key: cord-0902592-sxyo9umr authors: Allahyari, Abolghasem; Seddigh-Shamsi, Mohsen; Mahmoudi, Mahmoud; Amel Jamehdar, Saeid; Amini, Mahnaz; Mozdourian, Mahnaz; Javidarabshahi, Zahra; Eslami Hasan Abadi, Saeed; Amini, Shahram; Sedaghat, Alireza; Emadzadeh, Maryam; Moeini Nodeh, Mohammad; Rahimi, Hossein; Bari, Alireza; Mozaheb, Zahra; Kamandi, Mostafa; Ataei Azimi, Sajad; Abrishami, Mojtaba; Akbarian, Arezoo; Ataei, Parisa; Allahyari, Negin; Hasanzadeh, Sepideh; Saeedian, Neda title: Efficacy and safety of convalescent plasma therapy in severe COVID-19 patients with acute respiratory distress syndrome date: 2020-12-02 journal: Int Immunopharmacol DOI: 10.1016/j.intimp.2020.107239 sha: 411151c8de5113fb509d7c1b649a34728c33021f doc_id: 902592 cord_uid: sxyo9umr Since SARS-CoV-2 infection is rapidly spreading all around the world, affecting many people and exhausting health care resources, therapeutic options must be quickly investigated in order to develop a safe and effective treatment. The present study was designed to evaluate the safety and efficacy of convalescent plasma (CP) for treating severe cases of COVID-19 who developed acute respiratory distress syndrome (ARDS). Among 64 confirmed cases of severe COVID-19 with ARDS in this study, 32 patients received CP besides first line treatment. Their clinical response and outcome in regard to disease severity and mortality rate were evaluated and compared with the other 32 patients in the control group who were historically matched while randomly chosen from previous patients with the same conditions except for receiving CP therapy. Analysis of the data was performed using SPSS software. Patients with plasma therapy showed improvements in their clinical outcomes including a reduction in disease severity in terms of SOFA and APACHE II scores, the length of ICU stay, need for noninvasive ventilation and intubation and also showed an increase in oxygenation. They also showed reduction in mortality which was statistically significant in less severe cases with mild or moderate ARDS. Early administration of the convalescent plasma could successfully contribute to the treatment of severe COVID-19 patients with mild or moderate ARDS at risk of progressing to critical state. While most COVID-19 patients experience a mild clinical course, severe cases are at high risk of rapidly developing acute respiratory distress syndrome (ARDS) particularly within 7-14 days from onset of symptoms 6 which often leads to acute respiratory failure with high mortality rates in a short time (20 times higher than that of non-severe COVID-19 patients) 7, 8 . Healthcare systems are struggling to cope with the increasingly growing number of COVID-19 patients presenting to hospitals all around the world as the disease spreads rapidly 9 . Managing COVID-19 has been mostly supportive to date, such as providing supplemental oxygen to cases with mild disease and using extracorporeal membrane oxygenation for critically ill patients 10 . However, in fast evolving pandemics with no observable natural immunity in the population, effective treatment options must be quickly investigated and become available to alleviate the symptoms and reduce the mortality. Unfortunately, no specific treatment or vaccines have been proven to be effective for SARS-CoV-2 infection so far which makes it even more difficult for the pandemic to be contained. The efficacy of some specific drugs including anti-viral and anti-HIV agents (remdesivir, favipiravir 11 and Lopinavir /Ritonavir 12 ) and other medications and therapeutic strategies for COVID-19 such as antimalarial drugs (chloroquine and hydroxychloroquine ), a combination of hydroxychloroquine and azithromycin 13 and lopinavir + ritonavir + interferon-beta are still under investigation 9, 12, 14, 15 . While some of these medications have been shown to be beneficial, there is still not enough clinical evidence to prove their safety and efficacy. Therefore, the treatment remains challenging. Considering the absence of specific efficacy-proven preventative and therapeutic options, attention has also been given to classical and empirical interventions as an option to treat COVID-19 patients 16, 17 . Accordingly, researchers and physicians have resorted to use the plasma containing SARS-CoV-2 specific antibodies from convalescent donors to treat severe cases of COVID-19. In the late 19 th century, antibody transfer was first used to fight against bacterial toxins before the discovery of antimicrobials, however, it has been applied to confer passive immunity against many other infectious diseases associated with different microorganisms since then when there was no therapeutic agent available, high risk of infection or not enough time for the body to acquire active immunity due to rapid progression of the disease [18] [19] [20] [21] . Using convalescent plasma (CP) transfusion has also been recommended as an emergency intervention during the Spanish flu, H5N1 avian influenza, West Nile virus, SARS, MERS and Ebola outbreak which was shown to be beneficial in many cases by reducing the hospital stay and mortality rate 22-31 . Accordingly, a recent systematic review of 40 studies on CP treatment for infectious diseases including SARS, MERS, Ebola, H1N1, H5N1 and H7N9 also concluded that CP treatment could result in promoting antibody production while shortening the disease course, reducing viral load and mortality with low risk of adverse effects, suggesting that it could be potentially effective for treating COVID-19 patients 32 . Considering the value of time in the current pandemic, it is of great importance to promptly address the urgent question raised regarding CP efficacy in managing Sars-CoV-2 infection. Therefore, the present study was designed to investigate the efficacy of using convalescent plasma therapy to treat severe cases of COVID-19 suffering from ARDS. This clinical trial was carried out in Imam Reza hospital, the referral center for COVID- 19 and donors signed the written informed consent. A total number of 64 laboratory confirmed COVID-19 patients were included in the study whom met all of the Inclusion criteria which come as follows: Patients were excluded from the study if they did not consent to participate in the trial or were allergic to the plasma product. The control group were historically selected from the previous patients using the registry system which keeps record of the detailed clinical information and outcomes of the COVID-19 patients. In order to have a better comparison, they were matched with the patients in the intervention group considering factors such as disease severity (in terms of PaO2/FiO2 (± 30) on the similar day of hospitalization), age (± 10 years), gender, first line treatment, background diseases (Hypertension (HTN) and Diabetes Mellitus (DM) only) and symptom day. One hundred and eleven patients were randomly chosen by a blinded technician (totally unaware of the study design, purpose and possible results), using the advanced search tool in the registry system through which the first search results appeared according to the defined criteria applied on search filters were chosen. Propensity score (PS) was calculated regarding the abovementioned variables to remove the chronology bias and control the confounding factors. Ultimately, 32 patients who were more closely matched with the patients in the intervention group, were assigned to the control group. The day on which the convalescent plasma was transfused to the patients of the intervention group was defined as the day zero (day 0 ) and the following days were numbered accordingly. Recovered COVID-19 patients between 18 to 60 years old, with initial positive reverse transcriptase-polymerase chain reaction (RT-PCR) test for SARS-CoV-2 and absence of the symptoms of the disease for at least 14 days, who were eligible to donate blood considering the Blood Donor Organization standards (except for recent SARS-CoV-2 infection) were invited to participate in the study. The final donors participating in the study included asymptomatic men who tested negative for HBS Ag, HCV Ab, HIV Ab, HTLV1 Ab, VDRL and SARS-CoV-2 nucleic acid while positive for SARS-CoV-2 IgG and IgM at the time of donating blood. Other tests including CBC, CRP, blood group and Rh factor blood test were also carried out. Plasma contents of the donors were also subjected of quality control regarding the blood cells count in order to make sure that the number of red blood cells (RBC), white blood cells (WBC) and platelets were below 0.2×10 9 , 0.1×10 9 and 10×10 9 per liter, respectively. Moreover, plasma culture was performed for each donor to make sure there was no infection. The specimens including nasopharyngeal swabs and oropharyngeal swabs were taken and immediately placed in the tubes containing viral transport media. All sample processing and reactions were carried out under class II biological safety cabinets. Detection of SARS-CoV-2 nucleic acid was performed through real-time RT-PCR assays using commercial detection kit (DAAN Gene, China). Two independent primers and probes matching the open reading frame1ab (ORF1ab) and the nucleocapsid protein (N) fragments were used. RNase P gene was used as an internal control. Real-time RT-PCR was performed following the manufacturer's recommendations. Each amplicon provided a cycle threshold value (Ct value), which is the number of cycles required for the fluorescent signal. Ct values less than 40 were considered as positive results, while Ct values exceeding 40 were defined as negative results. Detection of anti-SARS-CoV-2 antibodies was carried out through two separate ELISA kits with similar procedures, each designed specifically to reveal anti-SARS-CoV-2 IgG and IgM using the plates coated by N proteins of SARS-CoV-2 (Pishtaz Teb, Iran). In each assay, diluted serum samples (1:101 for detection of IgG and 1;51 for detection of IgM) and controls were added to the plates. After a 30 min incubation at 37 o C, plates were washed and horseradish peroxidase (HRP)labeled antigens were added. Another 30 min incubation at 37 o C was applied before the plates were washed again so that the unbound components would be eliminated. Chromogen substrate was added afterward, which was followed by adding the stop buffer in 15 minutes. Absorbance was measured at 450nm and 630nm dual-wavelength using a microplate reader. The cutoff optical density (OD) was calculated as the mean OD of negative controls plus 0.25 in case of IgM and 0.15 in case of IgG. Results were reported as an index, calculated as the OD value of the samples divided by the cutoff OD value. The cutoff value recommended by the kit manufacturer was used according to which ratios higher than 1.1 were considered positive. However, to ensure providing recipients with higher levels of antibodies, the donors included in the study were chosen among those with highest cutoff index (between 10 to 32). Convalescent plasma was obtained from donors by apheresis and each patient received one cycle of 600 ml fresh ABO-identical and RhD-compatible convalescent plasma in the same day and as soon as possible (within 4 h). Transfusion was administered slowly with continuous monitoring of the patients' vital signs by the clinician in order to prevent adverse outcomes associated with plasma transfusion which mainly include volume overload and allergic response to plasma contents. In case of presenting volume overload signs, the transfusion would be paused and resumed slowly while reducing maintenance serum volume after patient's stability and administering diuretic and venous nitroglycerin. In case of mild allergic reactions such as rash, itching and mild fever to the plasma contents, the transfusion would be paused and resumed slowly after administering chlorphenamine and Corticosteroid. In case of severe hypersensitivity reactions such as hypotension and decreased oxygen saturation, the transfusion would be stopped and the complications would be controlled by chlorphenamine, epinephrine and Corticosteroid. No more plasma transfusion would be performed in such cases and the patients would be excluded from the study if they did not have two successful infusions before this one. Patients' conditions, clinical examinations and outcomes were precisely monitored in the intervention group and were compared with the control groups. Factors considered in this regard to evaluate and compare the outcome of the intervention included length of hospitalization, need for mechanical ventilation, disease severity based on PaO2/FiO2, Sequential Organ Failure Assessment (SOFA) score (ranging from 0 to 24 with 24 indicating the highest level of severity) and APACHE II (Acute Physiology And Chronic Health Evaluation II) score (ranging from 0 to 71 where higher scores correspond to more severity) on day zero, three and the day of discharge (last day of hospitalization leading to discharge or death) and finally the mortality rate in 4 weeks (day 28) as the main outcome. Analyzes were carried out using SPSS software (Version 22.0, SPSS, Inc., Chicago, IL, USA Quantitative variables were presented as mean and standard deviation while the qualitative variables were reported as frequency and percent. Comparison analysis between the two groups was performed using T-Test or Mann-Whitney U Test based on the variables' distribution. To compare the difference between the qualitative variables of the two groups, Pearson Chi-Square Test or Fisher's Exact Test were performed. Correlation analyzes were done using Spearman Correlation test. Differences were considered statistically significant where P values were less than 0.05. To assess the strength and direction of association between variables, Spearman Correlation Coefficient (r) was considered. Patients were matched regarding age, gender, background disease and PaO 2 /FiO 2 level on the similar day (the day of plasma transfusion, referred to as day zero) in both plasma and control groups, accordingly there were no significant differences between two groups regarding these variables. Patients receiving convalescent plasma did not present any immediate and noticeable adverse effect. Table 1 Table 4 also shows the quantity of the variables associated with the length of the disease and treatment of the patients in the plasma group including the number of days from patients' symptoms to their admission to the hospital, admission to the day of plasma transfusion and symptoms to the plasma transfusion day. The mean interval time from admission to receiving convalescent plasma by patients was assessed to be 4.41±2.28 days ranging from 3 to 11 days. Table 5 shows the mean, standard deviation, minimum and maximum of the quantitative variables associated with the disease severity in total and also in each group. According to the table, the length of hospitalization was more in the control group. The level of PaO 2 /FiO 2 on day of discharge was significantly higher in the plasma group (275.03) compared with the PaO 2 /FiO 2 level in control group (213.41) (P value:0.034 by Mann-Whitney U Test). SOFA scores were lower in plasma group both on the day three and the day of discharge compared with the scores of the control group. However, none of the abovementioned variables showed statistically significant difference between the two groups. APACHE II scores in the plasma group decreased over time and were estimated to be less than the scores in the control group on both days (day three and the day of discharge) which were significantly different (P values: 0.001 and 0.023 by Mann-Whitney U Test, respectively). No significant differences were observed between any of the abovementioned variables and gender neither with having diabetes mellitus in the patients in any of the two groups, however; having hypertension was shown to be statistically correlated with higher SOFA and APACHE II scores on day of discharge in the control group (P values: 0.034 and 0.040, respectively, calculated by Mann-Whitney U test) Spearman correlation analyses between different variables including IgM, IgG, CRP and Interleukin 6 on day zero and three, admission to discharge interval, PaO 2 /FiO 2 level, SOFA and APACHE II scores on day zero, three and day of discharge in the plasma group showed that there were statistically significant correlation between APACHE II on day of discharge and CRP on the day three (P value: 0.025, r: 0.456), an inverse correlation between CRP on day zero and PaO 2 /FiO 2 level on day of discharge (P value:0.033, r: -0.405) and also a stronger correlation (r: 0.607) between interleukin 6 on the day 3 and APACHE II score on the same day (P value: 0.013). The present study evaluated the safety and effectiveness of convalescent plasma therapy for severe COVID-19 patients with ARDS. The outcomes were compared with patients in the control group who shared the same characteristics and treatment protocol with the intervention group, except for the plasma therapy. No adverse effects or allergic responses associated with plasma transfusion were observed in recipients. The study results indicated that transfusion of convalescent plasma contributed to improvement of the patients' clinical outcomes including a decrease in the length of hospital stay, need for non-invasive mechanical ventilation and intubation and finally mortality rate which was significantly lower in the cases with less severe ARDS (PaO 2 /FiO 2 above 200). This could suggest that convalescent plasma therapy could be more beneficial if administered in early stage of the disease and before the patient is critically ill. The number of deaths and discharged COVID-19 patients with severe ARDS (PaO 2 /FiO 2 below 100) who were critically ill was equal in both intervention and control groups of the present study, strengthening the hypothesis of CP effectiveness in the less advanced stages of the disease. This is in accordance with the findings of the study done by Zeng et al. (2020) in which plasma transfusion in critically ill COVID-19 patients did not help with decreasing mortality (5 out of 6 patients died) 33 . The reason might lie in macrophage activation. Studies suggest that COVID-19 patients might undergo a macrophage activation associated with innate immune cells migration to lung tissues causing inflammation and pulmonary damage 18 . Inhibition of this immunological pathway might be helpful in averting cytokine storm and lung damage. This was also supported by a recent study reporting an up regulation of chemokines for innate immune cells mainly taking place within the first 7 days of the infection onset in COVID-19 patients 34 . The same pattern was observed in severe acute respiratory infections caused by other viruses such as H1N1 and SARS-CoV in which CP was transfused early after symptoms onset resulted in a reduction in mortality compared with those who received placebo or no therapy 22 . Moreover, a difference in case-fatality rate was observed in H1N1-infected patients who received CP earlier compared to those who received it later in their disease course 35 . Therefore, CP transfusion in early stages of COVID-19 disease may inhibit innate immune cells migration and lung damage. The mean interval time from admission to plasma transfusion in the present study was 4.41 days with a minimum and maximum of 3-11 days. In a recent similar study done by Abolghasemi et al. 36 in which plasma transfusion was performed within three days of hospitalization, higher percentage of the patients (98.2%) receiving CP were recovered and discharged in total, compared with the present study (78.1%). This could be due to earlier administration of plasma and also less severity of the cases. Considering the patients' mean and maximum of SpO2 which were 85.95% and 93% in the abovementioned study 36 , respectively, the patients included in the present study were more severe in comparison (SpO2≤85% and all the patients were suffering from ARDS). In this regard, the results of the present study also showed that all the patients (100%) with mild ARDS (200