Intravenous Immunoglobulin Gamma for Severe Cases of Coronavirus Disease of 2019: A Randomized Placebo-Controlled Double-Blinded Clinical Trial

Background: Coronavirus disease of 2019 (COVID-19) with high-transmission power has infected people in many countries around the world. Discovering an effective medication for the treatment of this disease, especially in severe cases, has become the subject of intense scientic investigations. Therefore, the objective of this study was to evaluate the ecacy of intravenous immunoglobulin (IVIg) in patients with severe COVID-19 infection. Methods: The study was conducted as a randomized placebo-controlled double-blinded clinical trial. Fifty-nine patients with severe COVID-19 infection who did not respond to initial treatment were randomly assigned into two groups. One group received IVIg (human) four vials every day for three days in addition to initial treatment, and the other group received placebo. Patients’ demographic, clinical, and selected laboratory test results, as well as the occurrence of in-hospital mortality, were recorded. Result: Among included patients, 30 patients received IVIg and 29 patients received placebo. Demographics, clinical characteristics and evaluated laboratory tests of two groups were not statistically different (P>0.05). The in-hospital mortality rate was signicantly lower in the IVIg group as compared to the control group (6 [20.0%] vs 14 [48.3%], respectively, p = 0.022). Multivariate regression analysis demonstrated that administration of IVIg had a signicant impact on mortality rate (aOR= 0.003 [95%CI: 0.001 - 0.815], p=0.042). Conclusion: Our study was the rst randomized double-blinded study that demonstrated that the administration of IVIg in patients with severe COVID-19 infection who did not respond to initial treatment could improve their clinical outcome and reduce the mortality rate. However, further multicenter studies with larger samples size are required to conrm the applicability of using this medication as the standard treatment. Trial registration: the registration 17 May 2020. registered.


Background
Coronavirus disease of 2019 (COVID- 19) outbreak has been announced as a pandemic by the World Health Organization on 11 March 2020 (1). The culprit virus known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly contagious and can spread mainly through respiratory droplets produced by coughing and sneezing (2). The mortality rate was reported to be about 2.5 percent at initial assessments; however, higher estimations were reported lately (3). Although a great deal of effort has been made to nd a proper medication against this disease, no speci c treatment has been established yet, and no vaccine is currently available. Therefore, the therapeutic strategies are mainly empirical and based on the experiences from treatment against other pathogens. Lopinavir/ritonavir, chloroquine phosphate, hydroxychloroquine, and alpha-interferon are of the most commonly used medications for COVID-19 (4). Moreover, preclinical studies have proposed remdesivir (an RNA polymerase inhibitor with in vitro activity against multiple RNA viruses, including Ebola) and tocilizumab (humanized IgG1 monoclonal antibody, directed against the IL-6 receptor) (5).
Intravenous immunoglobulin (IVIg) is a blood product that is obtained from healthy donors and contains polyclonal immunoglobulin Gamma. Since its emergence about 30 years ago, it has been administered as an effective immunomodulatory therapy in autoimmune or in ammatory diseases (6). Moreover, signi cant positive outcomes have been observed by the administration of IVIg in patients with SARS and Middle East respiratory syndrome (MERS) (7)(8)(9). Considering the presence of overwhelming immune response in COIVD-19 (10,11) as well as similarities between the pathogenesis of SARS and COVID-19, it seems that IVIg can be helpful to improve passive immunity and modulate the in ammation in patients with COIVD-19 (12). A case report in China described signi cant improvement of three patients with severe COVID-19 infection who received high dose IVIg (12). However, the lack of more inclusive studies still exists to propose this treatment as an effective therapeutic option for COVID-19. Therefore, the objective of this study was to evaluate the e cacy of IVIg in COVID-19 patients who did not respond to initial treatment.

Methods
This study was a randomized double-blinded placebo-controlled clinical trial on 59 patients with severe COVID-19 infection who did not respond to initial treatment. The study was conducted in accordance with the declaration of Helsinki protocol. Informed consent was obtained from the patients or their guardians after a short description of the study. The protocol of the study was approved by the medical ethics committee of the university (IR.UMSU.REC.1399.025). The study protocol is registered at the Iranian Registry of Clinical Trials with the registration number of IRCT20200501047259N1 (www.IRCT.ir).

Study Sample
The patients were consecutively included if they had developed acute respiratory syndrome and had a de nite diagnosis of COVID-19 which was made based on real-time reverse transcription-polymerase chain reaction (rRT-PCR) test and the chest computed tomography scan ndings from … (hided for blinded peer review) teaching hospital from May 9, 2020 to June 9, 2020. The inclusion criteria were age over 18 years, a de nite diagnosis of COVID-19, on admission involvement of more than 30 percent of both lungs in high-resolution computed tomography (HRCT) (con rmed by two radiologists), O 2 saturation (satO 2 ) less than 90 percent, lack of adequate response to initial treatment including at least one antiviral and chloroquine drugs. Exclusion criteria were pregnancy, coagulation disorders, history of hypersensitivity for IVIg, advanced heart failure (left ventricular ejection fraction less than 35%), pulmonary brosis or history of lung surgery, sarcoidosis and tuberculosis that may interfere with an accurate estimation of the severity of pulmonary involvement by COVID-19. Inadequate response to initial treatment was de ned as the lack of improvement of dyspnea and fever as well as hypoxemia (satO 2 less than 90%) and the need for oxygenation to maintain satO 2 above this level after 48 hours of starting the treatment.

Exposure
The included patients were randomly assigned into two groups of IVIg and control (1:1) using a computer-generated randomization schedule. IVIg group received IVIg (human) ebogamma 5% DIF GRIFOLS in addition to initial treatment. The patients received four vials every day for three days, and each vial had ve-gram IVIg. Those patients who expired before 72 hours since the initiation of IVIg were excluded from our study due to the incomplete course of treatment. The control group continued to receive the initial treatment and received a placebo. Neither the patients nor the physicians nor whom responsible for data analysis were aware of the types of treatment allocated, and only the pharmacist of the center had knowledge about IVIg or placebo. Placebo and IVIg vials had similar appearance and were labeled as A and B. The placebo vials contained a similar volume of normal saline solution.

Outcome
The study primary outcome was in-hospital mortality.

Data analysis
The normal distribution of the variables was evaluated using the Kolmogorov-Smirnov test. The majority of the continuous variables had not a normal distribution; therefore, the median and interquartile range (IQR, 25 percentile -75 percentile) of quantitative variables and frequency and percentage of qualitative variables were reported. Continuous variables were compared using the Mann Whitney U test. Qualitative variables were compared using Chi-square or Fischer exact test. Univariate logistic regression was used to model the mortality based on investigated variables. Signi cant variables in univariable logistic regression (p-value less than 0.2) were entered into multivariable logistic regression. The statistical analysis was conducted using Statistical Package for the Social Sciences version 22 (SPSS Inc., Chicago, IL).

Discussion
Our results showed that IVIg could improve clinical outcomes in COVID-19 patients with severe respiratory system involvement. Previously, Prohaska et al. showed that IVIg could not reduce the mortality of patients with ARDS undergoing extracorporeal membrane oxygenation (ECMO) therapy. In this study, patients with bacterial and fungal infection also included, and only 54% of patients in the IVIg group and 28% of patients in the control group had viral infection (13). Recently, a randomized controlled trial studied the hyperimmune IVIg (hIVIg) effect on patients with con rmed in uenza A or B infection. This study showed that hIVIg was not superior to placebo in treating patients with in uenza A or B. In this study the mortality rates of patients in hIVIg and control groups were 4% and 3% respectively (14). The two mentioned studies have investigated IVIg effect in very high-risk and very low-risk patients for mortality, and it is possible that the effectiveness of IVIg in our study could be due to using the drug in patients with intermediate-risk of mortality.  (16). Our results showed that IVIg 20 g/day for three days could be effective and safe.
Very recently, a multicenter retrospective cohort study conducted on 325 patients including 222 (68%) patients with severe type and 103 (32%) with critical type of con rmed COVID-19. In 174 patients, IVIg was administered and 151 patients did not take IVIg. Two groups had signi cantly different bassline characteristics, and the IVIg group were in more severe condition. This study showed a 28 days mortality rate of 13% in both groups. The primary analysis showed no difference between the IVIg group and control group in reducing in-hospital mortality. However, after adjusting the outcomes of two groups with the severity of illness, results showed that administration of IVIg signi cantly decreased the 60-day mortality. This study also showed that IVIg dosage > 15 g/d and starting the drug ≤ 7 days could be more effective (17). A meta-analysis showed that IVIg could be effective in patients with severe sepsis or septic shock and its e cacy in mortality reduction could be better in patients with higher baseline risk and those with lower plasma immunoglobulins (18). Justel et al. studied patients admitted with severe pandemic in uenza showed that lower level of IgG and IgM was associated with higher early mortality (19).
Our study showed that higher age, lower diastolic blood pressure, and higher LDH were also associated with higher mortality in COVID-19 patients with severe disease. Correspondingly, Du et al. also showed that age ≥ 65 years was associated with higher mortality in patients with COVID-19 pneumonia (20). Moreover, Henry et al. reported that elevated LDH was associated with 16 fold higher mortality among patients with COVID-19 infection (21).
To the best of our knowledge, our study is the rst randomized double-blinded control trial that suggests the effectiveness of IVIg in reducing mortality in patients with severe COVID-19 pneumonia. However, some limitations existed in our study. The study was conducted as a pilot study and included a relatively small sample size. Therefore, further multicenter studies with larger samples size should be conducted in this regard. Moreover, it was better to follow the patients for evaluation of intermediate and long term effects of IVIg treatment. However, due to the pressing requirement of physicians and health care systems for providing evidence-based medications for patients with COVID-19, we decided to report the follow-up data in future updates of the study. Furthermore, it should be noted that the cost of IVIg treatment is relatively high, and it may not be widely available, particularly in low and middle-income countries.

Conclusion
The result of our study demonstrated that the administration of IVIg in patients with severe COVID-19 infection who did not respond to initial treatment could improve their clinical outcome and reduce the mortality rate. However, further multicenter studies with larger samples size are required to con rm the applicability of using this medication as the standard treatment of these patients. Availability of data and materials: All Data and material collected during this study are available from the corresponding author upon reasonable request.