The role of interleukin‐6 in monitoring severe case of coronavirus disease 2019

A total of 69 severe cases were included. The median age was 56 years. Female accounted for 52.17% of the enrolled cases. The smoking rate was 11.59%. Fever (79.72%), cough (63.77%), shortness of breath (57.97%), and fatigue (50.72%) were the most common symptoms, while diarrhea (15.94%), vomiting (7.25%), and sore throat (5.80%) were relatively less common. 36.23% of patients had at least one comorbidity (e.g., hypertension, chronic obstructive pulmonary disease, coronary heart disease, and hepatitis B virus infection), and there were four cases of tumor. Another 11 non‐severe cases were also enrolled (Table 1).

Table 2 shows the radiologic and laboratory findings on admission. The most common CT patterns in severe cases upon admission were bilateral patchy shadowing (60.87%) and interstitial abnormalities (27.54%), while the common type mainly manifested as focal ground glass opacity and patchy shadowing (54.5%; Table 2).

Baseline neutrocytopenia, lymphocytopenia, and thrombocytopenia were observed in 13.04, 79.71, and 24.64% of the severe type patients, respectively. Lymphocyte count in severe case was significantly less than the non‐severe cases (Fig 1A). Increased creatine kinase (CK) was present in 14.49% of the cases. Elevated level of alanine aminotransferase and aspartate aminotransferase was more common, and both were detected in 37.68% of the cases (Table 2 and Fig 1B).

For inflammatory parameters, abnormal baseline PCT was not common in both non‐severe and severe patients. However, patients with severe disease showed significantly elevated level of D‐dimer, ESR, LDH, CRP, and ferritin than those with non‐severe disease (Fig 1C–E).

89.86% of the severe type patients received antibiotics, 63.77% received antiviral therapy (20.29% with oseltamivir, 52.17% with umifenovir, and 7.25% with lopinavir/ritonavir), 42.03% received glucocorticoids, and 50.72% received human immunoglobulin (Table 3). 55.07% of the severe cases needed oxygen therapy, among which 27.54% received high‐flow oxygen therapy, non‐invasive ventilation, or invasive ventilation (Table 3).

Among the enrolled patients with severe disease, 53.62% were recovered and discharged, 46.38% were still hospitalized, three patients (4.35%) needed transfer to the intensive care unit, and no death case occurred (Table 3). Seven patients developed acute respiratory distress syndrome (ARDS), and one got septic shock (Table 3). Compared with patients with non‐severe disease, the time from symptom onset to initial COVID‐19 diagnosis and to development of pneumonia in patients with severe disease was shorter, but it was not significant (P > 0.05; Fig 2A and B). There was no significant difference in the time from symptom onset to treatment, and the time from development of pneumonia to recovery in patients with severe disease was longer (P > 0.05; Fig 2C and D).

The baseline proportion of CD4⁺ T cells, CD8⁺ T cells, B cells, natural killer (NK) cells, and the CD4⁺ T cells/CD8⁺ T cells ratio are basically within the normal range (Fig 1F–H). The baseline level of IL‐2, IL‐4, IL‐10, TNF‐α, and IFN‐γ was within normal range, while IL‐10 was slightly increased (Fig 1I–K).

The IL‐6 level was increased significantly on admission in severe cases as compared with the non‐severe cases (Fig 1D). The baseline elevated IL‐6 in severe COVID‐19 cases was positively correlated with the maximal body temperature during hospitalization (r = 0.521, P < 0.001; Fig 3A). Meanwhile, it was positively correlated with the increase of CRP (r = 0.781, P < 0.001), LDH (r = 0.749, P < 0.001), ferritin (r = 0.606, P < 0.001), and D‐dimer (r = 0.679, P < 0.001; Fig 3B–E). Severe COVID‐19 patients who received high‐flow oxygen inhalation and mechanical ventilation during hospitalization showed significant higher baseline IL‐6 level than those who did not (Fig 3F). Patients who received administration of glucocorticoids also showed a tendency of higher baseline IL‐6 level, though the difference was not significant.

For the 30 patients whose IL‐6 was assessed before and after treatment, 26 patients, classified into three groups according to the baseline IL‐6 level of ≥ 40, ≥ 20, or < 20 pg/ml, showed significantly reduced IL‐6 after treatment (Fig 4A, left). Higher baseline IL‐6 was associated with more severe pulmonary damage as evidenced by chest CT scans, and the decrease in IL‐6 after treatment was accompanied with improved CT assessments after treatment (Fig 4A, right). Disease of four patients exacerbated progressively. Among them, three patients showed increased IL‐6 as compared with the baseline (Fig 4B), while the change in IL‐6 in one patient was not obvious. For this patient, there was an increase in PCT level, and the progression of lung disease was confirmed as bacterial, but not viral, pneumonia, as evidenced by sputum culture positive for bacteria growth (Fig 4C).

In another patient who presented with disease aggravation and subsequent alleviation evidenced by CT scans (Fig 5A), the IL‐6 and CRP varied correspondingly, and more importantly, the occurrence of abnormal IL‐6 and CRP was prior to the changes detected by CT assessment (Fig 5D and E). The procalcitonin (PCT) level had stayed within the normal range throughout the course of the disease, while the lymphocyte count began to decline 5 days after symptom onset and then returned to normal range (Fig 5B and C). The body temperature also correlated with the variation in IL‐6 and CRP, as the body temperature stayed abnormal during the rising phase while returned to normal during the decline phase of IL‐6 and CRP (Fig 5F).

COVID‐19 was diagnosed in accordance with the WHO interim guidance. A confirmed case of COVID‐19 was defined as positive for SARS‐CoV‐2 nucleic acid on high‐throughput sequencing or real‐time reverse transcriptase polymerase chain reaction (RT–PCR) assays of nasal and pharyngeal swab specimens. Only laboratory‐confirmed cases were included in this study, while disease diagnosed based on clinical presentation and radiologic findings, but not on SARS‐CoV‐2 detection, were excluded. The severity of COVID‐19 was classified according to the Guidelines for the Diagnosis and Treatment of Novel Coronavirus (2019‐nCoV) Infection (Trial Version 5) issued by the National Health Commission of the People's Republic of China. Severe case was defined when any of the following criteria was met: (i) dyspnea, respiration rate (RR) ≥ 30 times/min; (ii) oxygen saturation by pulse oximeter ≤ 93% in resting state; and (iii) partial pressure of arterial oxygen (PaO₂) to fraction of inspired oxygen (FiO₂) ratio ≤ 300 mmHg (l mmHg = 0.133 kPa). We collected data of 69 patients with severe type COVID‐19 hospitalized in the Department of Infectious Diseases, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, between January 21 and February 16, 2020. A retrospective study on the clinical characteristics and laboratory examination was conducted. Eleven non‐severe COVID‐19 patients were included for comparison. This study was approved by the Ethics Committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology and conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report. Verbal consent was obtained from patients before the enrollment. Written informed consent was waived due to the urgent need of data collection.

The clinical symptoms, physical signs, and results of laboratory examination of patients were recorded. Radiologic evaluation included chest CT scan. Laboratory tests included baseline whole blood cell count, blood chemistry, coagulation test, CRP, PCT, LDH, ferritin, ESR, CK, and lymphocyte subset and cytokine profile analysis on admission. In addition, cytokine profile follow‐up was conducted in 30 severe type patients after treatment. Treatment plan was recorded. Time from the symptom onset to initial treatment, to initial COVID‐19 diagnosis, and to development of pneumonia evidenced by CT scan was recorded, and time from development of pneumonia to discharge was recorded.

Nasal and pharyngeal swab specimens were collected and placed into a collection tube containing preservation solution for virus (Huang et al, 2020). Real‐time RT–PCR assay for SARS‐CoV‐2 was conducted by the viral nucleic acid detection kit according to the manufacturer's protocol (Shanghai BioGerm Medical Technology, Co., Ltd.). Laboratory confirmation of COVID‐19 was performed by local Center for Disease Control and Prevention (CDC) in accordance with the Chinese CDC protocol. Whenever needed, specimens, including sputum or alveolar lavatory fluid, blood, urine, and feces, were cultured to assess potential bacterial and/or fungal infection that might accompany with SARS‐CoV‐2 infection.

The lymphocyte test kit (Beckman Coulter Inc., FL, USA) was used for lymphocyte subset analysis by flow cytometry. Plasma cytokines (IL‐2, IL‐4, IL‐6, IL‐10, tumor necrosis factor [TNF]‐α, and IFN‐γ) were detected by ELISA with human Th1/2 cytokine kit II (BD Ltd., Franklin Lakes, NJ, USA). All tests were performed according to the product manual.

Randomization, blinding, or replication was not applicable in this retrospective study. Continuous variables were described as means and standard errors, or medians and interquartile range [IQR] values. Categorical variables were expressed as counts and percentages. Continuous variables that did not conform normal distribution were compared by the two‐tailed Mann–Whitney U‐test. Proportions for categorical variables were compared by the chi‐square test and Fisher's exact test as appropriate. Correlations were determined by Spearman rank correlation analysis. All statistical analyses were performed by GraphPad Prism (version 5.0) and SPSS 26.0 (IBM SPSS Statistics 26.0). For all statistical analyses, P < 0.05 was considered statistically significant. Respective statistical tests used are stated in the main text and figure legends.