Intravenous methylprednisolone pulse as a treatment for hospitalised severe COVID-19 patients: results from a randomised controlled clinical trial

Introduction There are no determined treatment agents for severe COVID-19. It is suggested that methylprednisolone, as an immunosuppressive treatment, can reduce the inflammation of the respiratory system in COVID-19 patients. Methods We conducted a single-blind, randomised controlled clinical trial involving severe hospitalised patients with confirmed COVID-19 at the early pulmonary phase of the illness in Iran. The patients were randomly allocated in a 1:1 ratio by the block randomisation method to receive standard care with methylprednisolone pulse (intravenous injection, 250 mg·day−1 for 3 days) or standard care alone. The study end-point was the time of clinical improvement or death, whichever came first. Primary and safety analysis was done in the intention-to-treat (ITT) population. Results 68 eligible patients underwent randomisation (34 patients in each group) from April 20, 2020 to June 20, 2020. In the standard care group, six patients received corticosteroids by the attending physician before the treatment and were excluded from the overall analysis. The percentage of improved patients was higher in the methylprednisolone group than in the standard care group (94.1% versus 57.1%) and the mortality rate was significantly lower in the methylprednisolone group (5.9% versus 42.9%; p<0.001). We demonstrated that patients in the methylprednisolone group had a significantly increased survival time compared with patients in the standard care group (log-rank test: p<0.001; hazard ratio 0.293, 95% CI 0.154–0.556). Two patients (5.8%) in the methylprednisolone group and two patients (7.1%) in the standard care group showed severe adverse events between initiation of treatment and the end of the study. Conclusions Our results suggest that methylprednisolone pulse could be an efficient therapeutic agent for hospitalised severe COVID-19 patients at the pulmonary phase.


Introduction
The world is experiencing the pandemic of a novel coronaviruses-induced respiratory illness named coronavirus disease 2019 . The diseases caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) belongs to the uneg s betacoronavirus [1]. Beta-coronaviruses are positive-single strand RNA (+ssRNA) viruses which have caused two other severe outbreaks, the middle east, and severe acute respiratory syndrome (MERS, SARS) just over the past decades [2]. COVID-19 has rapidly spread across the world, and the number of infected people is increasing since it was first discovered in China in late 2019. nin ihags soe n aoeseh nt nas e nas e nine he 2-14 days asymptomatic incubation period. The illness signs ranged from fever, dry cough, fatigue, myalgia, and mild respiratory tract symptoms to serve manifestations including breath shortness, pneumonia, and acute respiratory distress syndrome (ARDS) dependents on the patient's age, genetics factors, and the function of the immune system [3,4]. Extra-pulmonary involvements such as hepatic and gastrointestinal are also presented in some patients [5].
Typically, in the early phase of the disease, specific and proper immune system responses eliminate the virus reproduction and prevent disease progression into the hyper-inflammation phase. If the infection is not eliminated by the appropriate and strong immune responses, the disease enters to the severe inflammatory response phase when cytokine storm and elevated inflammatory markers produced by innate immune cells induced pulmonary fibrosis, shortness of breath, reduction in O2 saturation, and systemic injuries resulted in ARDS and patient's death [6]. Cytokine storm induction by SARS-CoV-2 was confirmed in COVID-19 patients at the intensive care unit (ICU), and elevated plasma levels of inflammatory cytokines have been associated with disease severity and prognosis [7,8].
ARDS is the main reason for death in COVID-19 patients [8], and there are no efficient specific treatment agents for the disease, therefore, it is suggested that glucocorticoids and immunosuppressive treatment can reduce the inflammation of respiratory system and prevents cytokine storm and ARDS induction [9]. Methylprednisolone is a glucocorticoid medication used to suppress the autoimmune and inflammatory responses in rheumatic diseases. [10] Previously, methylprednisolone was administrated in SARS and MERS patients, and the results were controversial [11][12][13], however glucocorticoid administration in COVID-19 patients in the hyper-inflammation stage are likely to have survival benefits due to cytokine storm suppression. Hence in this study, we investigated the methylprednisolone pulse effects as a glucocorticoid therapy on the treatment, clinical symptoms, and laboratory signs of hospitalized severe COVID-19 patients.

Study design
This study is conducted as a single-blind, two-arm parallel, randomized, controlled trial from April 20, 2020, till Jun 20, 2020. We enrolled 68 subjects from the Imam Khomeini Hospital,

Patients
The diagnosis of COVID-19 in subjects was performed based on the following criteria: 1.
Identification of SARS-CoV-2 via reverse transcription-polymerase chain reaction (RT-PCR) in nasopharyngeal swab or sputum samples and 2. Abnormal computed tomography (CT) scan finding (bilateral, subpleural, peripheral ground-glass opacities) with oxygen saturation <90% at rest. All patients had signed informed consent before enrolled in the study. The early pulmonary phase was defined as the start of the pulmonary involvement including hypoxia (SO2<93%) tachypnea (RR> 18) and little dyspnea and based on CT scan findings.

Inclusion criteria
Patients were included in our trial if they met the following requirments:1. Aged 18 years or older; 2. Confirmed COVID-19 with blood oxygen saturation <90%, elevated C-reactive protein (CRP >10), and interleukin (IL)-6 (>6) at the early pulmonary phase of disease before connecting to the ventilator and intubation and 3. agreed to give informed consent ( Figure 1).

Exclusion criteria
Individuals were excluded from the study if they met the following specifications: 1. Patients were intolerant or allergic to any therapeutic agents used in this research; 2. Pregnant or lactating women; 3. Patients with blood oxygen saturation <75%, positive pro-calcitonin (PCT) and troponin test, Acute Respiratory Distress Syndrome (ARDS), uncontrolled hypertension (HTN), uncontrolled diabetes mellitus (DM), gastrointestinal problems or gastrointestinal bleeding (GIB) history, heart failure (HF), active malignancies and received any immune-suppressor agents.

Randomization and masking
Once eligibility has been confirmed, (24- In this study, patients did not know which group of them used medicine. Physicians and clinicians team know about the medicine and intervention groups. Due to the emergency nature of this trial, placebos of methylprednisolone were not prepared.

Procedures and Outcome
The clinical and demographic characteristics of the study participants were obtained before enrolled in the study. All patients were followed-up from day 0 to day 3, improvement, hospital discharge, or death, and one week after hospital discharge, which was scheduled at three or four consecutive visit points. Clinical signs of the patients including heart rate, body temperature, blood pressure, oxygen saturation (SO2), and, dyspnea, cough, gastrointestinal involvement (GI) symptoms, myalgia, chest pain, and BORG score were assessed before and GI symptoms, myalgia, chest pain, and BORG score, were assessed one-week after discharge time.
All data were considered during the study and follow-up time and recorded on case report forms (CRFs) and the Excel database. The primary endpoint was the time of clinical improvement and discharge from the hospital or death whichever came first. Hospital discharge was determined according to the patients clinical and laboratory findings.
Improvement was defined as BORG score>3, improved dyspnea, stopped fever for 72 hours, SO2> 93%, tolerated oral regimen (PO), normal urinary output and reduced CRP level without any treatment side effects.

Adverse events
All undesirable effects (adverse events) experienced by patients during the study, whether or not related to methylprednisolone treatment, were defined and recorded.

Statistical analysis
In this study, all data were presented as the mean ± standard deviation for continuous variables. Categorical variables are presented as N (%). The Kolmogorov-Smirnov normality test was performed on all data. Repeated measures ANOVA was used for comparison of the trends over time between both groups in each studied variable. Moreover, Student's t-test (parametric) or the Mann Whitney test (non-parametric) was used to test for statistical differences (two-tailed) between two independent groups. Also paired t-test (parametric) or the Wilcoxon signed-rank test (non-parametric) was used to test for statistical differences between two-time points in each of intervention groups. Two-sided Chi square/Fisher's exact tests were used to assess the associations between intervention groups and the categorical variables. Kaplan-Meier survival curve analysis and the log rank test was used to analyze time-to-death between both intervention groups. After analyzing the baseline data, using the intention-to-treat (ITT) test, the multiple imputations were conducted by an expectation-maximization (EM) algorithm for making an unbiased comparison between intervention groups in handling missing data. The false discovery rate was corrected using the Benjamini-Hochberg correction method for multiple comparisons. All statistical analysis was analyzed using STATA software (Versions 11.2). Statistical significance was considered at p<005.

Patients
This study is conducted from April 20, 2020, until Jun 20, 2020. Of the 68 patients who  Table 1. RR and HR levels were significantly higher in the intervention group. Except for diabetic comorbidity, which was significantly higher in the standard care group, there were no major between-group differences in demographic and clinical characteristics at enrollment. The median interval time between disease symptom onset and hospitalization was 6.8 ± 2.97 days. The average blood oxygen saturation level and BORG score of patients were 82.7% ± 5.3 and 7.4 ± 2.14 respectively at baseline. The majority of patients had 30-50% (24 (38.7%)) and 50-70% (19 (30.6%)) pulmonary involvements respectively and all patients were receiving oxygen support. Table 2 shows patients' status and pulmonary involvement level at baseline of the patients in each group.
Except for the difference in pulmonary involvement zone, there were no between-group differences in patients' status and pulmonary involvement at enrollment.

Primary outcome
Patients assigned to the methylprednisolone group significantly have a reduced time to event (discharge, or death) compared to patients assigned to the standard care group (median, 11 (Figure 3).
The incidence of death was significantly lower in patients receiving NIV, reserve mask and nasal cannula in the methylprednisolone group (7.7%, 8.3%, and 0% respectively) compared to standard care group (60%, 57.1%, and 22% respectively) (Supplementary Figure 1). The CT scan findings from all of the dead patients in the methylprednisolone group (N=2) and 75% of patients in the standard care group (N=9) showed bilateral GGO at enrollment.

Secondary outcome
Blood SO2 level and the BORG score of patients was significantly improved after 3 days of treatment and at discharge time in the methylprednisolone group. While blood oxygen saturation level was significantly decreased in the standard care group after 3 days of treatment and the increase of SO2 at discharge time was not significant in this group.
Besides, the BORG score of patients did not change after 3 days of treatment in the standard care group and a significant decrease was only observed at discharge time in this group (Table 4).
Heart rate and temperature of patients were significantly decreased after 3 days of treatment and at discharge time only in the methylprednisolone group. Respiratory rate was also significantly reduced in the methylprednisolone group after treatment, while it is significantly increased in the standard care group after 3 days of treatment. The clinical characteristics of patients including GI Symptom, myalgia, chest pain, and cough were significantly improved in the methylprednisolone group after 3 days of treatment, and at discharge time, however, chest pain, and cough did not change significantly in the standard care group after treatment.
Clinical characteristics of patients before and after treatment are shown in Table 4.   (Table 5).

Safety and follow up
A total of two patients (5.8%) in the methylprednisolone group and two patients (7.1%) in the standard care group showed severe adverse events between initiation of treatment and the end of the study. There were one infection and one edema adverse event in the methylprednisolone group and two shock adverse events in the standard care group (Supplementary Table 1). All events and deaths during the study were judged by the site investigators to be unrelated to the intervention. In addition, no psychiatric or delirium events have been detected in patients. Following the use of high dose of corticosteroids, most of the patients required insulin due to their known or hidden diabetes, and the insulin requirement was increased in the intervention group especially in diabetic and overweight patients.  Table 2).

Discussion
The current study is the first randomized controlled trial that has evaluated changes in clinical symptoms and laboratory signs of COVID-19 patients by methylprednisolone therapy and found that methylprednisolone pulse administration at the beginning of the early pulmonary phase of illness decreased remarkably the mortality rate and improved pulmonary involvement, oxygen saturation, and inflammatory markers in COVID-19 patients. Given the increased incidence and mortality of COVID-19 across the world, the helpful and effective treatment for patients in the early pulmonary phase is still of paramount importance. There have been some reports, surrounding beneficial [1]  findings on the administration of corticosteroids in the treatment of COVID-19 [5]. Some studies did not find significant benefits of corticosteroid admission and reported that pulmonary involvements caused by the SARS-CoV-2 were not inhibited by corticosteroid treatment [6][7][8]. However, it was also reported that the administration of corticosteroid for patients with ARDS resulted in reduced risk of death [9]. The observed differences can be due to the difference in the amount and duration of treatment, small sample size, age of patients, and severity of the disease. The clinical and laboratory characteristics and pulmonary involvements of patients were not fully determined and reported in those observational studies. It seems that the administration time and pulmonary phase of patients are key factors in the corticosteroid treatment efficacy.
In our study, patients in the methylprednisolone group had a faster improvement in SO2 level, BORG score, and dyspnea. Improvement and worsening in oxygen-support status were observed in 55. 8% [11]. While in the methylprednisolone group, VBG markers did not change significantly.
The clinical characteristics of patients, including HR, RR, and temperature were also significantly improved in the methylprednisolone group while they did not change or worsen in the standard care group during treatment. While GI symptoms and myalgia were improved in patients from both groups, chest pain and cough were only significantly improved in methylprednisolone group patients. Intravenous methylprednisolone administration increased blood pressure in patients which is due to hypertensive side effects of glucocorticoids [12].
It is demonstrated that elevated serum level of IL-6 and CRP as an inflammatory marker is associated with the severity of COVID-19 and can be used as a predicted factor to disease risk [13]. Patients included in this trial had an increased CRP and IL-6 serum level at enrollment. A significant decrease in the serum level of these inflammatory markers was shown only in the methylprednisolone group after treatment.
Previous studies reported that corticosteroid administration can increase the risk of posttreatment infection in the viral disease, however, in our study the incidence of nosocomial infections is very low in both methylprednisolone and standard care group. Improved patients were followed up for seven days after treatment, and clinical symptoms remain unchanged.
We will continue to follow-up the patients and CT scans, spirometry, and pulse oximetry will perform six weeks after improvement to evaluate their long-term prognosis.

Conclusion
In this study, we assessed the intravenous methylprednisolone effect on the treatment of patients with severe COVID-19 patients. Clinical data showed that methylprednisolone administration at the beginning of the early pulmonary phase of illness improved remarkably pulmonary involvement, oxygen saturation, dyspnea, HR, RR, and temperature and inflammatory markers such as CRP and IL-6 serum level in patients, suggesting that methylprednisolone could be an efficient therapeutic agent for hospitalized severe COVID-19 patients at pulmonary phase. Unfortunately, we could not collect viral load data to assess the effects of methylprednisolone on the viral load changes between baseline and discharge time. Nevertheless, there are several limitations in this study, including the possible existed bias, single-blind design of the study, lack of follow-up to identify late adverse events, such as hip osteonecrosis. or tuberculosis re-activation, and limited sample size. Apparently, further studies need to be undertaken.

Ethics approval
This study was performed based on the Declaration of Helsinki guidelines and was approved by the ethics committee at the Tehran University of Medical Sciences (Approval ID: IR.TUMS.VCR.REC1399.54).

Funding
This study was supported by a grant from Deputy of Research, Tehran University of Medical Sciences (Grant No. 99-1-101-47282).

Role of the funding source
The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.

Consent to participate
The written informed consent was signed by all patients before enrolling in the study.

Data availability statement
Data are available upon request.

Competing interests
The authors declare that they have no competing interests