3+ T cells, CD4+ T cells, and CD8+ T cells) were measured by flow cytometry. Each comparison was computed for all clinical data collected.

Treatment regimen and efficacy studies

All enrolled patients received standard treatment according to the guidelines for diagnosis and treatment of COVID-19 issued by the 7^th edition of National Health Commission of the People’s Republic of China (NHCPRC). The clinical presentations, CT re-examination results, laboratory investigations (routine blood, serum biomarker indicators, total lymphocyte count, CD4+ and CD8+ T cells), inflammatory factors (IL-2R, IL-4, IL-6, IL-10, IFN-γ and TNF-α), Erythrocyte Sedimentation Rate (ESR), and C-reactive protein (CRP) levels were recorded before and every 7-10 days following treatment. Contrast analyses were carried out to evaluate the disease outcome. All patients were followed-up until the disease resolved or death occurred.

Study endpoint and outcome

The primary outcome was the disease remission time, defined as the time from the onset of treatment to the negative detection of COVID-19, and CT images of remission. The time for negative coronavirus nucleic acid detection (time from onset treatment to negative nucleic acid detection of coronavirus), and time to CT images remission (time from onset treatment to CT images showing the absorption of lung lesions > 20%) were analyzed. Lymphocyte subsets, inflammatory factors analysis, and their correlation with treatment efficacy were set as a secondary outcome.

Statistical analysis

All data were subjected to statistical analysis with SPSS version 17.0 statistical software and the results were presented as mean ± standard deviation (SD). Student’s t test was used to compare continuous variables and the Chi-squared test was performed to test for between-group differences among the categorical variables. Where the primary end point of this study was disease remission time, the Kaplan-Meier method and Log-rank test were performed for related data representation, comparison, and analysis. Cox regression was used for multivariate analyses to assess the relative risk for each factor including the clinical presentations, routine blood, serum biomarker indicators, inflammatory factors, etc. P < 0.05 was regarded as statistically significant.

Ethical approval and informed consent

This study was a retrospective observational study approved by the Ethics Committee of Huazhong University of Science and Technology, China. The ethical approval number was 0255 [2020].