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EClinicalMedicine 000 (2020) 100459

Contents lists available at ScienceDirect

EClinicalMedicine
journal homepage: https://www.journals.elsevier.com/eclinicalmedicine

Early use of low dose tocilizumab in patients with COVID-19: A

retrospective cohort study with a complete follow-up
Nicola De Rossia,*,1, Cristina Scarpazzab,1, Chiara Filippinia, Cinzia Cordiolia, Sarah Rasiaa,
Chiara Rosa Mancinellia, Damiano Rizzonia,c, Giuseppe Romanellia,c, Stefania Cossia,
Nereo Vettorettoa, Sergio Bovea, Silvano Manfredinia, Eva Andrea Beindorfa, Carlo Moscaa,
Vittorio Scipionea, Gigliola Flamminioa, Elena Albini Albinia, Paola Giansiracusaa,
Ruggero Capraa, on behalf of Montichiari COVID-19 Study Group
a
Covid 19 Unit, Spedali Civili di Brescia, Presidio Ospedaliero di Montichiari, Montichiari Hospital, via Giuseppe Ciotti, Montichiari 35018, Brescia, Italy
b
University of Padova, Via Venezia 8, Padova, Italy
c
Department of Medical and Surgical Sciences, University of Brescia, Italy

A R T I C L E I N F O A B S T R A C T

Article History: Background: Pneumonia with severe respiratory failure represents the principal cause of death in COVID-19,
Received 19 May 2020 where hyper-inflammation plays an important role in lung damage. An effective treatment aiming at reduc-
Revised 23 June 2020 ing the inflammation without preventing virus clearance is thus urgently needed. Tocilizumab, an anti-solu-
Accepted 29 June 2020
ble IL-6 receptor monoclonal antibody, has been proposed for treatment of patients with COVID-19.
Available online xxx
Methods: A retrospective cohort study at the Montichiari Hospital, Brescia, Italy, was conducted. We included
consecutive patients with COVID-19 related pneumonia at the early stage of respiratory failure, all treated with a
standard protocol (hydroxychloroquine 400 mg daily, lopinavir 800 mg plus ritonavir 200 mg per day). We com-
pared survival rate and clinical status in a cohort of patients who received additional treatment with tocilizumab
once (either 400 mg intravenous or 324 mg subcutaneous) with a retrospective cohort of patients who did not
receive tocilizumab (referred to as the standard treatment group). All outcomes were assessed at the end of the
follow-up, that correspond to death or complete recovery and discharge from the hospital.
Findings: 158 patients were included, 90 of which received tocilizumab. 34 out of 68 (50%) patients in the
standard treatment group and 7 out of 90 (7.7%) in the tocilizumab group died. Tocilizumab significantly
improved survival compared to standard care (multivariate HR: 0.057; 95% C.I = 0.017- 0.187, p < 0.001). No
differences between the two administration routes of tocilizumab were observed. No tocilizumab-related
infections and/or side effects were observed.
Interpretation: Early treatment with tocilizumab could be helpful to prevent excessive hyper-inflammation
and death in COVID-19 related pneumonia. Low dose administration of tocilizumab is not associated with
adverse events.
Funding: none
© 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license.
(http://creativecommons.org/licenses/by-nc-nd/4.0/)

1. Introduction Previous studies have shown that immunological hyper-activa-

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) tion, referred to as “cytokine storm” [2], can be a contributory cause
has severely affected northern Italy and especially the province of of interstitial damage in the lungs of COVID-19 patients, leading to a
Brescia. The disease has been characterized by a very high mortality more severe clinical course. During this state of immunological
rate being interstitial pneumonia with respiratory failure the main hyper-activation, peripheral CD4 and CD8 T cells counts were sub-
cause of death for COVID-19 [1]. stantially reduced while their status was hyper-activated. Further-
more, an increased concentration of highly pro-inflammatory CCR6+
* Corresponding author. T helper 17 (Th17) in CD4 T cells and a high concentration of cyto-
E-mail address: [email protected] (N. De Rossi).
1
toxic granules in CD8 lymphocytes in peripheral blood was found in
This author equally contributed to the work.

https://doi.org/10.1016/j.eclinm.2020.100459
2589-5370/© 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license. (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Please cite this article as: N. De Rossi et al., Early use of low dose tocilizumab in patients with COVID-19: A retrospective cohort study with a
complete follow-up, EClinicalMedicine (2020), https://doi.org/10.1016/j.eclinm.2020.100459
JID: ECLINM
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2 N. De Rossi et al. / EClinicalMedicine 00 (2020) 100459

19 disease. By blocking the IL-6 receptor interaction, tocilizumab

Research in context
inhibits the IL-6 mediated signal transduction, reduces the availabil-
ity of IL-6 and regulated immunological activity [16 18]. For this rea-
Evidence before this study
son, some authors recommended its use in critically ill COVID-19
Since the coronavirus disease 2019 (COVID-19) outbreak, evi- patients with significant elevated IL-6 [7]. However, the usefulness of
dence has emerged that patients may develop interstitial pneu- tocilizumab is still controversial. While some studies describing the
monia with severe respiratory failure. This represents the clinical and radiological improvement of patients with severe clinical
principal cause of death in COVID-19 related infection, where conditions due to COVID-19 related pneumonia treated with high
hyper-inflammation plays an important role in lung damage. dose of tocilizumab reported an increased survival [19], other studies
Tocilizumab, an anti-soluble IL-6 receptor monoclonal anti- failed to observe beneficial effect of tocilizumab in severe patients
body, has been proposed for treatment of patients with COVID- [20,21]. Furthermore, it should be noted that IL-6 inhibition has
19. However, data so far are limited, both in the term of efficacy potential hazards of inducing infectious diseases [22].
and safety and only small or uncontrolled studies using high Thus, the role of inhibiting the link between IL-6 and the soluble
dose of tocilizumab have been published on patients in severe receptor on COVID-19 evolution remains to be fully evaluated [23].
clinical conditions and the safety profile is still unknown. Interestingly, a recent paper suggested that treatment with tocilizu-
mab could be useful not only to target symptoms but also to modu-
Added value of this study late the disease itself in its early phase [24], preventing excessive
inflammation and lung damage [7]. In order to test this hypothesis, in
The current retrospective study describes the beneficial clinical
the current paper, we describe 158 patients with COVID-19, ninety of
effect of early IL-6 blockade with low dose tocilizumab in
whom were treated early in the disease course with single - low dose
patients with COVID-19 and respiratory failure. Furthermore,
of tocilizumab. Our a priori hypothesis was twofold. First, mortality
both the intravenous and subcutaneous administration routes
rate was expected to be lower in patients early treated with tocilizu-
demonstrated to be equally effective and safe.
mab compared to controls, who were treated with standard therapy
only. Second, a low dose of tocilizumab would is not expected to be
Implications of all the available evidence
associated with de novo infections.
Our study suggests that early administration of low dose tocili-
zumab, before the appearance of a respiratory distress requir- 2. Methods
ing assisted ventilation, modulates excessive hyper-
inflammation and reduces mortality caused by COVID-19. This 2.1. Patients
result, together with the excellent safety profile and the ease of
administration, suggests that the early administration of low We conducted a retrospective cohort study at the Montichiari
dose of this agent deserves consideration in controlled trials for Hospital, a tertiary health-care Centre in Brescia, Italy, which was
the treatment of COVID-19. designated as a COVID-19 hub by Italian health authorities. Patients
consecutively admitted to Montichiari Hospital were retrospectively
included in the study if they met the following inclusion criteria: 1)
confirmed COVID-19 infection as determined by a positive reverse-
dead patients infected with SARS-CoV-2 [3]. Importantly, as shown transcriptase-polymerase-chain-reaction (RT-PCR) assay of a speci-
by previous literature, increased circulating levels of pro-inflamma- men collected on a nasopharyngeal swab; 2) bilateral pulmonary
tory cytokines (e.g. interleukin IL- 1B, IL-6, IL-12) and chemokines interstitial opacities on chest imaging that were not fully explained
(CXCL10 and CCL2) are associated with pulmonary inflammation and by congestive heart failure or other forms of volume overload; 3) a
extensive lung involvement in SARS patients [4,5]. In this scenario, respiratory failure showing at least one of the following conditions:
Acute Respiratory Distress Syndrome (ARDS) is the ultimate result of respiratory rate
30 breaths/min; peripheral capillary oxygen satu-
cytokine storm [3]. ration (SpO2)  93% while breathing ambient air or ratio of the partial
Within the pro-inflammatory cytokines, Interleukin 6 (IL-6) plays pressure of oxygen in arterial blood to the fractional concentration of
a key role in the pathogenesis of the COVID-19 related cytokine storm oxygen in inspired air (PaO2/FiO2)  300 mmHg. In line with our
[2]. IL-6 seems to be also responsible for the activation of T helper 17 rationale of including only patients in the early phase of infection,
(Th17) cells in the dendritic cell-T cell interaction [6]. In COVID-19 the following exclusion criteria were applied: 1) presence of a critical
affected patients, a high Th17 cells activation could result from a respiratory syndrome that requires mechanical or invasive ventila-
virus-driven increased production of IL-6 by the immune system [2]. tion at hospital admission; 2) presence of severe clinical conditions
Several studies showed that the serum levels of IL-6 are increased in as revealed by transaminase 5 times the upper limit of the normal
COVID-19 patients and that its circulating levels are positively related value; neutrophils <500 mmc; platelets <50.000 mmc [17].
to disease severity [7 9]. Indeed, levels of IL-6 have been found to be This retrospective study has been conducted in accordance with
directly associated with severe lung damage [10,11], and a recent the declaration of Helsinki and its later amendments and was
meta-analysis of six studies investigating IL-6 concentration in approved by the Ethical Committee of the Spedali Civili of Brescia.
COVID-19 demonstrated 2.9 fold higher levels in patients with com-
plicated COVID-19 compared with patients with non-complicated 2.2. Study design
disease [12]. An excessive and dysregulated production of IL-6 is thus
considered a potential negative prognostic factor for survival during Due to the emergency situation in the province of Brescia, north-
COVID-19, being higher levels of IL-6 related to a higher mortality ern Italy, it was impossible to carry out a properly randomized con-
rate [10]. For this reason, high serum IL-6 levels were suggested to be trolled trial. Patients admitted to the hospital between February 26th
a reliable biomarker of COVID-19 progression [13 15]. The role of and March 13th underwent a standard therapy (hydroxychloroquine
hyper-inflammation in COVID-19 is so important that guidelines for 400 mg daily, lopinavir 800 mg daily plus ritonavir 200 mg per day)
the diagnosis and treatment of SARS-CoV-2 infected pneumonia rec- [25,26] as for standard protocol administered at our institution at the
ommended cytokine monitoring to reduce mortality [14,15]. time (hereafter defined to as “standard treatment group”). Patients
Tocilizumab, a humanized anti-soluble IL-6 receptor monoclonal admitted after March 13th patients received off-label a single low
antibody, has been proposed to be useful in the treatment of COVID- dose administration of tocilizumab in addition to standard therapy
Please cite this article as: N. De Rossi et al., Early use of low dose tocilizumab in patients with COVID-19: A retrospective cohort study with a
complete follow-up, EClinicalMedicine (2020), https://doi.org/10.1016/j.eclinm.2020.100459
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N. De Rossi et al. / EClinicalMedicine 00 (2020) 100459 3

(hereafter defined to as “tocilizumab group”). Inclusion of patients in for the following baseline variables: age, gender, diabetes, hyperten-
the standard treatment or in the tocilizumab group (400 mg intrave- sion or heart diseases; serum CRP and respiratory support needed at
nously -i.v.- or 324 mg subcutaneous -s.c.-) was determined by the hospital admission (both this variables included in the multivariate
availability of the drug at the moment, as in a previous study [23]. All model to correct for disease severity ad admission); time elapsed
patients receiving standard therapy only retrospectively full-filled from symptoms onset to hospital admission (hereafter referred as to
eligibility criteria for tocilizumab treatment. All patients gave written “time to hospitalization”, included in the multivariate model to cor-
informed consent for off-label use of tocilizumab. Administration of rect for treatment delay).
tocilizumab occurred upon worsening of the respiratory functions, as
described in the inclusion criteria, in accordance with the aim of the 2.5. Role of the funding source
study to treat patients early in the course of the respiratory distress.
This usually occurred the day of hospital admission or the day after. No funding was received for this study. All the authors had full
During hospitalization, patients in both groups were assisted with access to the raw data and to patient’s clinical records.
non-invasive (i.e. low flow nasal cannula; high flow mask; Continu-
ous Positive Airway Pressure CPAP-) or invasive (i.e. mechanical 3. Results
ventilation) oxygen therapy, according to their needs. Patients were
followed-up until the end of the clinical observation, defined as death The results are described in accordance with the STROBE guide-
or complete recovery and discharge from the hospital with lines [27].
SpO2>94% while breathing in ambient air. One hundred and fifty eight patients were included in the current
study: sixty-eight patients received standard care, while 90 patients
2.3. Data extraction were treated with tocilizumab in addition to standard care (43
(47.7%) received 400 mg i.v. once, whereas 47 (52.3%) received
The clinical record of each patient was retrospectively analyzed 324 mg s.c. once, according to the availability of the drug). Baseline
and, for each patient, the following information were extracted and demographic and clinical characteristics, including laboratory test
recorded in a dedicated database: patients anonymized ID; age at results, of the groups of patients in standard care or treated with toci-
admission, gender; inclusion criteria; comorbidities; date of first flu lizumab are reported in Table 1.
symptom; date of admission to the hospital; date of tocilizumab
administration (if pertinent); tocilizumab administration route (if 3.1. Effect of early administration of tocilizumab on mortality rate
pertinent); serum procalcitonin (both at the hospital admission and
at the time of discharge) to evaluate subclinical bacterial infections; Seven deaths were observed in the group of patients treated with
de novo infections and de novo respiratory infections; the need for tocilizumab (7 out of 90 patients, 7.7%, mean age=74) while 34 deaths
daily respiratory support (low flow cannula, high flow mask, CPAP; occurred in the control group (34 out of 68 patients, 50%, mean age=78).
SpO2; Peep; FiO2); daily body temperature; daily C Reactive Protein The Cox proportional hazard model (adjusted for age, gender, diabetes,
(CRP); complete laboratory test results; date of discharge or death; hypertension, heart disease; CRP; respiratory support needed at hospital
data of admission and of discharge from Intensive Care Unit, where admission; time to hospitalization) showed a significantly greater sur-
mechanical ventilation has been administered (if pertinent). vival rate of tocilizumab patients as compared to controls (multivariate
H.R. for death: 0.057; 95% C.I = 0.017- 0.187, p < 0.001, Fig. 1). The data
2.4. Statistical analyses revealed that the risk of death increases by 6% for each year of age, mak-
ing older age a risk factor for death in COVID-19. In addition, having dia-
Data were analyzed with SPSS version 24.0 (Chicago, IL, USA). We betes or heart disease increases the risk of death 3.2 or 3 times,
report categorical variables as number (%) and continuous variables as respectively. Results are reported in Table 2.
mean (standard deviation) or median (range) depending on whether
the data are normally distributed or not. Statistical significance was 3.2. Clinical longitudinal follow up in the two groups
assessed by means of chi-squared for dichotomous variables, or by
means of the two independent sample t-test or the Mann-Whitney U Considering the pharmacodynamic of tocilizumab [28], an imme-
test for continuous variable depending on whether the data are nor- diate effect on inflammatory indices (CRP and body temperature)
mally distributed or not. For longitudinal analysis, data were analyzed was expected. Patients were then closely monitored for the first five
using paired sample t test or Wilcoxon test depending on whether the days after the beginning of therapeutic interventions in both groups.
data are normally distributed or not. Regarding laboratory results, if one Fig. 2 (upper panel) showed the drastic reduction of fever and CRP in
or more laboratory test resulted to statistically differ between tocilizu- patients treated with tocilizumab but not in controls. As CRP was not
mab and standard treatment group with a potential clinical relevance, expected to fall within the normal range in few days, CRP was now
we were interested in understanding the effect of tocilizumab adminis- measured further.
tration route and posology on these laboratory test results. Thus, a As reported in the methods, all patients were provided with respi-
repeated measures ANOVA was performed on laboratory results with ratory support according to their needs. Respiratory support needed
potential clinical relevance using group (324 mg vs 400 mg) as indepen- at hospital admission is reported in Table 3. During the observation,
dent variable and Time (two levels: pre-therapy; 5 days post therapy six patients in the standard therapy group (8.82%) and 13 patients in
follow up) as dependent variable. the tocilizumab group (14.4%) needed mechanical ventilation.
The primary endpoint was the survival rate in patients treated During the longitudinal follow up, no mechanical ventilation asso-
with tocilizumab in addition to standard therapy (tocilizumab group), ciated pneumonia were observed in our cohort. Four patients within
and only with standard therapy (standard treatment group). The sur- the standard treatment group (5.9%) and six patients within the toci-
vival rate was assessed by Kaplan Meier (KM) plot using group (toci- lizumab group (6.6%) manifested de novo respiratory system infec-
lizumab vs controls) as between factor; death as event and time to tions. Details on the etiological agents are reported in Table 3. Twelve
death/discharge as time variable. Data were censored at the end of patients in tocilizumab group (13.3%) manifested pulmonary embo-
the observation, that corresponds to discharge from the hospital for lism, three of whom died. As during the first phase of the outbreak
patients with a complete recovery as the event (i.e. death) was not the association between COVID-19 and pulmonary embolism [29]
observed. Hazard Ratio (HR) with 95% confidence intervals (CI) were was not known and postmortem examinations were not done at our
calculated by means of the Cox proportional-hazard model, adjusting site, the prevalence of pulmonary embolism in the standard therapy
Please cite this article as: N. De Rossi et al., Early use of low dose tocilizumab in patients with COVID-19: A retrospective cohort study with a
complete follow-up, EClinicalMedicine (2020), https://doi.org/10.1016/j.eclinm.2020.100459
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4 N. De Rossi et al. / EClinicalMedicine 00 (2020) 100459

Table 1
Demographic and clinical characteristics of the two groups of patients. Number denotes mean (standard deviation) (a); raw number (percentages)
(b); median [interquartile range] (c). Statistical significance was evaluated using two independent sample t-test (a); chi square (b); Mann-Whitney U
test (c). P value reports the associated p value (statistical significance p<0.05). CRP = C-Reactive Protein; bpm = beats per minute.

Normative values Controls (n=68) Tocilizumab (n=90) Significance P value

Age (years)a 71 (14.6) 62.9 (12.5) -3.706 <0.001

Ethnicity (Caucasian)b 65 (95.5%) 85 (94.4%) 0.105 0.745
Gender (males)b 49 (72%) 64 (71.1%) 0.017 0.896
Diabetes (yes) b 21 (30.9%) 14 (15.5%) 5.276 0.022
Hypertension (yes) b 36 (42.9%) 41 (45.5%) 0.846 0.358
Heart disease(yes) b 22 (32.3%) 11 (12.2%) 9.500 0.002
Time to hospitalization (days)a 6 (3.1) 9.1 (8.1) 3.001 <0.001
Clinical characteristics at hospital admission
Heart rate (bpm) a 90.0 (16.0) 91.0 (16.0) 0.390 0.697
Systolic blood pressure (mm/Hg) a 127.0 (22.0) 130.0 (23.0) 0.918 0.360
Diastolic blood pressure (mm/Hg) a 73.0 (11.0) 74.0 (10.9) 0.285 0.776
Temperature (°C)c <37 37.5 [36 39.5] 37.5 [36-40] 0.460
Laboratory Results at hospital admission
CRP (mg/L)a <0.5 83.8 (64.1) 121 (77.5) 3.212 0.002
Procalcitonin (ng/L)c <0.1 0.14 [<0.1 3.2] 0.25 [<0.1-7.5] 0.980
White blood cells (x103 per mL) a 4.00-10.80 6.6 (3.9) 7.0 (3.7) 0.635 0.527
Lymphocytes (x103 per mL) a 0.90-4.00 1.1 (0.6) 9.9 (0.8) -0.491 0.624
Neutrophils (x103 per mL) a 1.50-8.00 4.7 (3.0) 5.6 (3.5) 1.444 0.151
Platelets (x103 per mL) a 130-400 188 (89) 224 (103) 2.252 0.026
Glycemia (mg/dl)b 76-115 118 [87 476] 114 [76-314] 0.061
Urea (mg/dl) b 17-49 38 [21 154] 35 [17-143] 0.140
Creatinine (mg/dl) a 0.60-1.00 1.1 (0.5) 1.0 (0.3) -2.369 0.016
Sodium (mmol/L) a 136-145 135.9 (3.6) 136.6 (5.3) 0.790 0.431
Potassium (mmoL/L) a 3.4-4.5 3.9 (0.5) 3.8 (0.4) -1.471 0.143
Chlorine (mmol/L) a 89-107 97.3 (4.1) 97.4 (4.2) 0.227 0.821
Bilirubin (mg/dL) a <1.20 0.5 (0.2) 0.6 (0.2) 0.982 0.328
Aspartate Transaminase -AST (U/L) a 18-34 52.7 (37.8) 56.7 (38.2) 0.623 0.534
Alanine amionotrasferase -ALT (U/L) a 10-35 35.4 (31.1) 46.9 (35.5) 2.015 0.046
Gamma glutamyl trasferase- gGT (U/L) b 6-42 34 [14 555] 45.5 [11-360] 0.053
Alkaline phosphatase -ALP (U/L) a 44-107 70.7 (51.1) 63.0 (34.3) -1.001 0.319
Lactate dehydrogenase- LDH (U/L) a 135-225 326.0 (146.7) 371.3 (131.1) 1.192 0.237
Creatine kinase- CK (U/L) b 26-192 102.5 [18 843] 113 [21 918] 0.048
Prothrombine time -Pt (sec) b 9.4-12.5 13.1 [10.9 18.4] 13.5 [10.8 22.2] 0.145
Activated partial thromboplastine time- aPTT (sec) b 24-38 32.2 (5.1) 33.1 (5.2) -1.033 0.303
International normalized ratio -INR c 0.9-1.2 1.2 [0.9 4.6] 1.2 [1.0 2.0] 0.165

group is not known. Both respiratory system infections and pulmo- patients treated with tocilizumab, one occurred in the 400 mg i.v.
nary embolism are known to be associated with COVID-19 [30 35], group (age=64) and 6 in the 324 mg s.c. group (mean age=76). The
thus the role of tocilizumab in their manifestation could be ruled out. Cox proportional hazard model (adjusted for age, gender, diabetes,
For this reason, these data will not be further discussed. No infections hypertension, heart disease; CRP, respiratory support needed at hos-
or additional safety concerns related to tocilizumab emerged. pital admission; time to hospitalization) revealed that survival rate
did not statistically differ between the two administration routes
3.3. Effect of tocilizumab on laboratory test results (multivariate H.R. for death: 5.234; 95% C.I = 0.241- 113.633,
p = 0.292). The two groups did not differ in the time to discharge
Furthermore, the short term effect of single low dose of tocilizu- (mean time to discharge: 18 days -95% C.I. [15.3-20.7]- in the 400 mg
mab administration was evaluated. Laboratory test results were com- i.v. group and 14.2 days -95% C.I. [12.1-16.3]- in the 324 mg s.c.
pared before tocilizumab administration and 5 days after tocilizumab group; baseline characteristics adjusted Cox proportional hazard
administration in 81 patients (n=7 dead patients were excluded; n=2 model: multivariate H.R.: 1.556; 95% C.I = 0.940- 2.479, p = 0.086).
patients had a follow up shorter than 5 days and laboratory test Furthermore, the quick decrement of body temperature and CPR after
results are not available). As shown in Table 4, few statistical differen- tocilizumab administration is similar between the two administra-
ces emerged between baseline and follow up, however only few of tion routes, as shown in the lower panel of Fig. 2. Finally, as an incre-
them are clinically relevant [36]. A decrement in heart rate (from ment in the ALT emerged as a potential safety concern, we were
90.5 at baseline to 73 bpm at follow up), in body temperature (from interested in understanding the potential impact on administration
37.5 to 36°C) and in CRP (from 108 to 22 mg/L) are indicative of route and posology on ALT increase. The repeated measures ANOVA
improving clinical conditions. An increment in alanine amionotras- revealed a non significant critical interaction Group x Time (F=2.18;
ferase (ALT) from 48 to 91 U/L has been observed. This is very com- p=0.14). Despite this, the Newman Keuls post hoc test revealed that,
monly observed as a result of tocilizumab and is clearly indicated in while the ALT results did not change significantly in the 324 s.c. mg
the patient information leaflet (PIL) [37]. group (from 50.1§40.9 to 74.0§46.3 U/L, p=0.15), ALT significantly
increase in patients in the 400 i.v. mg group (from 44.4§28.3 to
3.4. Effect of tocilizumab administration route 103.1§141.3, p=0.004).

We then considered only patients treated with tocilizumab (n=90) 4. Discussion

and we explored whether the two different administration routes
(400 mg i.v. or 324 mg s.c.) had an impact on patients outcome. Base- The current study describes the positive impact of a single low dose
line demographic and clinical characteristics of the two groups of of tocilizumab in addition to standard therapy in a relatively early phase
patients are reported in Table 5. Out of the seven deaths observed in of SARS-CoV-2 disease in a cohort of 90 patients compared to 68
Please cite this article as: N. De Rossi et al., Early use of low dose tocilizumab in patients with COVID-19: A retrospective cohort study with a
complete follow-up, EClinicalMedicine (2020), https://doi.org/10.1016/j.eclinm.2020.100459
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N. De Rossi et al. / EClinicalMedicine 00 (2020) 100459 5

Fig. 1. Kaplan-Meier survival curve for tocilizumab (blue line) and control (violet line) group. Analysis run using Group (tocilizumab vs controls) as factor; death as event and
time to death/discharge as time variable. Multivariate Hazard Ratio (H.R. for death: 0.057; 95% C.I = 0.017- 0.187, p < 0.001) is adjusted for baseline characteristics.

Table 2 ARDS [3]. A significant increase of white blood cells, in particular of neu-
Results from the Cox Proportional Hazard model. GROUP = main trophils, has been observed in patients with a severe disease, while a
variable (tocilizumab vs. controls); HR = Hazard Ratio; significant reduction of both CD8+ and CD4+ T lymphocytes cells was
CI = Confidence Intervals; CRP = C-Reactive Protein.
detected [38]. Among of immunologic biomarkers, the highest levels of
95% C.I. for HR IL-6 and serum ferritin were observed in severe subacute form of respi-
ratory disease and in non-survivors COVID-19 patients [9]; in the same
Variable HR Lower Upper p value
way, inflammatory cytokines as IL-2 and IFN-g show an high serum lev-
GROUP 0.057 0.017 0.187 <0.001 els in patient with severe course of SARS and MERS [5,39]. As cytokine
Age 1.069 1.026 1.114 0.001
storm has been proposed to have a pivotal role in organ injury in
Gender 1.727 0.797 3.743 0.166
Diabetes 3.272 1.477 7.245 0.003 COVID-19, tocilizumab has been suggested as a possible treatment in
Hypertension 1.634 0.710 3.758 0.248 SARS-CoV-2 infection. Indeed, anti-IL6 therapeutic strategy has been
Heart disease 3.001 1.422 6.332 0.004 shown to be effective in cytokine response syndrome (CRS) [40]. A rec-
CRP at admission 1.006 1.000 1.011 0.044 ommended protocol for tocilizumab used in COVID-19 [41], requires a
Time to hospitalization 1.001 0.913 1.098 0.978
first dose of 4-8 mg/kg and an additional infusion after 12 hours for
Respiratory support 1.728 1.141 2.619 0.010
patients with worsening or poor clinical response. This protocol, how-
ever, lead to inconsistent results. Previous literature indeed report small
patients treated with standard therapy only. All patients had laboratory or absent [20,21] effect of tocilizumab in a very small cohort of patients,
confirmed infection and were followed-up until the discharge from the making the results statistically unreliable. Additional studies provide
hospital or death. First and most important, this study found that the data of single arm of patients only (i.e. the control group was not avail-
risk of death for patients treated with tocilizumab is 94% lower than the able) [42-46], making the real efficacy of tocilizumab obscure. In the
one of patients treated with standard therapy only. Early treatment absence of specific treatment for COVID-19, the main objective of clini-
with a single low dose of tocilizumab is thus effective. Second, this effi- cians should be to promote virus clearance allowing immune system to
cacy profile is not related to the administration route, as the two groups over-ride viral infection, whereas preventing organ damage due to
(400 i.v. vs 324 s.c.) show the same survival rate. Third, the effect of toci- excessive inflammation. In this regard, early tocilizumab administration
lizumab on inflammatory indices is very quick and does not depend on was expected to be useful to modulate the cytokine storm and prevent
administration route. Finally, our study also revealed that low dose of the consolidation of lung damage, while the low dose was expected to
tocilizumab administration is safe, as no tocilizumab related infections be useful to reduce hyper inflammation without abolishing the immune
or safety concerns have been observed. Among COVID-19 patients, response to the virus [24]. In the current study, a single low dose of toci-
about 25% present severe complications including acute respiratory dis- lizumab (400 mg e.v. or 324 mg s.c., regardless of the patients’ weight)
tress syndrome (ARDS) with a rapid worsening of clinical conditions has been administered at the early stage of the respiratory failure. Our
leading to the need of mechanical or invasive ventilation to support study shows a higher survival rate in the treated group compared to the
respiratory functions [5]. Although the viral invasion and the direct patients who were treated with standard therapy only with a 94% treat-
cytopathic effect are critical for a worsening of the clinical course, there ment impact on the risk of death, fully supporting the efficacy of admin-
is evidence that a deranged immune response can be implicated in istration of low dose of tocilizumab early in the disease course.

Please cite this article as: N. De Rossi et al., Early use of low dose tocilizumab in patients with COVID-19: A retrospective cohort study with a
complete follow-up, EClinicalMedicine (2020), https://doi.org/10.1016/j.eclinm.2020.100459
JID: ECLINM
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6 N. De Rossi et al. / EClinicalMedicine 00 (2020) 100459

Fig. 2. Longitudinal data. Graphs show the longitudinal evolution of the body temperature and of the CRP (C Reactive Protein) after the hospital admission in the two groups of
patients. The first five days after admission to the hospital were reported, where Day 1 refers to the beginning of therapeutic intervention in both groups. The upper panel shows
the longitudinal body temperature and CRP data in tocilizumab and control groups, whereas the lower panel shows the longitudinal body temperature and CRP data in the tocilizu-
mab group divided in the two administration routes. Error bars denotes standard error of the mean.

In accordance with previous data [47], the current study also supports worse outcome Indeed, patients with diabetes have a 3.2 fold higher risk
that older age is a risk factor for death and that the probability to die of death compared to patients without diabetes, and patients with cardi-
increases by 6% for each year of age. Several reports also suggested that opathy have a 3 fold higher risk of death compared to patients without
concomitant chronic illness may have an impact on mortality rate [1,48]. cardiopathy [49,50]. In our cohort hypertension is not associated with an
The current results confirmed diabetes and cardiopathy as risk factors for increased risk of death but this could depend on the sample size.

Table 3
Respiratory data and Follow up data of the two groups. Numbers denotes row numbers (percentages) or median [interquar-
tile range]. SpO2 = peripheral capillary oxygen saturation; FiO2 = Fractional concentration of oxygen in inspired air;
CPAP = Continuous Positive Airway Pressure; Peep = positive end expiratory pressure.

Controls (n=68) Tocilizumab (n=90)

Respiratory support needed at hospital admision

1) No respiratory support 20 (29.4%) 18 (20%)
SpO2 (%) 95 [88 98] 93 [88 97]
2) Low flow cannula 13 (19.1%) 8 (8.8%)
SpO2 (%) 95 [92 97] 94 [92 98]
FiO2 (%) 28 [24 31] 31 [24 31]
3) High flow mask 24 (35.3%) 42 (46.6%)
SpO2 (%) 95 [91 98] 94 [88 100]
FiO2 (%) 60 [35 100] 60 [40-100]
4) CPAP 11 (16.2%) 22 (24.4%)
SpO2 (%) 94 [88 98] 95 [89 99]
FiO2 (%) 50 [40-60] 55 [40 100]
Peep (cm H2O) 13 [10 12.5] 13 [10 20]
Longitudinal follow up
Patients needing mechanical ventilation 6 (8.82%) 13 (14.4%)
Respiratory System Infections 4 (5.9%) 6 (6.6%)
Etiological agent for respiratory system infections Streptococcus Epidermidis (n=1) Mycoplasma Pneumoniae (n=2)
Clamydia Pneumoniae (n=1) Clamidya Pneumoniae (n=2)
Mycoplasma Pneumoniae (n=1) Staphilococcus Hominis (n=1)
Staphilococcus Hominis (n=1) Cytomegalovirus (n=1)
Pulmunary Embolism Not known 12 (13.3%)
Total deaths 34 (50%) 7 (7.7%)

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Table 4
Longitudinal clinical and laboratory results of patients treated with tocilizumab. Data are reported at baseline (before tocilizumab administration) and
at 5 days follow up. Number denotes mean (standard deviation) (a); median [interquartile range] (b). Statistical significance was evaluated using paired
sample t-test (a); Wilcoxon test (b). P value reports the associated p value (statistical significance p<0.05). Asterisk (*) denotes clinical significance.

Normative values Pre-tocilizumab 5 days post-tocilizumab Significance P value

treatment (n=81) treatment (n=81)

Clinical characteristics
Heart rate (bpm) a - 90 (15.0) 73 (13.0) 8.427 <0.001*
Systolic blood pressure (mm/Hg) a - 129 (22.) 131 (19.0) -0.458 0.648
Diastolic blood pressure (mm/Hg) a - 73 (11.0) 76 (10.0) -1.954 0.056
Temperature (°C)b <37 37.5 [36-40] 36.0 [36-37.5] -5.690 <0.001*
Laboratory Results
CRP (mg/L)a <0.5 108.4 (74.3) 22.2 (36.6) 9.950 <0.001*
Procalcitonin (ng/L)b <0.1 0.25 [<0.1 7.5] 0.1 [<0.01-0.56] -2.293 0.022
White blood cells (x103 per mL) a 4.00-10.80 6.9 (3.9) 7.4 (3.1) -0.790 0.432
Lymphocytes (x103 per mL) a 0.90-4.00 0.9 (0.4) 1.1 (0.5) -2.828 0.006
Neutrophils (x103 per mL) a 1.50-8.00 5.5 (3.7) 6.2 (5.6) -0.678 0.500
Platelets (x103 per mL) a 130-400 225 (106) 337 (282) -3.436 0.001
Glycemia (mg/dl)b 76-115 115 [76-314] 100.5 [64-323] -0.909 0.363
Urea (mg/dl) b 17-49 35 [16-143] 35 [16-88] -0.675 0. 500
Creatinine (mg/dl) a 0.60-1.00 0.9 (0.2) 0.8 (0.2) 7.108 <0.001
Sodium (mmol/L) a 136-145 136.5 (5.1) 140.1 (2.9) -4.707 <0.001
Potassium (mmoL/L) a 3.4-4.5 3.8 (0.5) 4.0 (0.5) -3.454 0.001
Chlorine (mmol/L) a 89-107 97.5 (3.9) 101.0 (9.1) -2.970 0.004
Bilirubin (mg/dL) a <1.20 0.5 (0.3) 0.5 (0.2) 1.622 0.115
Aspartate Transaminase -AST (U/L) a 18-34 58.6 (42.4) 58.9 (42.3) -0.057 0.995
Alanine amionotrasferase -ALT (U/L) a 10-35 48.6 (39.6) 91.2 (110.3) -3.322 0.002*
Gamma glutamyl trasferase- gGT (U/L) b 6-42 45 [11-360] 70 [8-609] -1.734 0.083
Alkaline phosphatase -ALP (U/L) a 44-107 65.1 (37.3) 67.0 (29.0) -0.373 0.711
Lactate dehydrogenase- LDH (U/L) a 135-225 358.6 (138.5) 324.7 (118.5) 0.415 0.681
Creatine kinase- CK (U/L) b 26-192 112 [11 878] 44 [7-1218] -3.176 0.001
Prothrombine time -Pt (sec) b 9.4-12.5 13.4 [10.8-22.2] 12.9 [11-15.8] -4.119 <0.001
Activated partial thromboplastine time- aPTT (sec) b 24-38 32.7 (4.0) 29.6 (5.4) 4.351 <0.001
International normalized ratio -INR c 0.9-1.2 1.2 [1.0-2.0] 1.2 [1.0-1.4] 0.504 0.674

Table 5
Demographic and clinical characteristics of the two groups of patients. Number denotes mean (standard
deviation) (a); raw number (percentages) (b); median [interquartile range] (c). Statistical significance was
evaluated using two independent sample t-test (a); chi square (b); Mann Whitney U test (c). CRP = C-Reac-
tive Protein.

324 mg s.c. (n=47) 400 mg i.v. (n=43) Significance P value

Age (years)a 66.8 (11.3) 58.7 (12.6) 3.182 0.002

Gender (males)b 34 (72.3%) 30 (69.7%) 0.072 0.788
Diabetes (yes) b 7 (14.9%) 7 (16.2%) 0.003 0.856
Hypertension (yes) b 24 (51%) 17 (39.5%) 1.203 0.273
Heart disease(yes) b 6 (12.7%) 5 (11.6%) 0.027 0.869
Time to hospitalization (days)a 10 (5.7) 8.1 (10.1) 1.068 0.289
CRP at admission (mg/L)a 109 (73.5) 134 (80.5) -1.550 0.125
Temperature at admission (°C)c 36 [36-39] 38 [36-40] 0.003
Procalcitonin (ng/L)c 0.19 [<0.1-5.2] 0.17 [<0.1-7.5] 0.822

The favorable outcome in the tocilizumab cohort is independent 324 mg. This elevation in ALT is known and expected during tocilizu-
from administration routes, as the survival rates and the respiratory mab therapy, is not associated with clinically relevant increases in
recovery are similar between patients receiving 400 mg i.v. and direct bilirubin and is not associated with clinical evidence of hepatic
patients receiving 324 mg s.c. The higher number of deaths observed insufficiency. Importantly, the PIL clearly indicate that this elevation
in the latter (6 deaths vs 1) is easily justified by the older average age did not result in permanent or clinically evident hepatic injury in
in the subcutaneously treated group compared to the intravenous clinical trials [37].
one. The equivalence between the two administration routes is fur- Critically, the treatment with a low dose of tocilizumab was safe.
ther supported by the the similar hospitalization time and by the During the follow-up period we did not observe any adverse drug
equally rapid pharmacological effect, as evidenced by the quick reactions; in particular, secondary infections or intestinal perfora-
decrease in CRP and body temperature analysis in both groups, in tions, both threatening complications of the administration of tocili-
accordance with previous studies [51]. These data confirm the phar- zumab [53,54], did not occur. Serum level of procalcitonin remained
macodynamic equivalence studies that analyzed the impact of tocili- stable and within the lower limits (<0.5 ng/mL), denoting the
zumab administration route and confirm what has already been absence of bacterial infections in our patients. These complications
found in the rheumatological field [28,52]. Despite the similar effect were instead observed using higher doses of tocilizumab in patients
of the two administration routes on tocilizumab efficacy, the current with COVID-19 [42].
data also provide preliminary support that the potential hepatotoxic- This study presented some limitations. The most important one
ity might be dose dependent, being the observed increment in ALT refers to the lack of randomization resulting in not matched groups.
higher in patients receiving 400 mg compared to patients receiving Indeed, patients treated with standard care were older and with
Please cite this article as: N. De Rossi et al., Early use of low dose tocilizumab in patients with COVID-19: A retrospective cohort study with a
complete follow-up, EClinicalMedicine (2020), https://doi.org/10.1016/j.eclinm.2020.100459
JID: ECLINM
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8 N. De Rossi et al. / EClinicalMedicine 00 (2020) 100459

higher prevalence of comorbidities compared to patients treated with Funding

tocilizumab. Contrarily, patients treated with tocilizumab were
admitted to the hospital later during the disease course, as supported This research received no funds.
by the longer time elapsed from symptoms onset to hospitalization.
Despite we are confident that the multivariate approach applied in Supplementary materials
the current paper (Cox proportional hazard model) removed the
potential biasing effect of these unmatched variables on the primary Supplementary material associated with this article can be found,
results, the current data need to be further supported. A second in the online version, at doi:10.1016/j.eclinm.2020.100459.
important limitation is that an additional control group, including
patients treated with tocilizumab during the late stage of respiratory References
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complete follow-up, EClinicalMedicine (2020), https://doi.org/10.1016/j.eclinm.2020.100459
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Please cite this article as: N. De Rossi et al., Early use of low dose tocilizumab in patients with COVID-19: A retrospective cohort study with a
complete follow-up, EClinicalMedicine (2020), https://doi.org/10.1016/j.eclinm.2020.100459