The effect of tocilizumab administration on inflammatory markers in COVID-19 patients

Background The COVID-19 outbreak was declared a worldwide emergency as a result of its rapid spread. The number of people infected with COVID-19 is increasing rapidly around the world, and pneumonia can develop in COVID-19 cases. The monoclonal antibody tocilizumab blocks the interleukin-6 receptor, which in turn reduces inflammation. Aim of the work The study aims to determine how tocilizumab affects inflammatory markers, laboratory indices, and oxygen therapy. Subjects and methods This retrospective observational study aimed to assess the effect of tocilizumab on inflammatory markers, laboratory parameters, and short-term outcomes in COVID-19 cases. Data was collected from 55 patients with COVID-19 who tested positive for SARS-CoV-2 using PCR. These patients were admitted to Ain Shams University Specialized Hospi-tal—Obour between June 1, 2021, and May 31, 2022. Results After tocilizumab administration, C-reactive protein levels decreased significantly, but there was no statistically significant change in hemoglobin, serum ferritin, or D-dimer levels. Following tocilizumab administration, the leukocyte counts, and platelet count increased significantly. There was a significant correlation between the presence of comorbidities in the studied patients (e.g., heart failure, post-renal transplantation, and hepatitis C virus) and the risk of mortality. The study’s final result showed a significant decrease in platelet count in dead patients compared to discharged patients after receiving tocilizumab. Regarding oxygen therapy following tocilizumab administration, the use of face masks and non-rebreather facemasks was high in dead patients, while nasal prong usage was high in discharged patients. After receiving tocilizumab, there was an increase in the mean liters of oxygen required in dead patients compared to discharged patients. Conclusion After administration of tocilizumab in COVID-19 hospitalized patients who have progressing disease, there was highly and significantly decrease in CRP level with no statistically significant alteration in the levels of hemoglobin, serum ferritin, and D-dimer and an increase in TLC and platelets was observed. Following tocilizumab administration, there was a decrease in oxygen demands, an improvement in oxygen therapy and oxygen saturation. Tocilizumab is a recommended therapy option.


Introduction
The coronavirus is an outbreak of a newly discovered virus that spreads quickly, resulting in systemic or local pneumonia consequences worldwide.The WHO declared the outbreak a pandemic on March 11, 2020 [1].Some inflammatory markers, including procalcitonin, C-reactive protein, ferritin, and D-dimer, have been identified in COVID-19 cases in research findings [2].
Leukocyte-mediated blood vessel destruction can occur during the active stages of some inflammatory conditions, leading to vasculitis.Destruction of blood vessel walls may also result in the activation of the coagulation process, the development of a thrombus, and the release of D-dimer into the bloodstream.D-dimer levels could be a good indicator of inflammation and damage to the blood vessels [3].
D-dimer is a metabolite of fibrin that is produced by a plasma fibrinolytic enzyme [4].The elevated D-dimer concentration can indicate secondary fibrinolysis [5].An increase in ferritin levels may contribute to inflammation during the onset of the disease [6].The cytokine storm causes macrophage activation syndrome (MAS), which in turn contributes to acute respiratory distress syndrome and multiple organ failure [7].Serum levels of cytokines, particularly interleukin-6 (IL-6), are elevated in critically ill COVID-19 patients [8,9].Reducing mortality appears to be feasible through reducing the cytokine storm [10].The monoclonal antibody tocilizumab suppresses signal transduction by targeting the interleukin-6 receptor.This antibody is used for the treatment of infection-induced cytokine storm [11,12].Tocilizumab is administered when there is an increase in the requirement for oxygen support, progression of CT chest, an increase in C-reactive protein, D-dimer, and ferritin, and a decline in the percentage of lymphocytes [13].

Aim of the work
To determine the effect of tocilizumab on inflammatory markers, laboratory indices, and short-term outcomes in patients with COVID-19.

Methods
This retrospective observational study aims to determine tocilizumab's effect on markers of inflammation, laboratory parameters, and short-term outcomes in cases with COVID-19.This was conducted using data from 55 patients with COVID-19 who tested positive for SARS-CoV-2 and were hospitalized at Ain Shams University Specialized Hospital-Obour between June 1, 2021, and May 31, 2022.Each patient received tocilizumab at a dose of 4-8mg/kg diluted in 100 milliliters of isotonic saline infused over 1 hour, followed by a second dose of 4 mg/ kg diluted in 100 milliliters of isotonic saline infused over 1 hour within 24-48 hours of the first dose.

Inclusion criteria:
-PCR positive for SARS-CoV-2 -Cases recently hospitalized (within the first 3 days of admission) and admitted to the intensive care unit (ICU) within the previous 24 hours, as well as those not admitted to the ICU but with rapidly increasing oxygen needs, regardless of gender.

Ethical consideration
The present study was done after the approval of the Research Ethics Committee of the Faculty of Medicine, Ain Shams University, with reference number FWA 00017585 and approval number FMASU MD 204/2021 on October 3, 2021.The study reviewed data from the files of patients with COVID-19 infection who tested positive for SARS-CoV-2 using PCR.

Study intervention
This retrospective observational study was done using data from 55 cases of COVID-19 with a positive PCR test for SARS-CoV-2 who were hospitalized at Ain Shams University Specialized Hospital-Obour between June 1, 2021, and May 31, 2022, and received tocilizumab as a part of their treatment.Patients who met any of the exclusion criteria were not included in the data collection.

All patients received
Full medical history and clinical examination, laboratory investigations (oxygen saturation, complete blood  Patients who meet these criteria: respiratory rate > 30 breaths per minute, oxygen saturation < 92% at room air, PaO2/FiO2 ratio < 300, and chest radiology showing > 50% lesion or progressive lesion within 24 to 48 hours, will be admitted to the intermediate care unit. Treatment will include antivirals: remdesivir or lopinavir/ritonavir; anticoagulant: prophylactic anticoagulation if d-dimer between 500 ng/ml and 1000 ng/ml; therapeutic anticoagulation if d-dimer > 1000 ng/ml or if severe hypoxia; anti-inflammatory: steroids (dexamethasone 6 mg or methylprednisolone 1 mg/kg/24 hours), tocilizumab 4-8 mg/kg/day followed by a second dose of 4 mg/kg within 24-48 hours from the first dose after the failure of steroid therapy to improve the case for 24 hours; convalescent plasma: before day 12 (under clinical trial) (after scientific committee approval); and antibiotics.

b)b)b)b)b) Critically ill
Patients with the following criteria: oxygen saturation <92%, or respiratory rate >30 breaths per minute, or PaO2/FiO2 ratio < 200 despite oxygen therapy will be admitted to the intensive care unit.
Treatment will include: antivirals (remdesivir or lopinavir/ritonavir); anticoagulant: therapeutic anticoagulation; anti-inflammatory: steroids (methylprednisolone (2 mg/ kg) or its equivalent, tocilizumab (4-8 mg/kg/day), followed by a second dose of 4 mg/kg within 24-48 hours from the first dose after the failure of steroid therapy to improve the case for 24 hours; and antibiotics.
Non-invasive ventilation or High Flow Nasal Cannula: patients who are conscious with minimal secretions, hypoxia (oxygen saturation less than 90%) on oxygen therapy, or PaCO2 above 40 mmHg as long as the pH is 7.3 or higher, a non-invasive ventilation trial shall be short with arterial blood gas measurement after 30 minutes.Any deterioration in arterial blood gases from baseline, oxygen saturation, or consciousness level shift to invasive mechanical ventilation.CPAP gradually increased from 5-10 cmH2O and pressure support from 10-15 cmH2O.HFNC can be an alternative to NIV.
Invasive Mechanical Ventilation: use personal protective equipment, especially eye goggles, during intubation and avoid bagging.This type of ventilation is indicated by failed non-invasive ventilation, or not available or practical; a PaO2 level less than 60 mmHg despite oxygen supplementation; progressive hypercapnia; respiratory acidosis (pH < 7.30); septic shock, whether progressive or refractory; and an alteration in consciousness level (Glasgow Coma Scale ≤ 8).
All patients received tocilizumab 8 mg/kg diluted in 100 ml isotonic saline infused over one hour followed by a second dose of 4 mg/kg diluted in 100 ml isotonic saline infused over one hour, within 24-48 hours from the first dose.
Patients will be assessed regarding clinical manifestations, markers of inflammation, and oxygenation 48 hours after the administration of tocilizumab.

Statistical analysis
The revised and coded data was submitted to the Statistical Package for Social Science (IBM SPSS) version 23.The quantitative data were presented as mean, standard deviations, and ranges when data were found to be parametric and median and inter-quartile range (IQR) when data were found to be non-parametric.So, the p-value was considered significant as follows: P>0.05: non-significant (NS), P<0.05: significant (S), and P<0.01: highly significant (HS) [14].

Results
Fifty-five cases were admitted to Ain Shams University Specialized Hospital-Obour in the period between June 1, 2021, and May 31, 2022, with ages ranging from 24 to   1 and 2).
There was a significant increase in the total leukocytic count and platelets after taking tocilizumab compared to before.Additionally, there was a significant decline in C-reactive protein levels after taking tocilizumab.However, there was no statistically significant alteration in the levels of hemoglobin, serum ferritin, and D-dimer after tocilizumab (Table 3).
There was a significant improvement in oxygen saturation (%) after taking tocilizumab compared to before, and there was a decrease in the flow of oxygen liters used after taking tocilizumab.Furthermore, the percentage of patients on nasal oxygen therapy increased, while the percentage of patients on a face mask decreased after tocilizumab compared to before (Table 4).
There was a statistically significant correlation between the risk of mortality and patients with comorbidities, including heart failure, post-renal transplantation, and the hepatitis C virus (Table 5).
There was a highly significant relationship found between the final outcome of the studied patients and oxygen therapy after tocilizumab was administered.The percentage of patients using a face mask or nonrebreather facemask was higher in the dead patients, while the percentage of patients using nasal prongs was higher in the discharged patients.There was a significant increase in the mean liters of oxygen used in the dead patients (7±3.46)compared to the discharged patients (5.0 ± 2.48).No significant relationship was identified between the outcome of the studied patients and oxygen saturation after tocilizumab was administered (Table 6).There was a significant relationship between the final outcome of the studied cases and platelets, with a decrease in platelet count observed after tocilizumab in the dead patients compared to the discharged patients.There was no significant relationship between the final outcome of the studied cases and other laboratory data, including total leukocyte count, hemoglobin, C-reactive protein, serum ferritin, and D-dimer, after tocilizumab was administered (Table 7) and (Fig. 1).

Discussion
A coronavirus is an outbreak of a newly discovered virus that spreads quickly, resulting in systemic or local pneumonia consequences worldwide.The WHO declared the outbreak to be a pandemic on March 11, 2020 [1].Tocilizumab is administered when there is a rising oxygen demand, progression of CT chest, an increase in C-reactive protein, D-dimer, and ferritin, and a decline in the percentage of lymphocytes [13].The purpose of this study is to describe the efficacy of tocilizumab as a beneficial treatment in patients with COVID-19 pneumonia using clinical, laboratory, and radiographic findings.The correlation between numerous inflammatory markers and the benefits of tocilizumab in COVID-19 hospitalized cases was shown in the study.
Tocilizumab delivery resulted in a significant decline in C-reactive protein levels when compared to the pretreatment level.This was in agreement with Broman et al. [15] in their study.Additionally, a study done by Conrozier et al. [16] found that CRP dropped dramatically after tocilizumab administration.Similar results were matched with Amin et al. [17] Consistent with this finding, Keske et al. [13] found that tocilizumab significantly reduced the CRP level after its administration.This didn't match with Tsai et al. [18], who observed that there was an increased CRP level after its administration, which has been attributed to a discrepancy in the tocilizumab dose.Of the 66 patients who took tocilizumab, a dose of 800 mg was provided to 10 patients (15.1%), a dose of 600 mg was provided to 3 patients (4.5%), and a dose of 400 mg was provided to 53 patients (80.3%).A second dose of tocilizumab has been prescribed to four patients.Hence, there was a possibility that the patients were vulnerable to becoming underdosed.
There was no significant alteration in hemoglobin level after tocilizumab administration in the current study.Also, the work done by Conrozier et al. [16] agreed with  our study.Also, this was in agreement with Amin et al. [17], who reported that the hemoglobin level was not significantly changed following tocilizumab therapy.This wasn't matching with Alattar et al. [19] who observed the presence of anemia after tocilizumab administration, which is commonly associated with a concomitant agent (ribavirin).
There was a statistically significant elevation in TLC after taking tocilizumab.Comparably, Mohamed et al. [20] found an elevation in total leucocytic count following tocilizumab administration in 100 patients due to secondary bacterial infection.Discordantly, Conrozier et al. [16] observed that there was no significant change in leucocyte count after giving tocilizumab to COVID-19 patients, and this may be due to the small size of the sample compared to the current study.Contrary to the current study, Grech et al. [21] showed no significant change in total leucocyte count after giving tocilizumab in the study, and this may be due to the unmentioned comorbidities, whether they were present or not, and the difference in the distribution of the sample size of the study in which 50 patients received tocilizumab, of which 41 patients were males (82%), ranging in age from 60 to 72 years, while in our study, 55 patients received tocilizumab, of which 31 patients were males (56.4%), ranging in age from 24 to 86 years.
There was no statistically significant change in D-dimer after tocilizumab.Similar results were matched with Conrozier et al. [16].Kaminski et al. [22] also described a non-significant change in serum levels of D-dimer after tocilizumab administration.On the contrary, Toniati et al. [23] reported that the D-dimer levels increased after tocilizumab administration, suggesting that tocilizumab partially acts on the process of inflammatory reactions while having a minor or no impact on active coagulation.
There was no statistically significant change in the levels of serum ferritin after tocilizumab administration.This was in accordance with Szabo et al. [24], who reported a non-statistically significant change in the ferritin level after tocilizumab in 30 cases of COVID-19.This was also consistent with Kaminski et al. [22].Opposingly, Toniati et al. [23] found that the ferritin serum levels decreased toward the normal range.This difference from the current study may be due to the large number of patients (100 patients).This wasn't matching with Akdeniz et al. [25], who observed a significant decrease in serum ferritin post tocilizumab administration, which may be due to the number of patients (92) and the fact that the number of patients with no comorbidities was 22 (23.9%) while in the current study was 8 (14.5%).The number of patients with hypertension and diabetes was 25 (27.2 %) and 13 (14.1%)respectively, while in the current study cases with hypertension and diabetes were 35(63.6 %) and 29 (52.7%)respectively.
There was a statistically significant rise in the levels of platelets after taking tocilizumab compared to before in the current study.This was matching with Conrozier et al. [16].These results imply that COVID-19 has been associated with increased production of large, immature platelets because megakaryocytes respond to increased platelet consumption.Remarkably, COVID-19 appears to correlate with a rise in the number of immature platelets, even at normal platelet levels [26].
There was a statistically significant increase in oxygen saturation in the present study.This significant improvement was matching with Yılmaz et al. [34].This was in accordance with Sharma et al. [27], in which a reduction in the oxygen requirement and a significant improvement in oxygen saturation after tocilizumab were found in the study.Hassan et al. [28] stated the same findings.
The Platelet count dropped significantly in the dead patients rather than the discharged patients.This was in matching with a study done by De Ardanaz et al. [29], in which the study showed that the platelets were lower in the dead cases compared to those that remained alive.
In the current study, post-renal transplantation, the hepatitis C virus, and heart failure were the comorbidities associated with an elevated risk of death.This was matched in a study done by De Ardanaz et al. [29], in which immunosuppression resulted in an elevated risk of mortality.
In the context of immunosuppression, no association with mortality was found in a large study of 1305 cases done by Imam et al. [30] in Michigan, United States, in which older age and comorbidities like hypertension, diabetes, and chronic kidney disease are independent mortality predictors.
The mortality rates tend to be high among patients who have undergone solid organ transplants, and autoimmune disease patients raise the risk of being infected with COVID-19 [31,32].
Obesity has been attributed to an increased risk of death.This was done in a study by Demeulemeester et al. [33].Nevertheless, because we obtained the data retrospectively from medical records in which obesity was not documented, we were unable to figure out how it affected the death risk estimation.
Our limitations were the small size of the population, the lack of randomization, and the fact that it was conducted in a single center.Additionally, the patients' comorbidities were not equally distributed.Because the study was conducted retrospectively, the impact of the time frame of tocilizumab delivery on patients' prognosis and death rate was unavailable.It is thought to be challenging to use laboratory parameters to define disease activity.The absence of a control group determined the adverse effects, the length of hospital stay, and death.Furthermore, our results should be evaluated cautiously, despite the fact that tocilizumab has a good response.
To further understand how tocilizumab affects cytokine release syndrome, the outcomes of ongoing studies and additional randomized large studies are required [34].

Conclusion
Following tocilizumab administration, there was a significant decrease in CRP level and a decrease in oxygen demand.Tocilizumab is a promising treatment for COVID-19 hospitalized patients with progressive disease and high oxygen requirements.Early assessment of disease severity using clinical, laboratory and radiological data will improve its outcome.

Table 1
Demographic data among the studied patients

Table 5
Relation between final outcome of the studied patients and their comorbidities P-value > 0.05: Non significant; P-value < 0.05: Significant; P-value < 0.01: Highly significant DM Diabetes mellitus, HTN Hypertension, HF Heart failure, DVT Deep vein thrombosis, SLE Systemic lupus erythematosus, CKD Chronic kidney disease, CML Chronic myeloid leukemia, HCV Hepatitis C virus, ISHD Ischemic heart disease, CLL Chronic lymphocytic leukemia * Chi-square test

Table 6
Relation between final outcome of the studied patients and oxygen therapy and oxygen saturation (%) after tocilizumab taken P-value > 0.05: Non significant; P-value < 0.05: Significant; P-value < 0.01: Highly significant * Chi-square test; •: Independent t-test