Second week methyl-prednisolone pulses improve prognosis in patients with severe coronavirus disease 2019 pneumonia: An observational comparative study using routine care data.

OBJECTIVE: To analyze the effects of a short course of methyl-prednisolone pulses (MP) during the second week of disease (week-2) in patients with severe coronavirus disease 2019 (COVID-19) pneumonia.
METHODS: Comparative observational study using data collected from routine care at Hospital Universitario Cruces, Barakaldo, Bizkaia, Spain in patients with COVID-19 pneumonia. We compared patients who received week-2-MP (125-250 mg/d x3) with those who did not, with the end-points time to death and time to death or endotracheal intubation.
RESULTS: We included 242 patients with COVID-19 pneumonia and elevated inflammatory markers at admission. Sixty-one patients (25%) received week-2-MP. Twenty-two patients (9%) died and 31 (12.8%) suffered death or intubation. The adjusted HRs for death and death or intubation for patients in the week-2-MP group were 0.35 (95%CI 0.11 to 1.06, p = 0.064) and 0.33 (95%CI 0.13 to 0.84, p = 0.020), respectively. These differences were specifically seen in the subcohort of patients with a SpO2/FiO2 at day 7 lower than 353 (adjusted HR 0.31, 95% CI 0.08 to 1.12, p = 0.073 and HR 0.34, 95%CI 0.12 to 0.94, p = 0.038, respectively) but not in patients with higher SpO2/FiO2. Patients receiving out-of-week-2-MP, non-pulse glucocorticoids or no glucocorticoids had an increased adjusted risk for both outcomes compared with week-2-MP group: HR 5.04 (95% CI 0.91-27.86), HR 10.09 (95% CI 2.14-47.50), HR 4.14 (95% CI 0.81-21.23), respectively, for death; HR 7.38 (95% CI 1.86-29.29), HR 13.71 (95% CI 3.76-50.07), HR 3.58 (95% CI 0.89-14.32), respectively, for death or intubation. These differences were significant only in the subgroup with low SpO2/FiO2.
CONCLUSIONS: Week-2-MP are effective in improving the prognosis of patients with COVID-19 pneumonia with features of inflammatory activity and respiratory deterioration entering the second week of disease. The recognition of this high-risk population should prompt early use of MP at this point.

Introduction

Beginning in December 2019, a novel coronavirus, designated SARS-CoV-2, has caused an international outbreak of respiratory illness termed COVID-19 [1, 2]. Glucocorticoids were banned in the initial recommendations for treating the disease [3]. However, severe lung and systemic inflammation may take place usually during the second week of disease, being the main cause of admission to intensive care units, need for mechanical respiratory support and death [4].

Admission of patients with COVID-19 started at Hospital Universitario Cruces in March 2020, reaching its peak between the 27th of March and early April. An initial period of no use of glucocorticoids [3] was followed by the administration of non-pulse glucocorticoids and later methyl-prednisolone pulses (MP) to selected patients. On April 3rd 2020, a joined protocol by the services of Infectious Diseases and Internal Medicine included MP in certain scenarios (see Methods).

The aim of this study is to analyse the effects of MP on the clinical course of patients with COVID-19 pneumonia admitted to the services of Infectious Diseases and Internal Medicine of Hospital Universitario Cruces. Our working hypothesis was that a short course of MP administered within the second week after the onset of symptoms (week-2-MP) would improve the outcome of patients with COVID-19 pneumonia with markers of inflammation and signs of progressive respiratory deterioration.

Methods

Study design and population

We conducted an observational study using data collected from routine care to assess the efficacy of week-2-MP to treat patients with COVID-19 pneumonia. The protocol was approved by the Basque Country Research Ethics Committee (code EPA2020032) in accordance with the Declaration of Helsinki’s guidelines for research in humans. This study has been registered in the EU PAS Register with the number EUPAS36287.

All patients admitted between March 1st and April 30th to the services of Infectious Diseases and Internal Medicine, Hospital Universitario Cruces, with a diagnosis of COVID-19 pneumonia, supported by chest X-ray and/or CT scan informed by skilled radiologists. All cases were confirmed by positive reverse-transcriptase–polymerase-chain-reaction (RT-PCR) assay for SARS-CoV-2 in nasopharyngeal swabs, were initially selected for the study. Patients were excluded if they had no inflammatory markers at admission (see Therapeutic protocols), died within the first week of symptoms, were admitted to hospital after the end of the second week, died of causes non-related to COVID-19 infection or were initially admitted to the Intensive Care Unit.

Therapeutic protocols

Initial management included supportive therapy with fluids and oxygenation with the goal of an O2 saturation ≥92%. The antiviral protocol was developed based on the recommendations by the Spanish Ministry of Health and the Spanish Agency for Medicines and Health Products (AEMPS) in the weeks prior to April 3, 2020 [5, 6].

Hydroxychloroquine for 5 days associated with lopinavir/ritonavir for 7–10 days were recommended for all patients with pneumonia. Selected patients with severe pneumonia -CURB65 ≥2 [7] and/or SpO2 in ambient air O2 <90%- could be treated with remdesivir for 5 to 10 days, at the discretion of the clinician, after enrolment in a clinical trial [8] or as an off-label drug. Following the recommendations of the World Health Organization (WHO) [3], glucocorticoids were not used during the initial weeks of the pandemic, unless needed for comorbid conditions.

After the recognition of the inflammatory phase of COVID-19 pneumonia [4], glucocorticoids were empirically given to selected patients with severe disease and positive inflammatory markers, defined as any of the following: lymphocyte count <800/mm3 (normal 1300–2900), platelet count <150000/mm3 (normal 135000–150000), ferritin >1000 ng/ml (normal 15–300), C-reactive protein >100 mg/l (normal 0–11), D-dimer >1000 ng/ml (normal 0–500). This was an empiric definition, in which the levels of ferritin, CRP and D-dimers well above the upper normal limit were set taking into account the marked elevation in such parameters seen in many patients with COVID-19 pneumonia.

Glucocorticoids were initially given at doses around 1 mg/Kg/d during several days and later as MP, 125 to 250 mg/d for 3 consecutive days, similar to the scheme used in our patients with systemic autoimmune diseases [9]. Our therapeutic protocol was updated on April 3rd 2020, including the recommendation of MP for patients with COVID-19 pneumonia with altered/worsening inflammatory parameters (lymphopenia, thrombocytopenia, rising ferritin, D-dimers and or C-reactive protein) and clinical deterioration, particularly those showing impending respiratory failure with decreasing SpO2/FiO2 values. MP were encouraged to be given during the second week after the onset of symptoms, always according to the attending physician best judgment. This therapeutic protocol remained unchanged until the end of the study period.

Study variables

All the clinical data were extracted from Hospital Universitario Cruces electronic medical records by the study investigators. Databases were accessed to extract the study data between 11th and 30th May 2020. All data were fully anonymized before the analysis and the Basque Country Research Ethics Committee waived the requirement for informed consent.

Baseline variables included age, gender, previous diagnosis of diabetes mellitus, obesity (body mass index ≥30), arterial hypertension, chronic pulmonary disease, active neoplastic, neurodegenerative disease or systemic autoimmune disease and immunosuppressive therapy. The CURB65 scale [7] was calculated at admission and divided into three categories (low, medium and high risk). In order to assess severity within the second week of disease, the SpO2/FiO2 at that point was included in the database. The definition of high inflammatory state at admission described in the therapeutic protocol in use (see above) was assumed for the purposes of this study.

Therapeutic variables included the following: lopinavir/ritonavir, hydroxychloroquine, non-pulse glucocorticoids (including the average daily dose and the number of days of treatment), low molecular weight heparin and MP (including the week of administration, counting after the onset of symptoms).

Outcomes

We used two primary outcomes: time to death (attributed to COVID-19) and time to death or endotracheal intubation.

Statistical analysis

Mean and standard deviation or median and interquartile range were used to describe continuous variables, according to their distributional characteristics. Counts and relative frequencies describe categorical variables. The time of onset of symptoms (fever and/or persistent cough), as reported by the patient, was the time origin for calculation of time to events. Life tables were used to describe risk of events over the follow-up period, which ended either at the occurrence of the event of interest, at discharge or at the end of the study follow-up on 20th May 2020. Kaplan-Meier failure curves with log-rank test were calculated for each of the two primary outcomes, using the dichotomous variable week-2-MP, yes/no, for group comparisons.

Adjusted Cox proportional risk models were fitted to assess the effect of the dichotomous variable week-2-MP as the predictor of main interest. The aforementioned variables were included in the full model. Likelihood ratio tests were used in a sequential fashion in order to find a reduced adjusted model containing statistically significant covariates. Hazard ratios (HR) with 95% confidence intervals were used to estimate the magnitude of the association between risk predictors and outcomes. Proportionality of hazards was tested through the use of comparison of adjusted and predicted survival curves and Schoenfeld residuals.

All the univariate and multivariate analyses were repeated after stratification by the median SpO2/FiO2 at week 2 (equal to 353), in two groups: SpO2/FiO2 ≤353 and >353 (designated as low SpO2/FiO2 and high SpO2/FiO2), as this variable showed a significant departure from the proportionality of hazards assumption.

Finally, the Cox analysis was repeated using a four-category predictor in order to better illustrate the effects of glucocorticoids according to the dose, duration and time of administration. Patients were divided into 4 groups: a) no-glucocorticoids, i.e. patients not receiving glucocorticoids in any form (n = 122); b) non-pulse glucocorticoids, i.e. patients receiving glucocorticoids at doses lower than 100 mg/d for periods longer than 3 days (n = 36, with 10 of them also receiving pulses); c) out-of-week-2-MP, i.e. MP at week 1 or 3, with no additional glucocorticoids at lower doses (n = 30); d) week-2-MP, i.e. patients receiving MP during week 2, with no additional glucocorticoids at lower doses (n = 54). As our proposed schedule consists of MP with no following tapering scheme, we decided to group all patients receiving non-pulse courses of glucocorticoids for longer than 3 days into the same category and keeping in the MP groups those patients receiving only pulses.

Stata 16.1 for Windows was used for all the analyses (StataCorp. 2019. Stata Statistical Software: Release 16. College Station, TX: StataCorp LLC.).

Patient and public involvement

Neither patients nor the public were involved in the conception or conduct of the study.

Results

Three hundred and forty-three patients with COVID-19 pneumonia were initially identified. Of these, 252 had an inflammatory state at admission; after excluding 2 patients with early death within the first week of disease course, 2 patients dying with COVID-19 but with death attributable to terminal cancer and 6 patients admitted after the end of the second week of disease, 242 patients were selected for the analysis of the primary and secondary outcomes.

Sixty-one patients (25%) received week-2-MP. In addition, 33 patients (14%) received out-of-week-2-MP (week 1 or 3). The remaining 148 patients (61%) did not receive MP.

Table 1 shows the clinical characteristics of the whole cohort and according to whether or not patients received week-2-MP.

Table 1

Clinical characteristics of the cohort according to treatment with MP.

VARIABLE	Overall (n = 242)	Week-2-MP (n = 61)	No week-2-MP (n = 181)	p-value1
Age (years), mean (sd)	64.4 (14.3)	65.0 (12.1)	64.2 (15.0)	0.702
Malen (%)	150 (62.0)	40 (65.6)	110 (60.8)	0.504
Follow-up* (days), mean (sd)	17.9 (7.3)	20.7 (6.0)	17.0 (7.5)	0.0007
Diabetes mellitusn (%)	51 (21.1)	9 (14.8)	42 (23.2)	0.162
Overweightn (%)	49 (20.3)	10 (16.4)	39 (21.6)	0.386
Hypertensionn (%)	117 (48.4)	33 (54.1)	84 (46.4)	0.299
Chronic bronchopathyn (%)	62 (25.6)	14 (23.0)	48 (26.5)	0.581
Active cancern (%)	26 (10.7)	7 (11.5)	19 (10.5)	0.831
Neurodegenerative diseasen (%)	9 (3.7)	3 (4.9)	6 (3.3)	0.567
Autoimmune diseasen (%)	9 (3.7)	3 (4.9)	6 (3.3)	0.567
Immunosuppressive therapyn (%)	21 (8.7)	5 (8.2)	16 (8.8)	0.877
CURB65, high risk n. (%)	19 (7.9)	5 (8.2)	14 (7.7)	0.461
Time of symptoms to admission (days) mean (sd)	6.6 (3.2)	7.4 (2.8)	6.3 (3.2)	0.021
Lymphocytes (count/mm3) median (iqr)	800 (580)	680 (480)	815 (575)	0.118
Platelets (count/mm3) median (iqr)	212,000 (144,000)	208,000 (156,000)	213,000 (135,500)	0.703
Ferritin (mg/dl) median (iqr)	543 (807)	824 (919)	481 (723)	<0.001
D-dimers (ng/ml), Median (iqr)	500 (1,171)	540 (870)	470 (1,171)	0.224
C-reactive protein (mg/dl)median (iqr)	79.5 (112.8)	112.3 (89.8)	73.4 (99.9)	0.024
SpO2/FiO2, median (iqr)	380 (160.0)	332 (201.0)	438 (125.0)	<0.001
SpO2/FiO2 < 353n (%)	117 (48.3)	41 (67.2)	76 (42.0)	0.001
Hydroxychloroquine n (%)	224 (92.9)	61 (100)	163 (90.5)	0.013
Days on hydroxychloroquine mean (sd)	6.25 (2.6)	6.8 (2.6)	6.1 (2.6)	0.24
Lopinavir/ritonavirn (%)	218 (90)	58 (95)	160 (88)	0.13
Betaferonn (%)	13 (5.3)	1 (1.6)	12 (6.6)	0.13
LMWHn (%)	225 (92.9)	60 (98.3)	165 (91.26)	0.057

Week-2-MP: methyl-prednisolone pulses in week 2; LMWH: low molecular weight heparin; sd: standard deviation; iqr: interquartile range.

* From disease onset to death, discharge or end of the study period.

Outcome: Death

Twenty-two patients (9.1%) died during the study period. The proportion of deceased patients was lower in the week-2-MP group: 4/61 (6.6%) vs. 18/181 (9.9%). The Kaplan-Meier failure curves (Fig 1A) showed a non-significant decreased risk of death of patients in the week-2-MP group (log-rank test, p = 0.102).

Fig 1

Kaplan-Meier failure curves, second week methyl-prednisolone pulses (2-MP) vs. no 2-MP.

Outcome: death. (a) Whole cohort (n = 242). Log-rank test, p = 0.102. (b) Patients with low SpO2/FiO2 (n = 122). Log-rank test, p = 0.041.

The final Cox model showed an adjusted HR for death of 0.35 (95%CI 0.11 to 1.06, p = 0.064) for patients in the week-2-MP group. Other independent predictors of death included a previous diagnosis of arterial hypertension, the use of non-pulse glucocorticoids, a high-risk CURB65 category and SpO2/FiO2 at week 2 (Table 2).

Table 2

Predictors of death: Final models.

Variable	HR (95%CI)	p
WHOLE COHORT (n = 242)
Week-2-MP	0.35 (0.11–1.06)	0.064
Non-pulse glucocorticoids	3.01 (1.28–7.14)	0.012
Hypertension	2.89 (0.91–9.17)	0.072
SpO2/FiO2	0.94 (0.91–0.98)	0.001
CURB65
Low risk	Reference
Intermediate risk	1.64 (0.55–4.88)	0.371
High risk	7.72 (2.56–23.26)	<0.001
PATIENTS WITH SpO2/FiO2 ≤353 (n = 122)
Week-2-MP	0.31 (0.08–1.12)	0.073
Non-pulse glucocorticoids	2.98 (1.09–8.17)	0.034
Hypertension	3.14 (0.84–11.75)	0.089
SpO2/FiO2	0.92 (0.87-0-97)	0.002
CURB65
Low risk	Reference
Intermediate risk	1.48 (0.41–5.34)	0.546
High risk	10.29 (2.72–38.94)	0.001

Week-2-MP: methyl-prednisolone pulses in week 2. HR: hazard ratio; CI: confidence interval.

*HR for SpO2/FiO2 is change in hazard for each increase of 10 units in its value.

In the subgroup with low SpO2/FiO2, 3/42 (7.1%) week-2-MP patients died vs. 14/80 (17.5%) non week-2-MP patients. The Kaplan-Meier failure curves depicted on Fig 1B showed a significantly lower mortality among week-2-MP patients (long-rank test, p = 0.041).

The final Cox model in this subcohort showed a protective effect of week-2-MP (HR 0.31, 95% CI 0.08 to 1.12, p = 0.073), with the rest of independent predictors being unchanged from the whole cohort model.

No differences were seen in the univariate analysis between patients with or without week-2-MP in the subpopulation with high SpO2/FiO2. Only 5/120 (4%) patients died in this subgroup. The multivariate Cox model could not identify any significant predictor of the outcome.

Outcome: Death or intubation

Thirty-one patients (12.8%) suffered death or intubation. Week-2-MP patients had a lower incidence of the combined outcome: 6/61 (9.8%) vs. 25/181 (13.8%). The Kaplan-Meier failure curves (Fig 2A) showed a non-significant largest time free of events in patients receiving week 2 MP, (log-rank test p = 0.125).

Fig 2

Kaplan-Meier failure curves, second week methyl-prednisolone pulses (2-MP) vs. no 2-MP.

Outcome: death or intubation. (a) Whole cohort (n = 242). Log-rank test, p = 0.125. (b) Patients with low SpO2/FiO2 (n = 122). Log-rank test, p = 0.032.

In the final Cox model, the adjusted HR for week-2-MP was 0.33 (95%CI 0.13 to 0.84, p = 0.020). The same independent predictors than in the model with death as the outcome variable, with the exception of arterial hypertension, were retained (Table 3).

Table 3

Predictors of death or intubation: Final models.

Variable	HR (95%CI)	p
WHOLE COHORT (n = 242)
Week-2-MP	0.33 (0.13–0.84)	0.020
Non-pulse glucocorticoids	3.87 (1.87–8.02)	<0.001
SpO2/FiO2	0.92 (0.89–0.94)	<0.001
CURB65
Low risk	Reference
Intermediate risk	1.93 (0.83–4.45)	0.125
High risk	3.65 (1.48–9.02)	0.005
PATIENTS WITH SatO2/FiO2 ≤353 (n = 122)
Week-2-MP	0.34 (0.12–0.94)	0.038
Non-pulse glucocorticoids	3.16 (1.36–7.37)	0.008
SpO2/FiO2*	0.88 (0.83-0-93)	<0.001
CURB65
Low risk	Reference
Intermediate risk	1.78 (0.69–4.57)	0.234
High risk	3.94 (1.44–10.78)	0.008

Week-2-MP: methyl-prednisolone pulses in week 2. HR: hazard ratio; CI: confidence interval.

*HR for SpO2/FiO2 is change in hazard for each increase of 10 units in its value.

In the subcohort with low SpO2/FiO2, the combined outcome was met by 5/42 (12%) vs. 20/80 (25%) patients receiving and not receiving, respectively, week-2-MP. The failure curves on Fig 2B showed a significantly better outcome in the former group (log-rank test, p = 0.032).

In the final Cox model, week-2-MP were beneficial (HR 0.34, 95%CI 0.12 to 0.94, p = 0.038). The model retained the same final variables than the whole cohort analysis.

Six patients suffered the combined outcome in the subcohort of patients with high SpO2/FiO2. Again, no differences were seen between patients with or without week-2-MP in the univariate or multivariate analysis. However, the use of non-pulse glucocorticoids was associated with an increased risk for the combined outcome (HR 10.5, 95%CI 0.94 to 116.84, p = 0.056).

Analysis of the four-level glucocorticoid variable

Table 4 depicts the clinical characteristics of the four subgroups.

Table 4

Clinical characteristics of the cohort according to type of glucocorticoid therapy.

VARIABLE	No glucocorticoids (n = 122)	Non-pulse-glucocorticoids (n = 36)	Out-of-week-2-MP (n = 30)	Week-2-MP (n = 54)	p-value
Age (years), mean (sd)	62.9 (14.7)	69.0 (13.1)	63.9 (16.4)	64.9 (12.5)	0.159
Malen (%)	63 (51.6)	29 (80.6)	23 (76.7)	35 (64.8)	0.003
Follow-up (days)*, mean (sd)	15.9 (5.0)	19.5 (10.7)	20.2 (10.7)	20.7 (5.9)	<0.001
Diabetes mellitus(n (%)	25 (20.9)	10 (27.8)	9 (30.0)	7 (13.0)	0.206
Overweightn (%)	29 (23.8)	5 (13.9)	5 (16.7)	10 (18.5)	0.536
Hypertensionn (%)	51 (41.8)	22 (61.1)	15 (50.0)	29 (53.7)	0.165
Chronic bronchopathyn (%)	29 (23.8)	15 (41.7)	6 (20.0)	12 (22.2)	0.116
Active cancern (%)	15 (12.3)	5 (13.9)	0 (0.0)	6 (11.1)	0.231
Neurodegenerative diseasen (%)	3 (2.5)	1 (2.8)	2 (6.7)	3 (5.6)	0.601
Autoimmune disordern (%)	4 (3.3)	3 (8.3)	0 (0.0)	2 (3.7)	0.339
Immunosuppressive therapyn (%)	10 (8.2)	7 (19.4)	0 (0.0)	4 (7.4)	0.041
Curb65:high riskn (%)	5 (4.1)	6 (16.7)	5 (16.7)	3 (5.6)	0.040
Time of symptoms to admission (days)mean (sd)	6.8 (3.1)	5.4 (3.1)	5.4 (3.6)	7.6 (2.7)	0.001
Lymphocytes (count/mm3)median (iqr)	950 (540)	670 (460)	625 (500)	690 (460)	<0.001
Platelets (count/mm3)median (iqr)	205,000 (116,000)	212,000 (154,500)	228,500 (135,000)	214,500 (165,000)	0.900
Ferritin (mg/dl)median (iqr)	402 (561)	444 (920)	1231 (1327)	818 (919)	<0.001
D-dimers ng/mlmedian (iqr)	410 (991)	565 (1,115)	750 (1670)	525 (890)	0.121
C-reactive protein (mg/dl)median (iqr)	60.1 (109.2)	78.2 (97.5)	110.8 (97.5)	120.6 (94.7)	0.001
SaO2/FiO2median (iqr)	450.0 (118.1)	445.5 (214.0)	299.5 (344.2)	330.5(206.3)	<0.001
SaO2/FiO2 <353n (%)	43 (35.2)	19 (52.8)	21 (70.0)	35 (64.8)	<0.001
Hydroxychloroquinen (%)	109 (89.3)	32 (88.9)	29 (96.7)	54 (100)	0.227
Days on hidroxicloroquinamean (sd)	6.0 (2.9)	6.8 (4.0)	6.2 (2.7)	6.9 (2.7)	0.290
Lopinavir-Ritonavirn (%)	109 (89.3)	31 (83.1)	27 (90.0)	51 (94.4)	0.602
Betaferonn (%)	8 (6.6)	3 (8.3)	1 (3.3)	1 (1.9)	0.472
LMWHn (%)	110 (90.2)	32 (88.9)	30 (100)	53 (98.2)	0.076

Week-2-MP: methyl-prednisolone pulses in week 2; LMWH: low molecular weight heparin; sd: standard deviation; iqr: interquartile range.

* From disease onset to death, discharge or end of the study period.

In the model with mortality as single outcome, week-2-MP patients showed lower mortality (HR 0.24, 95%CI 0.05 to 1.24, p = 0.088), whilst non-pulse glucocorticoid patients had an increased risk of death (HR 2.44, 95%CI 0.88 to 6.76, p = 0.087), both compared with patients with no glucocorticoids. The rest of the model included the same variables than the previous analysis (Table 5). In the subcohort with low SpO2/FiO2, week-2-MP decreased mortality compared with no glucocorticoids, HR 0.14, 95%CI 0.02 to 1.28, p = 0.082. The remaining subgroups showed non significant differences.

Table 5

Predictors of death: final models using the type of glucocorticoid therapy.

Variable	HR (95%CI)	p
WHOLE COHORT (n = 242)
Glucocorticoid therapy
No glucocorticoids	reference
Non-pulse glucocorticoids	2.44 (0.88–6.76)	0.087
Out-of-week-2-MP	1.22 (0.33–4.42)	0.767
Week-2-MP	0.24 (0.05–1.24)	0.088
Hypertension	2.96 (0.92–9.56)	0.069
SpO2/FiO2*	0.94 (0.91–0.97)	0.001
CURB65
Low risk	reference
Intermediate risk	1.75 (0.58–5.21)	0.318
High risk	7.66 (2.46–28.85)	<0.001
PATIENTS WITH SpO2/FiO2 ≤353 (n = 122)
Glucocorticoid therapy
No glucocorticoids	reference
Non-pulse glucocorticoids	2.30 (0.71–7.47)	0.169
Out-of-week-2-MP	1.40 (0.35–5.54)	0.636
Week-2-MP	0.14 (0.02–1.28)	0.082
Hypertension	3.11 (0.78–12.41)	0.108
SaO2/FiO2*	0.91 (0.86–0.96)	0.001
CURB65
Low risk	reference
Intermediate risk	1.67 (0.45–6.18)	0.441
High risk	10.85 (2.58–45.56)	0.001

MP: methyl-prednisolone pulses; Week-2-MP: methyl-prednisolone pulses in week 2. HR: hazard ratio; CI: confidence interval.

*HR here estimates change in hazard by 10 units increase in SpO2/FiO2.

In the model with the combined death or intubation outcome, 2-week-MP were protective (HR 0.28, 95%CI 0.07 to 1.12, p = 0.072), whilst non-pulse glucocorticoids increased the risk (HR 3.83, 95%CI 1.52 to 9.68, p = 0.004) vs. patients on no glucocorticoids. The results were similar in the subcohort with a low SpO2/FiO2: week-2-MP vs. no glucocorticoids, HR 0.20, 95%CI 0.04 to 1.00, p = 0.050; non-pulse glucocorticoids vs. no glucocorticoids, HR 3.06, 95%CI 1.06 to 8.85, p = 0.039 (Table 6).

Table 6

Predictors of death or intubation.

Final models using the type of glucocorticoid therapy.

Variable	HR (95%CI)	p
WHOLE COHORT (n = 242)
Glucocorticoid therapy
No glucocorticoids	reference
Non-pulse glucocorticoids	3.83 (1.51–9.68)	0.004
Out-of-week-2-MP	2.06 (0.71–6.00)	0.183
Week-2-MP	0.28 (0.07–1.12)	0.072
SpO2/FiO2*	0.92 (0.89–0.95)	<0.001
CURB65
Low risk	reference
Intermediate risk	2.38 (1.00–5.65)	0.049
High risk	3.98 (1.58–9.89)	0.003
PATIENTS WITH SpO2/FiO2 ≤353 (n = 122)
Glucocorticoid therapy
No glucocorticoids	reference
Non-pulse glucocorticoids	3.06 (1.06–8.85)	0.039
Out-of-week-2-MP	1.92 (0.61–6.00)	0.263
Week-2-MP	0.20 (0.04–1.00)	0.050
SpO2/FiO2*	0.88 (0.83–0.93)	<0.001
CURB65
Low risk	reference
Intermediate risk	2.49 (0.91–6.80)	0.075
High risk	5.00 (1.73–14.47)	0.003

MP: methyl-prednisolone pulses; Week-2-MP: methyl-prednisolone pulses in week 2. HR: hazard ratio; CI: confidence interval.

*HR here estimates change in hazard by 10 units increase in SaO2/FiO2.

None of the analysis in the subcohort with high SpO2/FiO2 at week 2 showed significant differences between the four therapeutic groups.

The results of the four-level glucocorticoid Cox analysis with 2-week-MP as the main comparator are presented in Table 7. Week-2-MP was superior to all the other therapies in order to prevent both main outcomes. The effects were more marked among patients with low SaO2/FiO2.

Table 7

Cox models for death and death or intubation by type of glucocorticoid therapy.

	Death				Death or intubation
	Whole cohort		SpO2/FiO2 ≤353		Whole cohort		SpO2/FiO2 ≤353
	HR (95%CI)*	p	HR (95%CI)*	p	HR (95%CI)**	p	HR (95%CI)**	p
Week-2-MP	reference		reference		reference		reference
Non-pulse glucocorticoids	10.09 (2.14–47.50)	0.003	16.20 (1.86–141.05)	0.012	13.71 (3.76–50.07)	<0.001	15.50 (3.14–76.38)	0.001
Out-of-week-2-MP	5.04 (0.91–27.86)	0.064	9.83 (0.78–12.41)	0.044	7.38 (1.86–29.29)	0.004	9.72 (1.91–49.31)	0.006
No glucocorticoids	4.14 (0.81–21.23)	0.088	7.04 (0.78–63.71)	0.082	3.58 (0.89–14.32)	0.072	5.07 (1.00–25.67)	0.050

MP: methyl-prednisolone pulses; Week-2-MP: methyl-prednisolone pulses in week 2. HR: hazard ratio; CI: confidence interval.

*Model adjusted by hypertension, SaO2FiO2 and Curb65 score.

**Model adjusted by SaO2FiO2 and Curb65 score.

Discussion

This study supports the utility of glucocorticoids to improve the outcome of patients with COVID-19 pneumonia. Glucocorticoid use, however, should not be indiscriminate, but rather restricted to patients with laboratory evidence of inflammation and progressing respiratory compromise, and best used as short-course pulse therapy (125–250 mg/d of methyl-prednisolone during 3 days) administered during the second week after the onset of symptoms, where the hyperinflammatory reaction takes usually place.

By the time the outbreak reached our region in late February 2020, the use of glucocorticoids was discouraged by the WHO [3]. This recommendation was based on a review of the clinical evidence of steroid therapy in other similar viral diseases, such as SARS-Cov-1, Middle East respiratory syndrome (MERS), influenza and respiratory syncytial virus, and on the idea that glucocorticoids could actually delay viral clearance [10]. However, an observational study of 201 patients with COVID-19 pneumonia admitted to Wuhan Jinyintan Hospital published in early March showed a reduced mortality among patients receiving methyl-prednisolone, although the dose, the duration and the time of administration were not specified [11].

Indeed, new insights into the pathogenesis of the disease seem to support immunoregulation of some kind in patients affected by SARS-Cov-2, with the recognition of an inflammatory phase of the disease that can lead to extensive lung and multisystemic injury [4]. This second phase usually takes place during the second week after the onset of symptoms.

Recent data also support the utility of glucocorticoids in COVID-19 disease [12-14]. In an observational study of 213 patients from Detroit [12], investigators compared patients of two groups, the standard of care (SOC) and early corticosteroid protocol groups (CP). Patients were given a similar median of 40 mg/d of methyl-prednisolone for 3 days, however, the proportion of treated patients (SOC 56.5% vs. CP 68.2%) and the time from symptoms to methyl-prednisolone administration (SOC 10 days vs. CP 8 days) were different. The combined composite end point (admission to intensive care unit/mechanical ventilation/death) occurred in 54.3% vs. 34.9% (OR 0.45, 95%CI 0.26–0.79) of patients in the SOC and CP, respectively.

A second observational study from Madrid compared 396 patients admitted for COVID-19 treated with glucocorticoids with 67 not treated [13]. In the glucocorticoid-treated group, methyl-prednisolone was given either at 1 mg/kg/d (78.3%) or as 2 to 4-day pulses, up to 500 mg/d (21.7%) with subsequent tapering in 25% patients. The duration of therapy with 1 mg/kg/d was not specified. The median time from disease onset to initiation of glucocorticoid was 10 days. This study showed a significant reduction in mortality among glucocorticoid users (13.9% vs. 23.9%, HR 0.51, 95%CI0.27–0.96), with differences being significant only in the subgroup classified as moderate-severe disease. There was no difference in mortality between patients receiving 1 mg/Kg/d or pulses, however, 70 patients received MP a mean of 5 days after the failure of the 1 mg/kg/d scheme.

The preliminary results from the RECOVERY trial have just been released [15]. Patients with suspected or confirmed COVID-19 infection were randomised to receive dexamethasone (n = 2104) or SOC (n = 4321). The dexamethasone protocol consisted of 6 mg/d for up to 10 days, although the actual median number of days was 6, and 7% of SOC patients also received dexamethasone. The primary outcome, all-cause mortality within 28 days of randomization, was met by 21.6% patients allocated to dexamethasone vs. 24.6% allocated to usual care (RR 0.83; 95% CI 0.74 to 0.92). The reduction of mortality was only significant in patients receiving respiratory support. Following the press release of these results, the WHO has already demanded an increase in dexamethasone production [15].

In contrast, a more recent Brazilian double-blind, randomized, placebo-controlled trial in patients with clinical or radiological suspicion of COVID-19 pneumonia, with SpO2 ≤ 94% at room air or need for non-invasive or invasive ventilation reported no benefit from the use of methyl-prednisolone [16]. They were treated with methyl-prednisolone, 0.5 mg/kg/12h, or placebo for five days. The 28-day mortality rates in the 393 patients who completed the follow-up was 37.1% vs. 38.2% in the intervention and placebo groups, respectively. Of note, the median number of days between disease onset and randomization was 13 days in both groups. Authors reported, in the subgroup of patients older than 60 years who also had higher levels of C-reactive protein, a lower mortality among patients treated with methyl-prednisolone.

Thus, a beneficial role of glucocorticoids in patients with severe COVID-19 disease has been generally found [12–14, 16]. Whether they should be used in short-term pulses (i.e. doses of methylprednisolone between 100–500 mg/d for 3–4 days) or at lower doses for more prolonged periods of time is still an open question. The advantages of dexamethasone over methyl-prednisolone are not well established; besides the large sample size and the randomised controlled trial design of the RECOVERY study [14], the difference between the treated and untreated groups was quantitatively smaller than in the studies using methyl-prednisolone [12, 13]. The time of administration of glucocorticoids has been variable, although in the Brazilian study, which concluded with mostly negative results, they were administered within the third week of disease in 50% of patients [16]. Thus, specific recommendations about which corticosteroid should be administered, at what dose, for how long and when in the clinical course, could not be yet made.

Our study confirms that patients with COVID-19 disease with a high inflammatory profile and respiratory compromise are most likely to benefit from glucocorticoid therapy. In addition, our study found that only in patients treated in the second week after the onset of symptoms seemed MP to be effective. We also observed a clear difference between the use of a short course of MP and more prolonged reduced doses of glucocorticoids, the former preventing and the second being associated to an increased risk of both unfavourable outcomes in all developed models. The presence of unidentified sources of bias cannot be excluded even after statistical adjustment for other prognostic factors and thus we cannot assure this deleterious effect on non-pulse methyl-prednisolone, moreover in the light of the results of previous studies. However, the superiority of week-2-MP over all the remaining options was clearly identified in our cohort. Our study does not support the use of non-pulse glucocorticoids in the setting of severe COVID-19 pneumonia, either alone or combined with MP.

These findings are actually in agreement with the results seen in patients with systemic lupus erythematosus and other autoimmune diseases. The activation of the non-genomic way by MP, given during a short period of time, maximises the anti-inflammatory activity of glucocorticoids without relevant side effects [17-19]. On the other hand, doses in the range of 40–90 mg/d during a period of one week or more are less effective and can increase the risk of infections. The reason for this is the full activation of the genomic way, which is responsible for most of the serious secondary effects of glucocorticoids [17-19].

Limitations

The most important limitation of this study is the observational design with a high variability in the therapeutic schemes, a consequence of the rapidly changing situation during this highly stressing pandemic. Indeed, the actual number of patients treated with MP was lower than expected after the approval of the revised protocol. Patients receiving MP also had a more severe disease. Thus, although the analysis included a high number of potentially predictive variables in the initial multivariate Cox models, it is possible that some unidentified confounder may have influenced our results. We also had a low number of patients meeting the final endpoints compared with other studies [12–14, 16]. A possible explanation for this is that, despite the high pressure attained, our hospital did not collapse as many others in Spain. Despite the consequent limited statistical power, the risk reduction among patients receiving week-2-MP was consistently between 60–70% for both endpoints in all the models, which supports a clinically relevant effect.

A critical point in the clinical application of our results is the correct identification of the point of disease onset, in order to make the correct evaluation and starting MP, if needed, early in the second week. This is not always easy, since non-specific symptoms may precede the fully symptomatic phase of the disease by several days and because some patients even with radiological features of severe pneumonia can be oligosymptomatic [20]. Thus, a thorough anamnesis must be made at admission with specific questions regarding the starting date of fever, persistent cough and/or dyspnea.

Conclusion

This study confirms that MP, 125–250 mg/d for 3 consecutive days given during the second week of disease without subsequent tapering, improve the prognosis of patients with COVID-19 pneumonia, features of inflammatory activity and respiratory deterioration. Our results open the door to a more rational and planned management of patients with COVID-19. Those with negative inflammatory markers and normal SpO2 seem to have a good prognosis, thus clinical observation monitoring oxygen saturation (even at home), could be appropriate. On the other hand, those approaching the second week of disease with worsening inflammatory markers and respiratory failure would greatly benefit from a short course of MP, which could not only be life-saving, but also help avoid the overload of critical care units.

COVID pulses.

(XLS)

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20 Aug 2020

PONE-D-20-23728

SECOND WEEK METHYL-PREDNISOLONE PULSES IMPROVE PROGNOSIS IN PATIENTS WITH SEVERE CORONAVIRUS DISEASE 2019 PNEUMONIA: AN OBSERVATIONAL COMPARATIVE STUDY USING ROUTINE CARE DATA.

PLOS ONE

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Reviewer #1: This study further supports the findings of recently published work evaluating the impact of steroids in patients hospitalised with COVID19 pneumonia. The concordance of the results presented here reinforce the evidence base, which although reassuring, does not really add anything to what has already been established in the RECOVERY trial. Indeed, the additional benefit of a small retrospective observational study compared to a large, prospective clinical trial that has clearly established the efficacy of steroids in patients with severe disease is questionable.

Issues:

Observational, retrospective nature of study and relatively small numbers (242 vs 6425 in RECOVERY). MP was given at the discretion of the attending physician and this is likely to have introduced bias. The characteristics of patients who received MP compared to those who did not differed making it hard to draw conclusions from the outcomes – for example, only 14.8% who had MP had diabetes compared to 23.2% of those who did not. Patients receiving MP had higher inflammatory markers (ferritin, CRP), and more were on LMWH, which may be of significance given the high incidence of pulmonary emboli in patients with acute infection. Table 2 compares the demographics of patients who received week 2 MP with those who did not but the latter group also includes those who had MP at some other point (33 patients). It would be useful to separate out these groups and to provide further information on the 14% of patients who had MP outside of week 2.

The secondary outcome was nosocomial infections but it is unclear how these were defined (and may have be difficult to differentiate from worsening primary disease). Details of the steroid regime and characteristics of patients receiving non-pulsed GC were also not clearly stated and there appears to be a wide dose range (could some patients be on pre-existing steroids?). The authors mention that pulsed MP was not associated with increased infection rate whereas non-pulsed GCs were. The numbers of patients in the GC subgroup was small and so I do not think conclusions can be drawn from the results. Furthermore, given that the duration of steroids was relatively short in both groups (only a mean of 6.5 days in the non-pulsed GC group), it would seem unlikely that infection risk would be significantly higher in the group receiving lower dose for marginally longer. I suspect that confounding factors are responsible but no information has been given on the non-pulsed GC subgroup. Whilst the authors confidently report that non-pulsed GC is associated with higher infection risk, they do not provide a possible explanation for their observation and place too much weight on this finding.

A lot of emphasis was put on timing of MP and the importance of short duration but little information was given regarding when patients had it outside this window i.e. how many had it at week 1 and how many at week 3. As the majority of patients who present to hospital have had symptoms for a number of days (often a week or so), then most would not have received it within the first week. Patients receiving MP in the third week, at the judgement of the attending physician, are likely to have a severe protracted form of the disease and therefore difficult to determine whether the steroids really were harmful (again, a separate demographic table looking at this group of patients would be helpful). The majority of patients in the RECOVERY trial received dexamethasone in the second week so this is not a new finding.

The first paragraph of the introduction states that no drug has been found to improve the outcome of severe COVID-19 infection. Clearly, this is no longer true with the publication of the RECOVERY trial (and other smaller studies), which are then referred to in the discussion.

The rationale for high-dose steroids for a short period of time is logical in the context of severe COVID19 infection where an exuberant immune response is thought to drive the later phase pathophysiology, although the authors mention that this is the case in many autoimmune diseases. This is not true however, and in many diseases (i.e. inflammatory interstitial lung disease (including organising pneumonia), vasculitis, rheumatoid arthritis etc) require long term immunosuppression which often takes the form of a high dose steroid regime that is weaned over time.

In summary, the data presented in this study is not new and the findings have recently published elsewhere. The secondary conclusions regarding infection risk and timing of MP are not adequately supported by the data presented.

Reviewer #2: PONE-D-20-23728

The authors conducted an observational study in which patients with COVID-19 pneumonia who received methyl-prednisolone pulse therapy during the second week of disease (week-2-MP) are compared with those who did not receive, in terms of time to death, and time to death or endotracheal intubation. They concluded that week-2-MP are effective in improving the prognosis of patients with severe COVID-19 pneumonia.

The authors do not set a significance level based on the p-value of the statistical analysis. Results are arbitrarily described to support the author's hypothesis. This study suggests that methyl-prednisolone pulse therapy may improve the prognosis of CODIV-19 patients depending on the timing of administration and the patient's condition. On the other hand, this study shows that non-pulse glucocorticoid treatment, and methyl-prednisolone pulse therapy other than the second week were harmful. In both respects, this study may be helpful for broad readers to treat COVID-19 patients, while several undescribed matters of the study should be clarified.

# The reviewer is not provided with supplemental table 1, 2 and 3, if any.

Questions;

1. The definition of pneumonia in this study is not described. How did the authors diagnose pneumonia, by chest CT scan, chest radiograph, or clinical impression?

2. The definition of onset of symptoms is not described. What are the symptoms that represent the onset of COVID-19 in this study?

3. Does “SaO2” in this manuscript represent arterial oxygen saturation?

Is this SpO2 (percutaneous arterial oxygen saturation), isn’t it?

4. How was the FiO2 for each patient who did not receive endotracheal intubation determined?

Was it really measured, or was it only speculated?

5. “inflammatory state at admission was defined as the presence of any of the following: lymphocyte count <800/mm3, platelet count <150,000/mm3, ferritin >1000 mg/dl, C-reactive protein >100 mg/dl, D-dimers >1000 ng/ml. “

The values of ferritin and C-reactive protein in this definition seem to be very high. What are the normal ranges of these five measurements at the hospital where this study was done?

6. page 9; “The Kaplan-Meier failure curves (figure 1a) showed a decreased risk of death of patients in the week-2-MP group with a trend for significance (log-rank test, p=0.102).”

How do the authors select significant results from this study based on the statistical p-value?

What does “a trend for significance” mean?

7. Figure 1b has no title, like "2b: Patients with low SaO2/FiO2 (n= 122). Log-rank test, p=0.032.”.

8. page 10 “In the model with mortality as outcome, week-2-MP patients showed a trend for lower mortality (HR 0.48, 95%CI 0.14 to 1.57, p=0.225), whilst out-of-week-2- MP patients had an increased risk of death (HR 2.49, 95%CI 0.87 to 7.11, p= 0.088), both compared with no MP patients.“

Comparison among 2-week-MP, out-of-week-2-MP and no MP patients is critical for this study. These data (probably in supplemental table 1, 2 and 3) should be presented as main tables.

9. Whole cohort (n=242) may contain the following four patient groups.

1, Week-2-MP patients (n=61).

2, Out-of-week-2-MP patients (week 1 or 3, n=33?).

3, Patients who did not receive any MP, but received any glucocorticoid (n=35?).

4, Patients who did not receive any glucocorticoid (n=113?).

The authors do not present the outcomes of these four patient categories clearly. Tables 2 and 3 should include HRs for these four patient groups. What is the outcome of patients who did not receive any glucocorticoid in Table 2 and 3 analysis?

10. Since out-of-week-2-MP patients had an increased risk of death, awareness of the onset of disease is critical. Actually, most people with SARS-CoV-2 infection have no recognized symptoms. Nevertheless, some of those people have asymptomatic pneumonia diagnosed by lung CT scan (CT features of SARS-CoV-2 pneumonia according to clinical presentation: a retrospective analysis of 120 consecutive patients from Wuhan city. Eur. Radiol. 30 (8), 4417–4426), and have even asymptomatic hypoxemia. It may be difficult to determine the actual onset of COVID-19 pneumonia. Therefore, clinically, it may be difficult to decide when the second week of disease begins and when week-2-MP therapy should be given. How do the authors think about it?

**********

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Reviewer #1: No

Reviewer #2: Yes: Tomohiko Aoe

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3 Sep 2020

RESPONSE TO REVIEWERS.

-We thank the reviewers for their comments. We feel that after the revision following their suggestions the study has improved in consistency and clarity of the results.

Reviewer #1: This study further supports the findings of recently published work evaluating the impact of steroids in patients hospitalised with COVID19 pneumonia. The concordance of the results presented here reinforce the evidence base, which although reassuring, does not really add anything to what has already been established in the RECOVERY trial. Indeed, the additional benefit of a small retrospective observational study compared to a large, prospective clinical trial that has clearly established the efficacy of steroids in patients with severe disease is questionable.

-We agree that the design and the size of our study are inferior to RECOVERY. However, we honestly feel that our work adds important complementary information regarding the use of corticosteroids in these patients: it supports the effective use of methyl-prednisolone, as an alternative to dexamethasone (opening the range of effective medications can be crucial in case of shortage of widely used drugs, as could be the future situation), of pulses instead of mg/kg/d doses, taking advantage of the non-genomic way activation and of short-term courses, potentially decreasing toxicity. This manuscript also contributes to answer two key related questions: when and to which patients should this therapy be given.

Issues:

Observational, retrospective nature of study and relatively small numbers (242 vs. 6425 in RECOVERY). MP was given at the discretion of the attending physician and this is likely to have introduced bias. The characteristics of patients who received MP compared to those who did not differed making it hard to draw conclusions from the outcomes – for example, only 14.8% who had MP had diabetes compared to 23.2% of those who did not. Patients receiving MP had higher inflammatory markers (ferritin, CRP), and more were on LMWH, which may be of significance given the high incidence of pulmonary emboli in patients with acute infection. Table 2 compares the demographics of patients who received week 2 MP with those who did not but the latter group also includes those who had MP at some other point (33 patients). It would be useful to separate out these groups and to provide further information on the 14% of patients who had MP outside of week 2.

-We deeply thank this comment. According to this remark and to reviewer 2 comments, we have added a more detailed analysis of patients within the four different therapeutic subgroups: no glucocorticoids; non-pulse glucocorticoids; week 2 MP and out of week 2 MP. We believe that with these results the final message of the study is reinforced.

The secondary outcome was nosocomial infections but it is unclear how these were defined (and may have be difficult to differentiate from worsening primary disease). Details of the steroid regime and characteristics of patients receiving non-pulsed GC were also not clearly stated and there appears to be a wide dose range (could some patients be on pre-existing steroids?). The authors mention that pulsed MP was not associated with increased infection rate whereas non-pulsed GCs were. The numbers of patients in the GC subgroup was small and so I do not think conclusions can be drawn from the results. Furthermore, given that the duration of steroids was relatively short in both groups (only a mean of 6.5 days in the non-pulsed GC group), it would seem unlikely that infection risk would be significantly higher in the group receiving lower dose for marginally longer. I suspect that confounding factors are responsible but no information has been given on the non-pulsed GC subgroup. Whilst the authors confidently report that non-pulsed GC is associated with higher infection risk, they do not provide a possible explanation for their observation and place too much weight on this finding.

-We agree with the reviewer that that we have attributed too much weight to infections. The diagnosis of the clinician was assumed by the investigators and we do not have much data to refine this. We agree that probably a number of confounders are playing a relevant role. After revising the data and the analysis, we are convinced that this section adds more shadows than light to the study, and, not being an essential research question, we have decided to drop this section. We thank the reviewer for his comments.

A lot of emphasis was put on timing of MP and the importance of short duration but little information was given regarding when patients had it outside this window i.e. how many had it at week 1 and how many at week 3. As the majority of patients who present to hospital have had symptoms for a number of days (often a week or so), then most would not have received it within the first week. Patients receiving MP in the third week, at the judgement of the attending physician, are likely to have a severe protracted form of the disease and therefore difficult to determine whether the steroids really were harmful (again, a separate demographic table looking at this group of patients would be helpful). The majority of patients in the RECOVERY trial received dexamethasone in the second week so this is not a new finding.

-We have added information regarding the out of week 2 MP group. The new by groups of glucocorticoid administration analysis shows that MP out of week two do not seem to be effective but do not confer a worse prognosis if not given with additional non-pulse glucocorticoids (see tables 5 and 6). However, we believe that the message that MP should be given during the second week in order to be beneficial is important, also suggested by RECOVERY and supported by current pathophysiological understanding.

The first paragraph of the introduction states that no drug has been found to improve the outcome of severe COVID-19 infection. Clearly, this is no longer true with the publication of the RECOVERY trial (and other smaller studies), which are then referred to in the discussion.

-Thank you for the comment, this sentence has been deleted.

The rationale for high-dose steroids for a short period of time is logical in the context of severe COVID19 infection where an exuberant immune response is thought to drive the later phase pathophysiology, although the authors mention that this is the case in many autoimmune diseases. This is not true however, and in many diseases (i.e. inflammatory interstitial lung disease (including organising pneumonia), vasculitis, rheumatoid arthritis etc) require long term immunosuppression which often takes the form of a high dose steroid regime that is weaned over time.

-The practice in our Autoimmune Diseases Unit is avoiding high-dose glucocorticoids and limiting the time on prednisone >5 mg/d, precisely by using MP as one of the main resources. We have large published experience on that, mainly on lupus, showing the efficacy and lack of toxicity of this approach. This has been summarised in reference 17.

In summary, the data presented in this study is not new and the findings have recently published elsewhere. The secondary conclusions regarding infection risk and timing of MP are not adequately supported by the data presented.

-We hope that after the modifications the reviewer feels happier with our study and considers that it contains useful information for treatment of forthcoming patients with COVID-19 pneumonia.

Reviewer #2: PONE-D-20-23728

The authors conducted an observational study in which patients with COVID-19 pneumonia who received methyl-prednisolone pulse therapy during the second week of disease (week-2-MP) are compared with those who did not receive, in terms of time to death, and time to death or endotracheal intubation. They concluded that week-2-MP are effective in improving the prognosis of patients with severe COVID-19 pneumonia.

The authors do not set a significance level based on the p-value of the statistical analysis. Results are arbitrarily described to support the author's hypothesis. This study suggests that methyl-prednisolone pulse therapy may improve the prognosis of CODIV-19 patients depending on the timing of administration and the patient's condition. On the other hand, this study shows that non-pulse glucocorticoid treatment, and methyl-prednisolone pulse therapy other than the second week were harmful. In both respects, this study may be helpful for broad readers to treat COVID-19 patients, while several undescribed matters of the study should be clarified.

# The reviewer is not provided with supplemental table 1, 2 and 3, if any.

-Supplemental tables have been modified and included as table 4, 5 and 6.

Questions;

1. The definition of pneumonia in this study is not described. How did the authors diagnose pneumonia, by chest CT scan, chest radiograph, or clinical impression?

-Pneumonia was diagnosed by chest X-ray and, in some cases, by CT scan. All patients had a confirmation of the diagnosis by image informed by skilled radiologists. .

2. The definition of onset of symptoms is not described. What are the symptoms that represent the onset of COVID-19 in this study?

-The clinical protocol in our hospital included the time of onset of symptoms in the admission clinical record. We considered fever and/or persistent cough as the clinically meaningful presenting symptoms.

3. Does “SaO2” in this manuscript represent arterial oxygen saturation?

Is this SpO2 (percutaneous arterial oxygen saturation), isn’t it?

-OK. Changed to SpO2.

4. How was the FiO2 for each patient who did not receive endotracheal intubation determined?

Was it really measured, or was it only speculated?

-It was estimated according to the flow rates of the Venturi masks.

5. “inflammatory state at admission was defined as the presence of any of the following: lymphocyte count <800/mm3, platelet count <150,000/mm3, ferritin >1000 mg/dl, C-reactive protein >100 mg/dl, D-dimers >1000 ng/ml. “

The values of ferritin and C-reactive protein in this definition seem to be very high. What are the normal ranges of these five measurements at the hospital where this study was done?

-This definition of a high inflammatory state was used to select patients candidate to MP in the therapeutic protocol of our hospital. It was an empirical definition, in which the levels of ferritin, CRP and D-dimers well above the upper normal limit were set taking into account the marked elevation in such parameters seen in many patients with COVID-19 pneumonia. Since this definition was used to guide the clinical indication for MP, we decided to keep it unchanged for the study. The prognosis of admitted patients who had a negative inflammatory profile actually proved to be very good, with only two deaths in 91 patients and no intubations. Thus, we decided to keep this definition for the purposes of the study and to limit the analysis to those patients with an inflammatory profile, where a decision about how to deal with their impending clinical deterioration had to be made.

6. page 9; “The Kaplan-Meier failure curves (figure 1a) showed a decreased risk of death of patients in the week-2-MP group with a trend for significance (log-rank test, p=0.102).”

How do the authors select significant results from this study based on the statistical p-value?

What does “a trend for significance” mean?

-We have substituted the term “a trend for significance” by “non significant”,

7. Figure 1b has no title, like "2b: Patients with low SaO2/FiO2 (n= 122). Log-rank test, p=0.032.”.

-Thank you, the title has been added.

8. page 10 “In the model with mortality as outcome, week-2-MP patients showed a trend for lower mortality (HR 0.48, 95%CI 0.14 to 1.57, p=0.225), whilst out-of-week-2- MP patients had an increased risk of death (HR 2.49, 95%CI 0.87 to 7.11, p= 0.088), both compared with no MP patients.“

Comparison among 2-week-MP, out-of-week-2-MP and no MP patients is critical for this study. These data (probably in supplemental table 1, 2 and 3) should be presented as main tables.

9. Whole cohort (n=242) may contain the following four patient groups.

1, Week-2-MP patients (n=61).

2, Out-of-week-2-MP patients (week 1 or 3, n=33?).

3, Patients who did not receive any MP, but received any glucocorticoid (n=35?).

4, Patients who did not receive any glucocorticoid (n=113?).

The authors do not present the outcomes of these four patient categories clearly. Tables 2 and 3 should include HRs for these four patient groups. What is the outcome of patients who did not receive any glucocorticoid in Table 2 and 3 analysis?

-We thank very much the reviewer for this useful comment. We have modified the analysis of the therapeutic subcohorts by dividing patients into 4 groups. As written in the text, the four groups were: “a) no-glucocorticoids, i.e. patients not receiving glucocorticoids in any form (n=122); b) non-pulse glucocorticoids, i.e. patients receiving glucocorticoids at doses lower than 100 mg/d for periods longer than 3 days (n=36, with 10 of them also receiving pulses); c) out-of-week-2-MP, i.e. MP at week 1 or 3, with no additional glucocorticoids at lower doses (n=30); d) week-2-MP, i.e. patients receiving MP during week 2, with no additional glucocorticoids at lower doses (n=54). As our proposed schedule consists of MP with no following tapering scheme, we decided to group all patients receiving non-pulse courses of glucocorticoids for longer than 3 days into the same category, keeping in the MP groups those patients receiving only pulses.”.

10. Since out-of-week-2-MP patients had an increased risk of death, awareness of the onset of disease is critical. Actually, most people with SARS-CoV-2 infection have no recognized symptoms. Nevertheless, some of those people have asymptomatic pneumonia diagnosed by lung CT scan (CT features of SARS-CoV-2 pneumonia according to clinical presentation: a retrospective analysis of 120 consecutive patients from Wuhan city. Eur. Radiol. 30 (8), 4417–4426), and have even asymptomatic hypoxemia. It may be difficult to determine the actual onset of COVID-19 pneumonia. Therefore, clinically, it may be difficult to decide when the second week of disease begins and when week-2-MP therapy should be given. How do the authors think about it?

-Once the effect of non-pulse glucocorticoids has been separated from MP, out of week 2 MP have no longer a significant detrimental effect, although the trend is towards a worse prognosis than no-glucocorticoids and week-2-MP. We agree with the reviewer that sometimes it is not easy to establish the clinical onset of the disease. However, as supported by these and other data and also by the physiopathology of the disease, every effort must be made to identify the starting date in order to assure that MP are not given too early (during the viral phase, in which it could reduce the clearance of the virus) or too late (when the inflammatory damage is already made). We have added a comment on this and the reference given by the reviewer.

-In addition to the changes made in response to the reviewers’ comments, we have introduced some modifications in the discussion. The reference to the GLUCOCOVID study has been deleted, since the study is not yet published since the outprint was launched in June and because the clinical management and outcomes of patients were confusing. Instead, we have added a reference to a recently published Brazilian RCT (METCOVID, reference 16).

Submitted filename: Response to reviewers-def.docx

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7 Sep 2020

SECOND WEEK METHYL-PREDNISOLONE PULSES IMPROVE PROGNOSIS IN PATIENTS WITH SEVERE CORONAVIRUS DISEASE 2019 PNEUMONIA: AN OBSERVATIONAL COMPARATIVE STUDY USING ROUTINE CARE DATA.

PONE-D-20-23728R1

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11 Sep 2020

PONE-D-20-23728R1

Second week methyl-prednisolone pulses improve prognosis in patients with severe coronavirus disease 2019 pneumonia: an observational comparative study using routine care data.

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