Efficacy and safety of Tocilizumab, plasmapheresis and their combination in severe COVID-19: A randomized clinical trial

Abstract

Background

This study sought to evaluate and compare the effectiveness of plasmapheresis, Tocilizumab, and Tocilizumab with plasmapheresis treatment on the removal of inflammatory cytokines and improvement clinically of patients with severe COVID-19 in Intensive Care Units (ICU) due to the association between increased cytokine release and the severity of COVID-19.

Methods

This clinical trial study was conducted in three treatment arms in Iran. All patients received standard care and randomization into one of three treatment groups; Tocilizumab (TCZ) alone, plasmapheresis alone, or a combination of Tocilizumab and plasmapheresis. Demographics, clinical evaluation, oxygenation status, laboratory tests and imaging data were evaluated in the three groups and re-checked 48 h after the end of treatment trials. Primary outcomes were oxygenation status, the need for mechanical ventilation and the rate of death.

Results

Ninety-four patients were included in the trial after meeting the eligibility requirements. Twenty-eight patients received Tocilizumab alone, 33 had plasmapheresis alone, and 33 received both Tocilizumab and plasmapheresis. Baseline characteristics did not differ between three groups that included demographic, clinical and laboratory parameters. Following therapy, there was no difference between the three groups for CRP, ferritin, d-dimer, IL-6, pro-calcitonin and neutrophil to lymphocyte ratio (NLR) (P > 0.05). While a significant reduction was found in CRP levels within each group (32.04 ± 42.43 to 17.40 ± 38.11, 51.28 ± 40.96 to 26.36 ± 33.07 and 41.20 ± 34.27 to 21.56 ± 24.96 in the tocilizumab, plasmapheresis, and combined group, respectively) (p < 0.05), procalcitonin levels were elevated significantly in the Tocilizumab group (0.28 ± 0.09 to 0.37 ± 0.11) (p < 0.05). Clinically there was no difference between the three groups following treatment for O₂ saturation levels with supplementary oxygen at discharge, endotracheal intubation rate, use of NIVPP, mortality, mean hospital and ICU length of stay (p > 0.05).

Conclusion

Study results showed that the reduction of serum inflammatory markers, the rate of intubation and therapeutic complications including death were no different between the three groups; however, CRP levels were significantly reduced in all three groups, indicating that the interventions reduced inflammation likely through a reduction in the cytokine storm, though clinical outcomes were unaffected.

1. Introduction

Coronavirus disease-2019 (COVID-19) was first diagnosed in December 2019 and quickly became a pandemic as it spread globally [1], [2]. Patients with this condition may present with a variety of clinical symptoms, ranging from asymptomatic or moderate indications of acute upper respiratory tract infection to highly serious and life-threatening symptoms including acute respiratory distress syndrome (ARDS) and multi-organ failure [3], [4]. According to studies, 3 to 5 percent of patients need admission to intensive care units (ICU) [5]. Pro-inflammatory cytokine production is increased in a significant number of ICU patients, resulting in cytokine storm and multi-organ failure [1], [5], [6], [7], [8]. The primary source of pro-inflammatory cytokines such as interferon gamma (IFN-γ), interleukin-6 (IL-6), interleukin-12 (IL-12), tumor necrosis factor (TNF), Interleukin-1 receptor antagonist (IL-1Ra), and C-X-C motif chemokine ligand 10 (CXCL10) is macrophage activation, which initially occurs in the lungs and then spreads throughout the body [9], [10]. An escalating inflammatory response that causes thrombotic events and bleeding in the lungs and other organs, activates other systems such the coagulation cascade [10], [11], [12], [13], [14]. This multi-organ injury is now known as a critical factor in the pathogenesis clinical impact of COVID-19. Inhibition of the inflammatory cascades in patients is an important strategy to treat severe disease and prevent the increased mortality [10], [15], [16].

Based on the effect of cytokines on the severity of this disease, Tocilizumab may be effective in the treatment of severe cases. Tocilizumab a recombinant human monoclonal antibody that specifically acts by inhibiting the IL-6 receptor, but its role in patients with COVID-19 has not been proven [17]. However, it has been shown that treatment with anti-inflammatory drugs, such as Dexamethasone reduces the risk of death in patients with COVID-19, who are on respiratory care. This finding suggests that treatments targeting the cytokines involved in the inflammatory cascades may play an important role to reduce severe inflammation associated with lung damage. Considering the association of high levels of IL-6 and the severity of COVID-19, it would suggest that inhibition of IL-6 could be a promising treatment for COVID-19 [16].

Plasmapheresis reduces inflammatory factors in the blood and may be considered for the treatment of cytokine storm. Plasmapheresis has been used for previous Corona virus strains, including severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) and in septic shock [18], [19], [20]. Plasmapheresis is an extracorporeal treatment that removes abnormal substances, especially larger molecules, from the blood. There are two main methods for plasmapheresis that are centrifuge or membrane filtration and apheresis that uses centrifugation: the main advantage of this method is that there are no limits on the size of filtration molecules. The alternative method is membrane filtration plasmapheresis that is limited by molecule size limited to the size of filter pores, with the disadvantage that this method may cause activation of complexes and leukocytes by artificial membranes, and there is the need to insert a central catheter to obtain an adequate blood flow. Nowadays, plasmapheresis is a treatment method that plays a role in the treatment of diseases, such as myasthenia gravis, Guillain-Barre syndrome and thrombotic microangiopathy. Therefore, this treatment may reduce the hyper-inflammatory response by removing pro-inflammatory cytokines and may be effective to treat patients with COVID-19 disease [21].

Tocilizumab or plasmapheresis are both clinically used to treat severe cases of COVID-19 patients hospitalized in intensive care units, but their efficacy and possible complications have not been accurately studied. Therefore, this study aimed to compare the efficacy and side effects of plasmapheresis alone, Tocilizumab alone or Tocilizumab in combination with plasmapheresis in severe cases with COVID-19 admitted to ICU.

2. Methods

This was a randomized clinical trial that was conducted in 2021 in patients with COVID-19 hospitalized in the intensive care unit of Yazd University of Medical Sciences, between 22/05/2021 and 13/12/2021. All procedures were performed in accordance with relevant guidelines and regulations. Based on a table of random numbers with an allocation ratio of 1:1:1, patients, were divided among the 3 arms. Blinding the groups was not feasible given the constraints of the study. Inclusion criteria were ICU patients between the ages of 18 and 65, serum CRP and interleukin-6 levels twice normal or higher; O2 saturation < 90 % without the use of supplemental oxygen; respiratory rate (RR) >24; no clinical improvement after at least 96 h of standard treatment. COVID-19 was diagnosed through PCR (polymerase chain reaction) of nasopharyngeal secretions. Exclusion criteria included a history of drug allergy to Tocilizumab; platelets below 50,000; absolute neutrophilic count (ANC) <500; any clinical and imaging suspicion of bacterial, fungal and viral (non-corona) infections; serum pro-calcitonin level above 0.5 ng/dl; any clinical suspicion of the possibility of gastrointestinal obstruction, renal failure or hepatic insufficiency with Child-Paugh category C; pregnancy and lactation; primary endotracheal intubation or the need to use NIPPV (Non Invasive Positive Pressure Ventilation) on arrival in the ICU; patient choice not to participate.

In addition to routine treatment, patients were allocated to one of three groups of Tocilizumab alone, plasmapheresis alone, or Tocilizumab with plasmapheresis. Patients underwent interventional therapy as follows: 1. Patients in the Tocilizumab group received two doses of 400 mg Tocilizumab (24 h interval). 2. Patients in plasmapheresis group underwent three sessions of plasmapheresis every 48 h. 3. Patients in the Tocilizumab & plasmapheresis group, after 48 h of receiving two doses of 400 mg Tocilizumab, underwent plasmapheresis three times every 48 h. Routine treatment included 4 mg dexamethasone three times a day intravenously, 2 g vitamin C intravenously, prophylaxis dose 5000 IU of UFH Three times a day, vitamin-D3 at a dose of 2000–4000 IU daily and Remdesivir (200 mg on the first day and 100 mg on days 2–5).

At baseline demographic, clinical, initial oxygenation and respiratory status, laboratory tests and imaging were determined in all studied groups. Laboratory markers were ESR (Erythrocyte Sedimentation Rate), CRP (C-Reactive Protein), pro-calcitonin (PCT), d-Dimer, ferritin, and serum levels of interleukin-6, which were re-checked for patients 48 h after completion of the trials. The severity of lung involvement in CT scan was assessed according to Fleischner Society glossary scoring system that was based on lobar involvement of each lung, scoring from 0 to 5. The final score of lung involvement was made from the total involvement of all lobes of the lungs, which ranged from 0 to 25. Clinical manifestations included: status of oxygenation during admission and discharge (O2 saturation with and without supplemental oxygen), mortality rate, the need for invasive mechanical ventilation, mean hospital and ICU length of stay and side effects of treatment, were evaluated in the study groups.

This study was reviewed and approved by the local ethics committee of Yazd University of Medical Sciences (IR.SSU.MEDICINE.REC.1400.023) and performed in accordance with the Declaration of Helsinki and Good Clinical Practice Guidelines. The study was registered on Iranian Registry of Clinical Trials (IRCT20220123053807N1 (29/01/2022). All patients gave written informed consent before enrollment into the study.

3. Statistics

Based on an effect size of 0.8, the probability level of the first and second type of error 5 % and 10 % respectively, the sample size in each group was calculated to be 27 people per group that was increased to 32 people to account for a 20 % dropout rate. The sample size was performed using he G * Power software.

SPSS 22 for windows was used to do the statistical analysis. The mean, SD and frequency of the quantitative and qualitative variables determined. Analysis of variance was performed to assess the efficacy across the three groups, and paired t-tests were employed to examine the effectiveness of treatment approaches (with normal data scattering) in each group before and after the intervention. A significant difference was defined as p < 0.05.

4. Result

94 patients were enrolled into this investigation between 22.05.2021 and 13.12.2021; 28 patients in the Tocilizumab group, 33 patients receiving plasmapheresis, and 33 patients receiving both Tocilizumab and plasmapheresis. A CONSORT flow diagram of the study is presented in Fig. 1 .

Fig. 1.

CONSORT flow diagram.

Table 1 displays the baseline characteristics of the patients in each group are shown in Table 1 with no difference between groups.

Table 1.

Demographic, clinical and imaging characteristics of the study groups.

Variable		Tocilizumab(N = 28)	Plasmapheresis(N = 33)	Plasmapheresis & Tocilizumab (N = 33)	p. value
age	Mean (SD)	53.71(11.13)	57.33(12.57)	51.21(10.86)	0.10
Sex, F (%)	male	19(67.9)	18(54.5)	22(66.7)	0.48
	female	9(32.1)	15(45.5)	11(33.3)
BMI, F (%)	<30	13(46.4)	19(57.6)	19(57.6)	0.61
	>30	15(53.6)	14(42.4)	14(42.4)
O2 saturation ranges at baseline	Mean (SD)	71.07(11.30)	73.85(11.65)	74.64(10.93)	0.30
Comorbidities, F (%)
Smoking	yes	8(28.6)	10(30.3)	21(36.4)	0.76
Opium		9(32.1)	12(36.4)	8(24.2)	0.56
HTN		14(50)	18(54.5)	16(48.5)	0. 88
DM		12(42.9)	13(39.4)	7(21.2)	0.15
IHD		8(28.6)	5(15.2)	3(9.1)	0.12
CKD		3(10.7)	3(9.1)	2(6.1)	0.80
COPD		5(17.9)	1(3)	2(6.1)	0.10
CVA		0	1(3)	1(3)	0.65
DLP		3(10.7)	4(12.1)	6(18.2)	0.66
Brain. hemorrhage		0	0	1(3)	0.39
Cancer		1(3.6)	1(3)	0	0.57
Hypothyroidism		4(14.3)	1(3)	1(3)	0.12
Seizure		0	0	1(3)	0.39
Others*		3(10.7)	4(12.1)	2(6.1)	0.57
Drug History, F (%)
ARB/ACEI	yes	12(42.9)	15(45.4)	15(45.4)	0.57
Insulin		11(39.2)	10(30.3)	7(21.2)	0.51
Biguanide		5(17.9)	4(12.1)	4(12.1)	0.74
Levothyroxine		4(14.3)	1(3)	1(3)	0.12
Statin		10(35.7)	11(33.3)	7(21.2)	0.17
Imaging Result at baseline
Score of lung involvement at baseline	Mean (SD)	16.11(3.42)	16.03(4.16)	17.85(3.70)	0.10
percentage of lung involvement at baseline	Mean (SD)	59.85(19.33)	58.48(19.82)	68.24(19.50)	0.10

N: Number; SD: Standard Deviation; F: Frequency; BMI: Body Mass Index; DM: Diabetes Mellitus; HTN: Hypertension; IHD: Ischemic Heart Disease; DLP: Dyslipidemia; COPD: Chronic Obstructive Pulmonary Disease; CVA: Cerebrovascular accident; O2Sat: Oxygen Saturation; ACEI: Angiotensin-converting enzyme inhibitor; ARB: Angiotensin receptor blockers

*Others: syndrome down, Wegener’s syndrome, Rheumatoid arteritis, Multiple sclerosis, lymphangioleiomyomatosis (LAM).

Changes in blood levels of inflammatory markers and interleukin 6 at baseline and after intervention were performed that showed no difference between groups for any of the parameters from the ANOVA analysis for changes in CRP, ferritin, d-Dimer, IL-6, PCT, and NLR. Within each group, CRP levels fell significantly following the intervention (Table 2 ).

Table 2.

Comparison of laboratory tests and complication before and after the intervention in the study groups.

variable	group	Pre. intervention	Post. intervention	*P-value
		Mean(SD)	Mean(SD)
CRP	Tocilizumab(N = 28)	32.04 (42.43)	17.40 (38.11)	0.002
	Plasmapheresis(N = 33)	51.28 (40.96)	26.36 (33.07)	<0.0001
	Plasmapheresis & Tocilizumab (N = 33)	41.20 (34.27)	21.56 (24.96)	0.001
	**P-value	0.166	0.555
Ferritin	Tocilizumab	559.86 (459.46)	737.62(752.03)	0.24
	Plasmapheresis	979.23 (916.32)	793.92(764.32)	<0.0001
	Plasmapheresis & Tocilizumab	752.30 (699.02)	536.62(428.12)	0.003
	**P-value	0.085	0.260
d-Dimer	Tocilizumab	671.01 (796.04)	536.03 (630.65)	0.14
	Plasmapheresis	299.39 (451.32)	294.29 (364.91)	0.92
	Plasmapheresis & Tocilizumab	478.82 (765.09)	427.99 (568.43)	0.52
	**P-value	0.111	0.206
IL-6	Tocilizumab	64.87(147.50)	129.27 (194.75)	0.01
	Plasmapheresis	103.79(138.72)	120.22(175.81)	0.22
	Plasmapheresis & Tocilizumab	96.24 (119.26)	113.12 (178.43)	0.436
	**P-value	0.503	0.942
Pro-calcitonin	Tocilizumab	0.28 (0.09)	0.37 (0.11)	<0.0001
	Plasmapheresis	0.29 (0.12)	0.29(0.11)	0.92
	Plasmapheresis & Tocilizumab	0.23 (0.11)	0.29(0.19)	0.09
	**P-value	0.06	0.116
NLR	Tocilizumab	12.32(7.61)	14.06(9.95)	0.38
	Plasmapheresis	11.65(10.99)	16.83(14.30)	0.08
	Plasmapheresis & Tocilizumab	8.34(6.49)	14.08(12.22)	0.01
	**P-value	0.151	0.592

*Paired t-test, ** Analysis of variance (ANOVA).

Clinical comparisons between the three groups were made based on in-hospital mortality, treatment side effects, and oxygenation status upon discharge. There was no difference in the duration of hospital and ICU stay, the mortality rate, the rate of endotracheal intubation, NIVPP rate or the arterial oxygen saturation at discharge between the three groups. There was no difference between the three groups, for bacterial secondary infection, gastrointestinal (GI) bleeding, or vascular thrombosis, being the most common major problems seen in patients (Table 3 ). Infection seen for the Tocilizumab group (2 positive blood cultures, 3 positive sputum cultures), for the plasmapheresis group (2 positive blood cultures, 8 positive sputum cultures) and for the plasmapheresis & Tocilizumab group (1 positive blood cultures, 5 positive sputum cultures).

Table 3.

Comparison of Outcomes & Complications before and after the intervention in the study groups.

Variable	Tocilizumab	Plasmapheresis	Plasmapheresis & Tocilizumab	p-value
Hospital length of stay, Mean (SD)	12.29(4.71)	13.30(4.57)	13.12(4.90)	0.68
ICU length of stay, Mean (SD)	9.82(4.06)	11.15(4.07)	10.48(4.52)	0.48
O2 saturation ranges at discharge with supplementary o2, Mean (SD)	91.68(1.64)	92.20(2.12)	92.88(2.12)	0.12
Intubation, F (%)	6(21.4)	9(27.3)	8(24.2)	0.87
NIPPV, F (%)	15(53.6)	15(45.5)	13(39.4)	0.54
Death, F (%)	7(25)	9(27.3)	8(24.2)	0.96
Discharge, F (%)	22(78.6)	25(75.8)	26(77.7)	0.95
Complication
Infection, F (%)	5(17.9)	10(30.3)	6(18.2)	0.40
GI. Bleeding, F (%)	3(10.7)	5(15.2)	2(6.1)	0.49
Thrombosis, F (%)	1(3.1)	3(9.1)	0	0.18

5. Discussion

Despite the time from the onset of the COVID-19 pandemic, the lack of effective treatment for these patients remains an important issue. At present, despite the production of differing vaccines, due to their unavailability to specific populations in the world or the reluctance to be vaccinated by some individuals, there are still cases of severe and life-threatening diseases in ICUs. Recognising the role of the inflammatory and cytokine cascade in causing hypoxemia and multi-organ failure in the patients, using Tocilizumab with the aim of inhibiting interleukin 6 receptors and plasmapheresis with the aim to remove inflammatory factors from the body has been studied [22], [23], [24]. The biggest limitation of previous studies has been the retrospective analysis in the design of most studies as well as the few existing RCTs (randomized controlled trial). To the best of our knowledge, this is the first prospective interventional study that compared the three treatments of Tocilizumab, plasmapheresis and a combination of these two treatments.

Based on oxygenation levels and the involvement score from the CT scan, it was determined that most of the patients in our study fell into the category of severe and life-threatening disease with high levels of respiratory involvement. The reduction in blood levels of inflammatory markers and interleukin-6 were not different across the treatment groups. The use of these therapies all led to a significant reduction in CRP level within each group, which may be promising to use this inflammatory marker to assess the response to these treatments. Decreased CRP in the therapeutic groups indicated positive anti-inflammatory and cytokine-lowering effects in these patients. Furthermore, the results of this study showed an increase in serum interleukin-6 levels in the treated groups, though this was statistically significant in the Tocilizumab-only group. This increase in interleukin-6 levels in treatment with Tocilizumab has been previously reported in rheumatoid arthritis (RA). In patients with RA, an increase in serum interleukin 6 levels was observed after treatment with Tocilizumab, despite clinically significant improvement [25]. As a interleukin-6 receptor inhibitor, Tocilizumab increases the amount of free interleukin-6 in the blood and promotes its accumulation. Previous investigations assessing the blood level of interleukin 6 following therapy with Tocilizumab in COVID-19 patients found that the level first increased and then gradually decreased with time [26]. Because inflammatory markers and interleukin 6 levels were re-measured 48 h after the end of the intervention, a significant increase in the Tocilizumab group was seen than in the other two groups. Therefore, it seems that the increase in interleukin 6 levels in the first days after treatment with Tocilizumab is normal and should not be considered as a treatment failure. Decreased levels of inflammatory markers in treatment with Tocilizumab or plasmapheresis have been shown in previous studies [26], [27], [28], [29].

In the subgroup analysis performed, serum levels of inflammatory factors and primary interleukin 6, after their intervention and differences, were compared in each group between deceased patients and discharged patients and did not differ. However, when the levels of Pro-calcitonin were determined in the groups treated with Tocilizumab alone or in combination with plasmapheresis, the dead patients in the Tocilizumab-treated groups had greater pro-calcitonin levels after the intervention, or at least an additional rise in serum levels. Tocilizumab therapy has been reported to have an increased incidence of secondary infection in patients [30], [31], and here the incidence of secondary infection was greater in the Tocilizumab-treated group than in the plasmapheresis-treated group alone, though did not differ statistically. The increase in infection in the group receiving Tocilizumab alone, was associated with a considerable rise in neutrophilia and white blood cell levels, likely due secondary infections that were the primary cause of the majority of fatalities. Using Tocilizumab in the treatment of COVID-19 may lead to higher mortality in these patients by increasing the incidence of secondary infection. Therefore, follow-up of patients is recommended in terms of possible secondary infections and their early treatment.

In this study there was no difference between the study groups in terms of the progression toward mechanical ventilation, the need for endotracheal intubation, and the rate of mortality. The duration of hospital or ICU stay did not significantly differ between the study groups. Accordingly, it seems that these therapies have the same therapeutic effectiveness in patients with severe COVID-19 based on the findings of this research. Other studies reported the efficacy of plasmapheresis treatment or Tocilizumab [16], [17], [21] The use of plasmapheresis in the treatment of COVID-19 patients was linked to clinical improvement in patients with severe illness and lowered blood levels of inflammatory markers [32]. In another study, 22 patients with severe coronary artery disease were treated with three plasmapheresis sessions and compared with the control group that showed clinical improvement and further reduction in serum levels of inflammatory factors were seen in the group treated with plasmapheresis [33]. In the study of F. Khamis et al.[34], the effect of plasma replacement therapy in patients with severe COVID-19 infection was investigated in 31 patients who underwent 5 sessions. The results of the study showed that the length of stay of patients in the ICU in the plasma replacement treatment group was longer than the non-plasma replacement treatment group, on the other hand, the probability of extubation was higher in the plasma replacement group. Mortality was slightly lower in the plasma exchange group, and patients in the plasma exchange group showed lower levels of IL.6, d-dimer, ferritin and CRP levels. In this study, three plasmapheresis sessions were performed and only ferritin and CRP levels were reduced and it had no effect on other biomarkers, likely-three sessions of plasmapheresis sessions were too few to effect a result. In general, plasmapheresis or Tocilizumab treatment appears to be associated with decreased inflammatory markers or clinical improvement in patients with severe COVID-19; however, there are reports to the converse for Tocilizumab therapy[1], and in a multicenter clinical trial study, Tocilizumab therapy was associated with increased mortality in severe COVID-19 patients [26].

The most frequent adverse effects seen in patients were secondary infection, gastrointestinal bleeding and venous thrombosis; however, there was no difference between treatment groups. The complication of venous thrombosis was more likely to be seen in the plasmapheresis group alone, due to the greater placement of the catheter to perform plasmapheresis through the femoral vein. In two patients in the plasmapheresis group alone and one patient in the Tocilizumab and plasmapheresis group, iatrogenic pneumothorax due to subclavian vein catheterization occurred, with full recovery. Studies on plasmapheresis in the treatment of patients with COVID-19 or other diseases have not reported excess complications and in many studies were introduced as a safe treatment [21]. Hypotension, cardiac arrhythmia and cold sensation were some of the possible complications mentioned in previous studies, which necessitated careful follow-up [35].

The most important limitation of our study was the lack of a comparator group with standard treatment alone to accurately compare the effectiveness of the three treatment methods. Due to the seriousness of the clinical condition of patients it was not possible to get informed consent four treatment arms, one of which potentially was going to be less effective Therefore, due to the conditions imposed on us, study was conducted without a control group of standard treatment. In addition, the study population in this study was relatively small and a larger prospective study is needed. Another limitation was that the information relating to the time delay between the onset of clinical symptoms of the patients and admission to the ICU in different groups was not precisely available; therefore, this data is not stated in our study that this may have affected the results of the study. Finally, it should be mentioned that in our study, patients were examined and follow-up for secondary complications such as infections only during hospitalization. One of the other limitations of this study was the lack of follow-up of patients after discharge.

6. Conclusion

There was no difference between the three study groups of Tocilizumab alone, plasmapheresis alone or a combination of both on the inflammatory or clinical response; however, all three treatments decreased CRP as a general marker of a reduction in inflammation due to a decrease in the inflammatory cytokines. Without the documentation of an improvement against a control group, it is not possible to firmly recommend the use of these treatment methods in current practice.

Declaration

Ethics Approval and Consent to Participate: This study was reviewed and approved by the local ethics committee of Yazd University of Medical Sciences (IR.SSU.MEDICINE.REC.1400.023) and performed in accordance with the Declaration of Helsinki and Good Clinical Practice Guidelines. The study was registered on https://irct.ir/ IRCT20190810044500N9 (29/01/2022). All patients were given informed written consent before enrollment in this study.

Consent for publication: Consent for publication was agreed upon in the written consent forms signed by the patients.

Availability of data and materials: All data generated or analyzed during this study are included in this published article.

Competing interests: The authors declare that they have no conflicts of interest.

Funding: This study was approved by Clinical Research Development Center at Shahid Rahnemoon Hospital, Yazd, Iran.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

No data was used for the research described in the article.