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Current views on the potentials of convalescent plasma therapy (CPT) as Coronavirus disease 2019 (COVID‐19) treatment: A systematic review and meta‐analysis based on recent studies and previous respiratory pandemics

Summary Convalescent plasma therapy (CPT) has been investigated as a treatment for COVID‐19. This review evaluates CPT in COVID‐19 and other viral respiratory diseases, including severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS) and influenza. PubMed and Google scholar databases were used to collect eligible publications until 8 December 2020. Meta‐analysis used Mantel–Haenszel risk ratio (RR) with 95% confidence interval (CI) and pooled analysis for individual patient data with inverse variance weighted average. The study is registered at PROSPERO with the number of CRD4200270579. Forty‐four studies with 36,716 participants were included in the pooled analysis and 20 studies in the meta‐analysis. Meta‐analysis showed reduction of mortality (RR 0.57, 95% CI [0.43, 0.76], z = 3.86 [p < 0.001], I 2 = 44% [p = 0.03]) and higher number of discharged patients (RR 2.53, 95% CI [1.72, 3.72], z = 4.70 [p < 0.001], I 2 = 3% [p = 0.39]) in patients receiving CPT compared to standard care alone. A possible mechanism of action is prompt reduction in viral titre. Serious transfusion‐related adverse events were reported to be less than 1% of cases, suggesting the overall safety of CPT; nevertheless, the number of patients participating in the studies was still limited. It is also important to notice that in all the studies, the majority of patients were also given other medications, such as antivirals, antibiotics and corticosteroid; furthermore, randomized controlled studies involving more patients and in combination with other treatment modalities are urgently needed.


| INTRODUCTION
Severe acute respiratory syndrome-coronavirus-2 (SARS-  infections emerged at the end of January 2020, leading World Health Organization (WHO) to declare COVID-19 as a Public Health Emergency of International Concern, and later updated the status into pandemic. Up to 2 October 2020, 216 countries were affected with 1,023,522 (4%) deaths and 25,634,071 (96%) recovered among 26,657,593 confirmed cases. 1 The disease, later known as COVID-19, is mainly characterized by myalgia, fever, cough, dyspnoea, sore throat, dizziness and confusion. Laboratory and radiological examinations often reveal decreased albumin, high C-reactive protein (CRP), lymphopenia and pneumonia. 2 These clinical symptoms are similar with the previous cases of severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS) and influenza. 3 Further genetic findings confirmed that a significant proportion of genetic sequences in SARS-CoV (79%) and MERS-coronavirus (MERS-CoV) (50%) are identical with SARS-CoV-2. 4 Up until now, no specific and efficient pharmacological therapy has been validated. Chloroquine, a drug commonly used to treat malaria, is suggested to be effective against SARS-CoV-2 in vitro. 5 Hydroxychloroquine, one of the chloroquine derivatives, is suggested to be more potent than chloroquine, with less toxicity 6 ; however, recent systematic reviews based on 19 studies have shown antiviral treatments, including ribavirin, favipiravir, lopinavir/ritonavir, umifenovir, interferon and hydroxychloroquine, which exhibit no beneficial effects in both mild and severe COVID-19 patients when compared to control. 7 Remdesivir, a monophosphoramidate prodrug, has a broad antiviral activity against human coronaviruses, including SARS-CoV, MERS-CoV, CoV-OC43, CoV-229E and SARS-CoV-2 in vitro.
Remdesivir reduces viral titre, thus improving pulmonary lesions and respiratory function in SARS-CoV MA15-infected mice and SARS-CoV-2-infected rhesus macaques. 8 Two meta-analyses showed that remdesivir is associated with better overall clinical recovery 9,10 ; however, no evidence suggests any differences in terms of mortality between remdesivir versus standard care. 10,11 Another therapeutic approach having been intensively investigated is immunotherapies. Immunotherapies, such as convalescent plasma therapy (CPT) (polyclonal antibody), monoclonal antibodies, hormone for T-cell maturation and ACE2 immunoadhesins, focus on promoting patients' immune system against viral infection. 12 In CPT, blood plasma from recovered individuals is expected to contain high titre of neutralizing antibody, thus transplanted into newly infected patients. 13,14 CPT has been used in previous outbreaks of viral infections, such as Ebola virus, 15 Lassa fever, 16 Junin virus of Argentinian haemorrhagic fever, 17 Spanish flu influenza, 18 H1N1 influenza, 19 H5N1 avian influenza, 20  Encouraging results from CPT application in other severe acute respiratory infection cases suggest the potential of this therapy in COVID-19 patients. 13 This study aims to evaluate the potentials of CPT to COVID-19 patients by performing systematic review and meta-analysis on the published application of CPT in COVID-19, influenza, SARS and MERS patients. Since the data on CPT in COVID-19 patients are not abundant yet, the inclusion of studies in other viral respiratory diseases is important to obtain an objective overview of this treatment method, including patients' characteristics, infection states, adverse effects and outcomes.

| Literature search and identification
This systematic review and meta-analysis were conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) reporting guidelines. 23   including original data, such as reviews, systematic reviews, comments or editorial letters; (c) not written in English; (d) using monoclonal antibody therapy or manufactured immunoglobulin; (e) using vaccination to enhance immune response; (f) using other intervention other than standard care as control; and (g) performed on animals.

| Inclusion and exclusion criteria
For meta-analysis synthesis, case reports, case series or studies which were not reporting comparison standard care group were excluded.

| Data collection and analysis
Two authors (JKA and DH) independently reviewed all titles and abstracts. Abstracts fulfilling the inclusion criteria underwent full-text screening. The following information were obtained: authors, country, publication year, number of patients, diseases, type of study, patients' ages, gender, plasma dose, comorbidities and clinical outcomes.
Cochrane Collaboration's tool was used for assessing risk of bias for the included randomized clinical trials (RCTs), 24 while Risk of Bias in Non-Randomized Studies (ROBINS-I) was used for non-RCTs. 25

| Data synthesis
Baseline characteristics were compared with primary outcomes describing the efficacy and safety of CPT in patients. For pooled analysis, the parameters considered as primary outcomes included status at 7 and 30 days after intervention and serious transfusionrelated adverse effects. Status after intervention is classified into four groups: discharged, hospitalized, deceased and alive. Outcomes are defined as additional data used to assess patients' improvement after intervention, including laboratory findings, time to negative viral titre and oxygenation.
In meta-analysis, primary outcomes included mortality and discharge rates, while secondary outcomes included clinical improvement and viral nucleic acid negative rates in the treated groups (CPT-receiving patients) versus control groups (standard care alone). Mortality is defined as a cumulative number of deaths after 30-day intervention. Discharge rate is defined as the number of patients discharged from the hospital after 7 or 28 days after intervention. Clinical improvement is defined as an increase by 6 or 8 points in WHO disease severity scale, 26    Only RCTs, match-control observational studies or single-arm studies with subgroup analysis were included for meta-analysis. One study, Joyner et al. (2020) 49 was excluded from the meta-analysis as there was duplication of data with Joyner et al.. 48 Characteristics of the included studies are presented in Table S1.

| Baseline characteristics
Baseline characteristics of patients receiving CPT are shown in Table 1. The majority of patients receiving CPT in COVID-19 group were 40-70 years old. There were more male patients compared to female ones, which were especially observable in COVID-19 cases    aspartate aminotransferase (AST) and total bilirubin levels were normal prior to and after CPT transfusion. Lactate dehydrogenase (LDH) and ferritin levels were elevated above normal range before and after transfusion; however, the levels were decreased within 7-14 days after transfusion (Table 3).

| META-ANALYSIS OF COVID-19, INFLUENZA, SARS AND MERS PATIENTS RECEIVING CPT COMPARED TO STANDARD CARE ALONE
Meta-analysis was performed on studies comparing patients receiving CPT with patients treated with standard care (control). RCTs are presented on Table S2 and Figure S1, while the risks of bias in non-RCTs are presented on Table S3 and

| Meta-analysis on 7-and 28-day discharge rate
The comparison of number of discharged CPT-treated patients to untreated patients was reported during 7 and 28 days after transfusion in five 27 (Table S4).
Subgroup analysis for diseases severity and antibody titre was not possible for these particular outcomes as very limited studies reported the subgroup analysis.    (Table S4). Subgroup analysis for diseases severity and antibody titre was not possible for these outcomes since only few studies reported the subgroup analysis.  Figure 6). No publication bias or small-study effect was found (Table S4). Subgroup analysis for disease severity and antibody titre was not possible for these outcomes as subgroup analysis was reported in very few of the studies.

| META-ANALYSIS ON FACTORS AFFECTING CPT OUTCOMES
There were some known conditions affecting the effect of CPT Biologically, SARS-CoV-2 uses angiotensin converting enzyme 2 (ACE2), which is highly expressed in heart, lungs, kidney and gastrointestinal tract as an initial binding receptor for infection.
Interestingly, patients with hypertension and cardiovascular diseases demonstrated elevated ACE2 expression; therefore, they could have a risk to acquire more severe symptoms of COVID- 19. 81 In influenza cases, cardiovascular diseases, especially atherosclerosis, is associated with an increased risk of influenza infection since atherosclerosis plaque can function as carriers of influenza viruses for a long period of time. 82 Obesity had been known to be correlated with the COVID-19 and influenza infection as it reduced and delayed capacity to produce interferons, thereby allowing more viral RNA replication. 83 Obesity even impairs functions of the mucociliary cells, therewith reducing the clearance of the viruses. 84 Our results showed that diabetes is the second most common major comorbidity in COVID-19, but not in influenza. This might be related to the elevated circulating level of furin, a cellular protease involving in facilitating viral entry by cleaving S1 and S2 domains of SARS-CoV-2 spike protein in patients with diabetes mellitus. 85 Underlying respiratory disease is another major comorbidity aside from hypertension in influenza patients, but not in COVID-19 pa- remarkably higher than mortality rates in influenza cases. Thirty-day mortality rates were reported just in COVID-19 and influenza cases.
Mortality in COVID-19 cases was more than twofold higher compared to influenza cases. Our results are in line with previous reports where overall in-hospital mortality rates were higher in COVID-19 cases compared to influenza and SARS-CoV cases. [86][87][88] Clinical improvements were seen in COVID-19 patients receiving CPT. Improvement of oxygenation was seen within 7-day posttransfusion. Reduced lymphocyte counts were noticed before transfusion which was significant in severe/critical patients. 89 The levels of IL-6 and CRP increased significantly in patients on admission. IL-6 is a pro-inflammatory cytokine elevated during bacterial or viral infection. It is the primary trigger for cytokine storms. CRP is induced by IL-6 in the liver whose level increases dramatically during acute inflammatory responses. A cohort study involving 140 patients showed patients with IL-6 >32.1 pg/ml or CRP >41.8 mg/L were more likely to have severe complications. 90 Ferritin levels were also elevated during admission. Ferritin is a key mediator of immune dysregulation contributing to cytokine storm which indicated the admitted patients had severe inflammatory conditions. 91 CPT transfusion was associated with resolved inflammatory conditions in 1-2 weeks mean time (Table 3). ALT, AST and total bilirubin levels were normal before and after CPT transfusion indicating that the majority of the patients did not experience kidney function impairment. 92 LDH level was elevated indicating that most of the patients were in severe to critical conditions. 93 The meta-analyses showed that the differences in mortality between COVID-19 patients who did and did not receive CPT were significant ( Figure 2). The same finding was also identified in influenza

F I G U R E 7
Meta-analysis of factors affecting convalescent plasma therapy outcomes as indicated with mortality rate and length of hospital stay. Only COVID-19 cases can be subgroup analysed for disease severity: severe versus critical. The outcome assessed here were comparison of (a1) mortality rate and (a2) length of hospital stays, while analysis of mortality for comparison of antibody titre was carried out for COVID-19 and influenza cases (b) antivirals, antibiotics, corticosteroid and immunomodulatory or immunosuppressive agents; therefore, it remains plausible to assess the possibilities to use CPT in combination with other medications for patients with viral respiratory diseases, such as COVID-19.

| CONCLUSION
Based on the results from meta-analyses, it is safe to conclude that CPT is a potential therapy to accelerate the decrease of viral titres, thus expecting to increase the rates of hospital discharge and decrease the rates of mortality. Since CPT is used as a part of multimodality treatment for COVID-19 patients, development of other drugs included in the treatment will also affect the post-CPT outcomes.

ACKNOWLEDGEMENT
The authors would like to thank Alya Mardhotillah Azizah and Ika Adhani Sholihah from Universitas Pendidikan Indonesia for giving some valuable inputs at the initiation of this study.
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

CONFLICT OF INTEREST
The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT
The data supporting the findings of this study are available in the supplementary material of this article.

ETHICAL APPROVAL
Not required.