Abbreviations ALT alanine aminotransferase ARDS acute respiratory disease syndrome AST aspartate aminotransferase CPT convalescent plasma therapy IL-6 interleukin 6 LDH lactate dehydrogenase MERS Middle East respiratory syndrome MERS-CoV MERS-coronavirus nAb neutralizing antibody PaO2/FiO2 partial arterial oxygen pressure to fractional inspired oxygen ratio RR risk ratio S/Co Signal to cut-off ratio SARS severe acute respiratory syndrome SARS-CoV SARS-coronavirus SARS-CoV-2 SARS-coronavirus-2 TACO transfusion-associated circulatory overload TRALI transfusion-related acute lung injury 1 INTRODUCTION

Severe acute respiratory syndrome–coronavirus-2 (SARS-CoV-2) 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 toxicity6; 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 recovery9, 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 SARS21 and MERS22 cases. 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.

2 METHODS 2.1 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 PubMed and Google Scholar databases were used to collect publications up to 8 December 2020. The following search term were used in searches: convalescent plasma (title) AND (influenza OR SARS OR MERS OR Coronavirus OR SARS-CoV-2 OR COVID-19).

2.2 Inclusion and exclusion criteria

Studies are included if they have (a) reported clinical evaluations of convalescent plasma or hyper-immune plasma; (b) reported viral respiratory diseases; and (c) reported response in severe to critically ill patients.

Studies are excluded if they are (a) not fully accessible; (b) not 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. For meta-analysis synthesis, case reports, case series or studies which were not reporting comparison standard care group were excluded.

2.3 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

2.4 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 transfusion-related 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 and improvement of oxygenation at 14 days after intervention. Viral nucleic acid negative rate is defined as the number of patients with undetectable viral load via polymerase chain reaction (PCR) assay during 1, 2, 3 or 7 days after intervention. The above-mentioned analysis was performed for all diseases severity, time-to-transfusion, and antibody titre; furthermore, we also did meta-analysis of possible confounding factors which might affect CPT outcomes, including diseases severity and convalescent plasma antibody titre. Disease severity was classified into mild, moderate, severe and critical based on its clinical manifestations. Mild symptoms in COVID-19 patients are characterized by fever <38°C, with or without cough, no dyspnoea, no gasping, no chronic disease and no imaging findings of pneumonia. Moderate symptoms of COVID-19 are when patients developed fever, respiratory symptoms, with imaging findings of pneumonia. Severe signs of COVID-19 in patients are characterized by respiratory distress, suggested by tachypnoea of ≥30 breaths per minute in resting state, oxygen saturation of 93% or less in room air, or arterial partial pressure of oxygen (PaO2)/fraction of inspired oxygen (FiO2) of 300 or less. Critical symptoms of COVID-19 are characterized by respiratory failure requiring mechanical ventilation, shock or other organ failure (apart from lung), leading to the necessity of intensive care unit (ICU) monitoring.27, 28 Subgroup analysis was also performed according to the types of diseases (COVID-19, influenza, SARS and MERS).

2.5 Statistical analysis

Meta-analysis used Mantel–Haenszel risk ratio (RR) for dichotomous data and mean difference (Mean diff) for continuous data with 95% confidence interval (CI). RevMan version 5.3 software (Cochrane Collaboration) was used for these purposes. Pooled analysis for individual patient's data was performed with inverse variance-weighted average. Heterogeneity across studies was assessed using inconsistency index (I2) test, with p-value <0.10 indicating a significant heterogeneity. Risk of publication bias was evaluated with Egger's statistics. Habord's and Peter's statistics was used to assess small size bias. This study is registered to PROSPERO with the number of CRD4200270579.

3 RESULTS

The literature searches identified 3710 studies, and an additional 20 studies were found through bibliographical search (Figure 1). After removing duplicates and filtering all titles and abstracts, 169 full-text articles were reviewed, of which only 53 articles met the inclusion criteria. There were only 44 studies eventually included in the qualitative synthesis and pooled analysis, including 7 RCTs,19, 29-34 9 non-RCTs or matched-control observational studies,27, 35-42 15 single-arm studies21, 43-55 and 14 case reports,20, 22, 30, 56-66 while 8 studies reporting the use of manufactured immunoglobulin as the main intervention67-74 and 1 study reporting the use of fresh frozen plasma control75 were excluded. Five studies were performed on influenza cases,19, 20, 29, 30, 56 4 studies were of SARS,21, 35, 57, 58 2 case reports were of MERS,22, 76 and 33 studies reported trials in COVID-19 cases.27, 31-34, 36-55, 59-66 Data from studies in SARS and MERS were grouped together, and further termed as SARS and MERS group in the text since studies in MERS only consisted of two case reports. 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.

Study selection based on PRISMA flow diagram. PRISMA, Preferred Reporting Items for Systematic Reviews and Meta-Analyses.

4 CHARACTERISTICS AND OUTCOMES OF CPT RECEIVING PATIENTS IN COVID-19, INFLUENZA, SARS AND MERS CASES 4.1 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 (60.23% vs. 39.63%). Major comorbidities in the studies were hypertension and respiratory system diseases among COVID-19 and influenza patients, while diabetes and cardiovascular diseases were identified among COVID-19 patients. In addition to CPT, all patients received standard treatment of antiviral therapy and corticosteroid. The commonly used antiviral in influenza cases was oseltamivir, in SARS and MERS cases was ribavirin, while in COVID-19 cases was remdesivir. Hydroxychloroquine was only used in COVID-19 patients. Antibiotics and/or antifungal treatments were also given as secondary bacterial and/or fungal infections were indicated in the cases of 42.50% of COVID-19 patients and 76.64% of SARS and MERS patients. At the time of admission, the majority of patients were identified as having severe or critical illness.

TABLE 1. Baseline characteristics of CPT-treated patients admitted to the studies Characteristics COVID-19 (n = 36,401) (%) SARS and MERS (n = 107) (%) Influenza (n = 208) (%) Age ≤ 40 3486 (9.58) 5 (4.67) 0 40–60 12,209 (33.54) 3 (2.8) 1 (0.48) 60–70 8,984 (24.68) 0 0 ≥70 10,841 (29.78) 0 0 Not stated or uncategorized 881 (2.42) 99 (99.52) 207 (99.52) Gender Male 21,874 (60.23) 5 (4.67) 108 (51.92) Female 14,395 (39.63) 3 (2.80) 100 (48.08) Not stated 51 (0.14) 99 (2.52) 0 Comorbidities Diabetes 318 (0.87) 1 (0.93) 9 (4.33) Hypertension 352 (0.97) 0 22 (10.58) Cardiovascular diseases 102 (0.28) 0 10 (4.81) Respiratory system diseases 86 (0.24) 0 22 (10.58) Chronic kidney diseases 43 (0.12) 0 8 (3.85) Immunocompromised 19 (0.05) 0 0 Obesity 79 (0.22) 0 19 (9.13) Othersa 87 (0.24) 0 24 (11.54) Not stated 35,403 (97.26) 106 (99.07) 138 (66.35) Management before CPT Medications Antibiotics/antifungalb 15,472 (42.50) 82 (76.64) 10 (4.81) Antiviral therapy 10,999 (30.22) 107 (100) 166 (79.81) Arbidol 31 (0.09) 0 0 Lopinavir-ritonavir 220 (0.60) 3 (2.80) 0 Oseltamivir 7 (0.02) 0 127 (61.06) Ribavirin 8 (0.02) 104 (97.20) 0 Favipavir 28 (0.08) 0 0 Remdesivir 10,672 (29.32) 0 0 Unspecified/othersc 33 (0.09) 0 39 (18.75) Hydroxychloroquine/chloroquine 7597 (20.87) 0 0 Corticosteroid 17,933 (49.27) 102 (95.33) 0 Immunosuppresive drugs 140 (0.38) 0 9 (4.33) Immunotherapyd 77 (0.21) 3 (2.80) 0 Not stated 96 (0.26) 2 (1.87) 0 Oxygenation Low-flow nasal cannula 89 (0.24) 0 65 (31.25) High-flow nasal cannula 156 (0.43) 0 24 (11.54) Mechanical ventilation 9778 (26.86) 3 (2.80) 70 (33.65) Extracorporeal membrane oxygenation 14 (0.04) 1 (0.93) 10 (4.81) No requirement on oxygen supplement 8 (0.02) 0 34 (16.35) Not stated 26,344 (72.37) 103 (96.26) 0 Renal replacement therapy 13 (0.04) 0 0 Severity before CPT Mild 1 (<0.01) 0 0 Moderate 13 (0.04) 0 34 (16.35) Severe 25,935 (71.25) 0 89 (42.79) Critical 9889 (27.17) 8 (7.48) 82 (39.42) Severe or critical 99 (92.52) Abbreviations: CPT, convalescent plasma therapy; MERS, Middle East respiratory syndrome; SARS, severe acute respiratory syndrome. a Other comorbidities including gastro-oesophageal reflux disease, sleep apnoea, cancer, mental disorders and other neurological diseases. b Antibiotics or antifungal used were azithromycin, trazodone, moxifloxacin, cefoxatime, levofloxacin, clarithromycin, meropenem, cefoperazone sodium, linezolid, imipenem-sitastatin sodium, cefoperagone sodium, tazobactam sodium, fluconazole and caspofungin. c Other antivirals used including peremivir, zanamivir and darunavir. d Immunotherapy used including interferon (IFN)-alpha-2b, IFN-alpha-1b, IVIG (intravenous immunoglobulin) and monoclonal antibodies. 4.2 Primary outcomes of CPT in COVID-19, SARS and MERS and influenza patients

Mortality in COVID-19, SARS and MERS and influenza patients reached as high as 10.50% (n = 3725), 12.05% (n = 10) and 2.23% (n = 4) in 7 days after transfusion, and 24.26% (n = 8820), 0% (n = 0) and 7.69% (n = 15) in 30 days after transfusion, respectively. Serious transfusion-related adverse events are in the form of anaphylactic shock, deep vein thrombosis, sepsis, transfusion-related acute lung injury (TRALI), transfusion-related circulatory overload (TACO) and transfusion-related mortality were reported in ≤0.2% COVID-19 cases and only 1 reported in MERS cases (0.93%). Urticaria was reported in 9 (0.04%) COVID-19 and 4 (1.92%) influenza patients, while no SARS and MERS and influenza patients experienced this mild adverse effect. Febrile non-haemolytic transfusion reaction was reported in one COVID-19 case (<0.01%). Transfusion reaction symptoms of haematuria and dyspnoea were reported in two COVID-19 cases (<0.01) (Table 2).

TABLE 2. Primary outcome of patients receiving CPT Outcomes COVID-19 (n, %) SARS and MERS (n, %) Influenza (n, %) Status during 30 days after transfusion Discharged 528 (1.46) 19 (79.17) 154 (78.97) Hospitalized 75 (0.21) 5 (20.83) 26 (13.33) Deceased 8802 (24.26) 0 15 (7.69) Lived, not specified 26,875 (74.08) 0 0 Total 36,280 (100.00) 24 (100.00) 195 (100.00) Status during 7 days after transfusion Discharged 20 (0.06) 33 (39.76) 94 (51.51) Hospitalized 43 (0.12) 40 (48.19) 81 (45.25) Deceased 3725 (10.50) 10 (12.05) 4 (2.23) Lived, not specified 31,679 (89.32) 0 0 Total 35,467 (100.00) 83 (100.00) 179 (100.00) Transfusion-related adverse effectsa Anaphylatic shock 28 (0.13) 0 0 Urticaria, mild effects 9 (0.04) 0 4 (1.92) Deep vein thrombosis 42 (0.20) 0 0 Febrile non-haemolytic 1 (<0.01) 0 0 Haematuria 1 (<0.01) 0 0 Transfusion-associated dyspnoea 1 (<0.01) 0 0 Sepsis 3 (0.01) 0 0 TRALIb 23 (0.11) 1 (0.93) 0 TACOc 37 (0.18) 0 0 Transfusion-related mortality 16 (0.08) 0 0 Abbreviations: MERS, Middle East respiratory syndrome; SARS, severe acute respiratory syndrome; TACO, transfusion-associated circulatory overload; TRALI, transfusion-related acute lung injury; . a Transfusion-related adverse effects were reported for 21,079 Covid-19 patients, 107 SARS and MERS patients and 208 influenza patients b TRALI, Transfusion related acute lung injury c TACO, Transfusion-associated circulatory overload 4.3 Secondary outcomes of CPT in COVID-19 patients

Secondary outcomes are measured as time to hospital discharge, time to negative viral titre, improvement of oxygenation and laboratory findings. None of the influenza, SARS and MERS studies included in this systematic review reported any secondary outcomes. The mean time to discharge after CPT in COVID-19 patients was 14.78 days, while the mean time to negative viral titre was 3.04 days. Oxygenation baseline indicated that patients experienced moderate level of AR