Convalescent Plasma to Treat Covid-19: a Randomized Double Blind 2 Centers Trial

doi:10.21203/rs.3.rs-1277990/v1

In SARS-CoV-2, there is an overactivation of the immune system that triggers systemic hyperinflammation that causes lung damage; therefore, the use of convalescent plasma (CP) has been considered for its immunological mechanisms that could benefit patients in moderate and severe stages of the disease. This study evaluated the safety and efficacy of the use of convalescent donor plasma for COVID-19 to reduce mortality in patients with SARS-CoV-2 stage II (moderate) and stage III (severe) disease.

Material and methods

A double-blind, randomized controlled clinical trial was conducted from May 20 to December 10, 2020. Thirty-nine participants with moderate (II) and severe (III) stage COVID-19 confirmed by RT-PCR and tomography were included. The study randomization rate was set at 3:1. Convalescent plasmas were chosen for application with a neutralizing antibody titer of ≥ 1:32. Patient follow-up included assessment by Sequential Organ Failure Assessment (SOFA) score and use of the Oxygenation Index (PAO₂/FIO₂) index and monitoring of blood markers, such as C Reactive Protein (CRP), D-Dimer (DD), ferritin, IgG antibody titers against SARS-COV-2, and inflammatory cytokines at days three and seven post-treatment.

Results

We observed a significantly lower 21-day post-transfusion mortality HR: 0.17 [95.0% CI 0.07-0.45, p<0.001] in the group receiving convalescent plasma compared to the control group; protective units (PU) in the group receiving convalescent plasma after seven days were significantly higher 512 (32-16384) vs 96 (32-256), p=0.01; the SOFA scale decreased to 3.7 ± 2.02 vs 7.1 ± 2.8, p<0.001 and the PAO₂/FIO₂ index showed a significant improvement in the group receiving convalescent plasma 251.01 ± 109.4 vs 109.2 ± 62.4, p<0.001, vs the control group. In terms of safety, no adverse events related to the transfusion of convalescent plasma were observed.

Conclusion

Convalescent plasma is safe and effective, as it decreases mortality in the convalescent plasma group compared to the control group.

Convalescent plasma treatment

COVID-19

neutralizing antibodies

SARS-CoV-2

Safety and efficacy of the use of convalescent plasma in patients with COVID-19.
The presence of neutralizing antibodies ensures the efficacy of the convalescent plasma.
Decrease in mortality in patients treated with convalescent plasma.

Treatment of the disease is a challenge because of the lack of clinical evidence regarding the effectiveness and safety of specific antiviral agents for the management of COVID-19. When facing an emergency, different treatment regimens have been used, such as the lopinavir/ritonavir combination, which did not result in a reduction in overall mortality. [1] Another randomized controlled trial using hydroxychloroquine showed a reduction in body temperature and remission of cough in the intervention group compared to controls; however, these studies were not robust due to short periods and small sample sizes.

In the absence of evidence for specific treatment against COVID-19, classical and historical interventions have emerged as options for disease control. A passive immunization strategy with CP has been used to prevent and manage emerging infectious diseases since the early 20th century. In 2015, a protocol for the use of CP to treat Middle East respiratory syndrome (MERS) [2] and in 2009 for pandemic H1N1 influenza was conducted. In this prospective cohort study by Hung et al., a significant reduction in the relative mortality risk odds ratio 0.20 [95% CI 0.06-0.69) was observed in patients treated with CP (p=0.01). [3]

CP is obtained by apheresis from survivors with previous infections caused by pathogens of interest, in which antibodies develop against the causative agent of the disease. Since its rapid acquisition, CP has been considered an emergency intervention in different pandemics: Spanish flu, West Nile virus, and Ebola virus. [4] Therefore, the strategy of using CP has been proposed as a treatment option for SARS-CoV-2 infection. To date, several studies have reported controversial results regarding the use of CP. [5] Based on the scientific evidence obtained so far, CP was granted emergency use authorization to treat hospitalized patients with COVID-19 on August 23, 2020, by the Food and Drug Administration (FDA). [6] Its application met the "maybe effective" standard for emergency use, and it is reasonable to consider the already known potential benefits of CP. [7] There have been a lot of different results, some of them showed positive results, like Lili L et al [8] or Yogiraj R et al whom, a single center assay open label and randomized controlled by standard treatment (PICP19), reported 25.0 % versus 35.0 % mortality in a CP arm (RR= 0.71, CI 95.0 %: 0.36-1.41). [9] In another study, Avendaño C. et al, in a multicenter randomized clinical trial (ConPlas-19), they found 0.0 % of mortality versus 9.3 % in a standard treatment arm (p=0.07). [10]

This study aimed to evaluate the safety and efficacy of using plasma from convalescent donor COVID-19 to reduce mortality in patients with moderate (II) and severe (III) stage SARS-CoV-2 disease.

The study was conducted by the Blood Bank of the Centro Médico Naval (CEMENAV) and the Hospital General de México "Dr Eduardo Liceaga" (HGM) in Mexico City, from May 20, 2020, to October 10, 2020. The study was approved by the Research Ethics Committee and the Research and Biosafety Committee of both institutions. Written informed consent was obtained from each patient or responsible family member, as well as from convalescent plasma donors. The protocol was authorized by the Comisión Federal para la Protección contra Riesgos Sanitarios (Federal Commission for the Protection against Health Risks, COFEPRIS) with the registration number: CE/168/20. Trial Registration. ClinicalTrials.gov Identifier: NCT04405310.

Study design.

A parallel, double-blind, randomized phase II clinical trial compared CP and placebo (albumin 0.5%) for patients with stage II and II SARS-COV-2 pneumonia. Four treatment groups were formed: group A, patients with stage II pneumonia who received CP and conventional therapy; group B, patients with stage II pneumonia who received albumin solution and conventional therapy, group C: patients with stage III pneumonia who received convalescent CP and conventional therapy; and group D, patients with stage III pneumonia who received albumin solution and conventional therapy. Conventional therapy consisted of azithromycin and hydroxychloroquine.

CP donor selection criteria

Diagnosis of COVID-19 confirmed by RT-PCR, complete resolution of symptoms, with subsequent RT-PCR test with a negative result, male, no history of transfusion in the last 15 days, age 18-55 years old, weight > 50 kg, and authorization of informed consent, comply with the requirements for blood donors established in the Mexican Official Standard (NOM-253-SSA1-2012).

CP receiver selection criteria

Adults ≥18 years of age with a diagnosis of COVID-19 confirmed by RT-PCR stages II and III. An Hscore ≥169 points and the presence of severe acute hypoxemia with SpO2 <90% on room air and PaO₂/FiO₂ <300 mmHg. Meeting imaging criteria for SARS-CoV-2 stage II and III pneumonia (plain chest CT scan or plain chest X-ray), with the requirement for supplemental oxygen either via face mask plus reservoir bag, high-flow nasal prongs or advanced airway management, and invasive mechanical ventilatory support, C-reactive protein (CRP) value increased by 3.5 the baseline or greater than 18 mg/dL. Stage II was defined as evidence of lower airway disease, either by clinical assessment or imaging, and oxygen saturation (SpO2) ≥94%. Stage III was defined as a respiratory rate >30 breaths per minute, SpO2 <94%, ratio between the arterial partial pressure of oxygen and the fraction of inspired oxygen (PaO₂/FiO₂) <300 mmHg, or pulmonary infiltrates >50%.

Interventions

Recipients assigned to CP received two units of 300 mL intravenously, one on day one and the second unit on day three, with a neutralizing antibody titer ≥ 1:32, equivalent to 256 UP/mL. The recipients in the control group received two units of 300 mL of 20% albumin in Hartman's solution intravenously on day one and day three.

Inactivation of convalescent plasma

Inactivation of CP with riboflavin and UV light from each CP donor, 600 mL was collected by apheresis. The plasma was divided into two units (A and B) of approximately 300 mL for subsequent pathogen inactivation with riboflavin and UV light.

Two sterile disposable kits for plasma were used (Ref. 10390 Terumo BCT), the plasma was transferred to the illumination bag provided in the kit using a hose coupling equipment (T-SEAL II, Terumo Tube Sealing Device) and mixed with a riboflavin solution (500 µm in 0.9% sodium chloride solution, pH 3.5–5.1). After the riboflavin-plasma mixture was inoculated with the virus, the samples were placed into the Mirasol Illuminator (Terumo BCT) for UV treatment. The plasma units were exposed to 6.24 J/ml of energy.

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR).

From nasopharyngeal samples, extraction of total genetic material from potential donors and recipients was performed using the QIAamp Viral RNA kit (Qiagen, Germany), using the equipment (QIAcube-classics, Germany). The amplification of specific genes (Rd, Rp, E, N) for SARS-CoV-2 was performed using a GeneFinder COVID19 PLUS realAmp kit, and qRT-PCR was performed using the Applied Biosystems 7500 FAST kit (Applied Biosystems, USA). All samples were inactivated in a class A-II biosafety cabinet following the Biosafety and Good Laboratory Practice protocols issued by the WHO and the Instituto de Diagnostico y Referencia Epidemiological (Institute of Diagnosis Epidemiological Reference, INDRE) in Mexico.

IgG nucleocapsid determination.

Detection of IgG nucleocapsid antibodies from donor and recipient were analyzed in an Abbott Architect (Abbott Diagnostics®), according to the manufacturer's instructions. The chemiluminescent assay detects IgG raised against the SARS-CoV-2 nucleocapsid protein. A signal to cut-off (S/CO) ratio of ≥1.4, was interpreted as reactive. Calibration was performed, and the positive quality control S/CO 1.65-8.40 and negative quality control S/CO ≤ 0.78 were met prior to performing the studies.

IgG S1/S2 spikes protein determinants.

The LIAISON SARS-CoV-2 S1/S2 IgG antibody (quantitative assay) was performed using a Liaison-XL (DiaSorin®). The cut-off was > 15.0 AU, which included a negative (<3.8 AU) and positive (>31.9 AU) control. The assay was performed according to the manufacturer's instructions.

Neutralizing antibody assays.

An in vitro neutralization assay was developed similar to that reported by Beales et al.5 For this, serial dilutions were made with minimum essential medium and 50 µL of donor and recipient sera. 5ml of SARS-CoV-2 virus (MOI=0.1) was added for each dilution, which was previously titrated using a lytic plate assay. The dilutions were incubated for one h at 37 °C. Each dilution was inoculated into a 96-well plate, seeded with VERO.E6 cells, and incubated at 37 °C and 5% CO2 for two days. Plates were washed with PBS IX, fixed with an ethanol/acetone mixture (1:1) for 15 min, and stained with crystal violet for 20 min. The titer was obtained by considering the dilution at which the first lytic plaques appeared. The dilution titer was converted to protective units. The inverse of the dilution was divided by the final volume of each well (0.125 mL), and its titer per final volume of 1 mL was considered, which is equivalent to the protective units/mL (PU/mL). The lowest transfusion titer was 256 PUs.

Determination of inflammatory cytokines.

Inflammatory cytokine levels were determined in plasma samples from recipients on the day before transfusion and on days 3 and 7 post-transfusion. A flow cytometry panel was analyzed, where 13 human cytokines were quantified as described in the test methodology (IL-1β, IFN-α2, IFN-γ, TNF-α, MCP-1, IL-6, hIL-8, IL-10, IL-12p70, IL-17A, IL-18, IL-23, IL-33 Multi-analyte flow assay kit LEGENDplex (BioLegend® USA), and samples were acquired using a FACS Canto II flow cytometer (BD®, USA), obtaining 4000-5000 events in was sample using the LEGENDplexv8 software performed cytokine analysis.

Generation and concealment of random allocation.

Random sequence generation was performed by an external monitor who assigned a code to each patient who met the inclusion criteria. The staff who recruited the patients had no way of knowing which intervention each participant was assigned. A 3:1 allocation of patients was chosen because of the ethical issues involved in not providing a potential clinical benefit to a population with a high fatality rate. Figure 1 shows the process of patient selection and allocation to the two treatment arms.

The CEMENAV and HGM blood banks provided the monitor with a list of convalescent plasma and potential recipients selected by the intensivist in charge of the intervention. Once the random allocation had been performed, staff assigned by the Blood Bank, who was not involved in the randomization of treatments, placed the CP and albumin solution bags in a metal bag before taking them to the COVID hospitalization area for administration. The staff involved in the infusion of plasma and solutions and the patients and staff who treated them were blinded to the treatment received. Figure 1.

Statistical methods

As this was a pilot study and considering the health emergency caused by the pandemic, we chose to use a convenience sample size to evaluate the efficacy and safety of CP in patients with COVID-19 pneumonia.

Survival at 15 and 21 days was estimated by the Kaplan-Meier method (95% confidence interval [CI]) and comparisons by group using the log-rank test and hazard ratios (HRs). Measures of central tendency and dispersion were calculated according to the type of variable. The t-test was performed for differences between standard variables and non-parametric tests for non-normal variables. Multivariate analysis was performed to identify variables with significant relationships in univariate analysis.

General

Thirty-nine patients were included in the trial: 16 in group A (moderate + CP), 3 in group B (moderate + albumin solution), 13 in group C (severe + CP), and 7 in group D (severe + albumin solution). Of the 39 patients (Figure 1), 9 (23.1%) were women, 35.9% had type II diabetes mellitus, 51.3% had systemic arterial hypertension, and only 2.6% had chronic obstructive pulmonary disease. The distribution of these comorbidities did not differ significantly between treatment groups. There were no cases of liver disease or ischemic disease among the selected patients.

Table 1 shows the demographic characteristics of the patients included in the treatment group and the mean values of the variables assessing the clinical status and IgG anti-nucleocapsid antibody titers, anti-spicule proteins, and neutralizing antibodies at screening (baseline). In these values, only the PAO₂/FIO₂ index showed a significant difference between treated patients and controls; however, both results reflected the same clinical status in the two groups.

Table 1

Description of the population at the time of selection.

DAY BEFORE TRANSFUSION
	Totals	Convalescent plasma	Control	P
Age (years old)	55.9 ±9.6	55.5 ±10.3	56.9± 7.5	0.7*
BMI (kg/cm2)	30.7 ±5.5	31.5 ±5.5	28.7 ±5.24	0.17*
SOFA	5.1 ±2.6	4.8 ±2.7	6.1 ±2.23	0.2*
PAO₂/FIO₂	146.7 ± 69.6	160.6 ±74.4	107.8 ±31.9	0.004*
Ferritin	855.6 (139-2302)	855.6 (73.2-2607.5)	933.5 (321-1532)	0.56^#
CRP (mg/dL)	33.5 (1.3-377)	22.5 (1.7-372.0)	86.3 (0.68-377)	0.4^#
DD (ng/mL)	788 (135-7299)	784 (154.8-5924.5)	956 (135-7285)	0.64^#
IgG (NC) (OJ)	7.3 (3.6-8.8)	7.4 (3.2-9.0)	6.6 (4.2-7.9)	0.09^#
IgG (Spike) (OD)	188.13 ± 88.7	190.7 ± 98.7	181.8 ± 62.5	0.804*
UP	128 (16-2048)	128 (16-2048)	128 (32-512)	0.94^#

BMI, body mass index; SOFA, C-reactive protein; DD, Dimer D; IgG (NC), IgG anti-nucleocapsid antibodies; OD, optical density; IgG (spike), IgG anti-spike antibodies; UP, protective units. T-test* and Mann-Whitney^# U-tests were applied for normal and non-normal distributions, respectively.

The changes in the variables studied in the patients at seven days post-transfusion are shown in Table 2, where the value obtained for the SOFA scale was significantly lower in the CP group, whereas in the case of the PAO₂/FIO₂ index and the PU titer, both results were significantly higher in the CP group than in the control group.

Table 2

7-day post-treatment follow-up.

DAY 7 POST-TREATMENT
	Total	Convalescent plasma	Control	P
SOFA	4.8 ±2.8	3.7 ±2.02	7.1 ± 2.8	0.001*
PAO₂/FIO₂	202.1 ± 116.8	251.01 ±109.4	109.2 ± 62.4	<0.001*
Ferritin	733.6 (207.2-2973.6)	704.7 (182.9-2813)	1057.6 (237-3105)	0.34
CRP (mg/dL)	7.8 (0.6-251.1)	6.5 (0.51-157.4)	89 (0.65-382)	0.07
DD	1590.5 (215.1-7399)	1666.5 (278-9977.3)	1300.5 (142-5040)	0.61
IgG (NC) (OJ)	7.7 (4.9-8.9)	7.8 (4.8-9.14)	7.23 (5.10-8.4)	0.39
IgG (Spic) (OD)	280.3 ± 97.7	269.2 ± 100.5	304.1 ± 94.3	0.448
Protective Units (PUs)	256 (32-16384)	512 (32-16384)	96 (32-256)	0.01*

BMI, body mass index; SOFA, C-reactive protein; DD, Dimer D; IgG (NC), IgG anti-nucleocapsid antibodies; OD, optical density; IgG (spike), IgG anti-spike antibodies; UP, protective units. T-test* and Mann-Whitney^# U-tests were applied for normal and non-normal distributions, respectively.

Although CRP, ferritin, and DD values in the plasma group were lower at seven days post-transfusion, no significant differences were observed.

When comparing the change in SOFA and PAO₂/FIO₂ index values in the study groups between baseline and seven days after treatment, it was found that in the CP group vs. control group, the decrease in SOFA score was statistically significant p=0.014 in the CP group than in the control group (p = 0.168). In the case of the PAO₂/FIO₂ ratio, the increase observed at seven days in the CP group vs. the control group showed a significantly higher value, p=0.001 vs. p=0.946 (Tables 1 and 2).

In terms of the ability to prevent the development of severe disease or to prevent the use of invasive mechanical ventilation, no significant differences were found between the plasma and control groups, neither the number of days after transfusion hospitalization (data not shown).

When comparing IgG antibody titers against SARS-COV2 (anti-spike and anti-nucleocapsid) at day seven post-transfusion, no significant difference was found (Table 2); however, the UP/mL titer at day 7 in the CP group was higher than that in the control group: 512(32-16384) vs 96 (32-256), p=0.01.

Quantification of inflammatory cytokines

In the patients who received CP, an increase at day three post-transfusion was observed in 10 cytokines (IL-1β, INF-α2, INF-γ, MCP-1, IL-8, IL-10, IL-12p70, IL-18, IL-23, and IL-33), which in most cases was statistically significant. Nevertheless, there was a downward trend at day 7 for most cytokines compared to the baseline and day 3. The latter was expected, but it was not statistically significant because some patients could not be analyzed due to a lack of samples. In contrast, in the control group, we observed a different behavior with a decrease in some cytokines at day three but an increase at day 7, of which only IL-12p70 and IL-23 showed statistically significant differences (Table 3).

Table 3

Measurement of inflammatory cytokines by groups (CP and control) on the day before, day 3, and day 7 post-transfusion.

pg/mL

CONVALESCENT PLASMA

CONTROL GROUP

Basal

D-3

D-7

p

Basal

D-3

D-7

p

IL-1β

8.1*

(8.1- 410)

17.5*

(8.1-1542)

8.1

(8.1-114.6)

0.059

8.1

(8.1-57)

8.1

(8.1-37.4)

29.4

(8.1-61.4)

NS

INF-α2

2.7*

(1.9-498)

5.1*

(2.2-3029)

5.1

(2.2-23.5)

0.043

11.1*

(1.9-19.6)

6.2

(2.2-16.4)

9.8*

(3.1-23.5)

NS

INF-γ

5.9*

(5.9-942)

19.9*

(5.9-4677)

7.2

(5.9-110)

0.017

5.9

(5.9-34)

7.2

(45.9-34)

23.4

(5.9-49)

NS

TNF- α

146.7

(14.3-20513)

133.7

(16.0-8381)

107.0

(16.6-260)

NS

151

(14.3-11654)

120.6

(54.7-198)

94.3

(55-160)

NS

MCP-1

988.8*

(25.4-6496)

6004*

(91.7-5636)

493

(33.5-1186)

0.013

908

(189-9427)

844

(339-5798)

733

(136-1685)

NS

IL-6

89.8

(12.0-722)

60

(12-18835)

45

(12-823)

NS

104.7

(12.1-4162)

119.0

(45-1116)

60

(12.1-488)

NS

IL-8

80.0*

(69-706)

181*

(69-4402)

169

(69-1359)

0.007

326

(69-3672)

217

(90-3536)

267

69-665)

NS

IL-10

2.7*

(2.7-261)

27.9*

(1.7-479)

25

(2.7-142)

0.006

2.7

(2.7-217.0)

13.4

(2.7-6323.)

64

(13.0-89)

NS

IL-12p70

3.2

(3.2-24)

11.3

(3.2-20.1)

8.0

(3.2-31.4)

NS

3.2*

(2.2-21.4)

9.6

(7.-12.5)

14.9*

(8-27)

0.028*

IL-17a

2.7*°

(2.7-20.5)

8.9*

(2.7-645)

8.9°

(2.7-22.1)

<0.001*

0.017

2.7

(2.7-108)

8.3

(2.7-16.2)

11.9

(4-22)

NS

IL-18

427*

(27-1062)

549*

(55.4-2082)

572

(111-2436)

0.036

620

(250-2684)

476.0

(206-2868)

472

(45-1305)

NS

IL-23

7.1*°

(7.1-78.6)

26.6*

(7.1-125)

41.2°

(7.1-95.8)

0.002*

0.047°

7.1*

(7.1-51.6)

36.2

(7.1-67.8)

46.4*

(26.6-90.0)

0.018*

IL-33

3.8*

(3.8-154)

19*

(3.8-5967)

22.5

(3.8-95)

0.007*

3.8

(23.8-40.7)

15.7

(3.8-37)

29.6

(3.8-60.1)

NS

The Wilcoxon test was applied.

Mortality

When considering the treatment group, in the CP group, it was 13.8% (4/29), while in the control group, it was 80% (8/10), RR 0.17 [95% CI; 0.07-0.45].

Considering severe (III) vs. moderate (II) patients, mortality was lower in both groups, in those who received CP vs. those who received the albumin solution (Table 4).

Table 4

Mortality analysis by treatment and comorbidities. N=39

Condition 1	% Mortality	Condition 2	% Mortality	p
Woman	12.5	Man	35.48	0.2
Plasma	13.8	Control	80.0	<0.001*
Plasma (III)	30.7	Control (III)	100	0.003*
Plasma (II)	0.0	Control (II)	33.33	NS
Intubated	57.1	Non-intubated	0.0	0.0001*
Diabetic	27.27	Non-diabetic	32.1	0.76
AHT	36.8	No AHT	25.0	0.42
Smoker	0.0	Non-smoker	34.3	0.15
With Tocilizumab	100 (1/1)	Without Tocilizumab	29.7	0.135
With steroids	42.3	Steroid-free	8.3	0.036*
Steroids (III)	52.7	Steroid-free (III)	100 (1/1)	0.35
Steroids (II)	14.3	Steroid-free (II)	0.0	0.197
With antibiotics	36.4	No antibiotics	25.0	0.46
Antibiotics (III)	50.0	No antibiotics (III)	66.7	0.49
Antibiotics (II)	12.5	No antibiotics (II)	0.0	0.25

AHT: Hypertension. II: Covid-19 in the moderate stage; III: Covid-19 in the severe stage.

Of the 19 patients included in COVID-19 stage II, 16 received plasma. Mortality in the plasma group was 0.0% (0/16) and 33.33% (1/3) in the control and control groups, respectively. No statistically significant difference was found in the mortality of stage II patients receiving plasma compared to the control group RR 0.08 [95% CI; 0.00-1.59]. Of the 20 patients included in stage III, 13 received plasma. Mortality in the plasma group was 30.76% (4/13), and in the control group was 100% (7/7), RR 0.31 [95% CI; 0.14-0.7] (Table 4). Mortality analysis under different conditions is also shown. There was no association between sex and mortality, Diabetes Mellitus (DM), Arterial Hypertension (AHT), smoking, use of broad-spectrum antibiotics, or tocilizumab. The mortality rate of patients who received steroids was 42.3% compared to those who did not (8.3%) (p= 0.036). In the sub-analysis by severity, statistical significance was lost.

For the thrombosis prevention scheme, three grades of anticoagulation were used: prophylactic doses (40 mg/24 h), intermediate doses (60 mg/24 h), and total doses (1 mg/kg body weight every 12 hours). Higher mortality was found in patients with intermediate and full doses than in those who received only prophylaxis (Table 5).

Table 5

Mortality and thromboprophylaxis.

Anticoagulant treatment	% Mortality	Anticoagulant treatment	% Mortality	Anticoagulant treatment	% Mortality	P
Prophylaxis	7.14	Intermediate dose	50.0	Anticoagulation	45.5	0.015
Prophylaxis + moderate	0.0	Intermediate + moderate dose	0.0	Anticoagulation + moderate	20.0	0.097
Prophylaxis + severe	100.0 (1/1)	Intermediate + severe dose	50.0 (1/2)	Anticoagulation + severe	52.9	0.35

Prophylaxis: 40 mg/24 h, intermediate dose: 60 mg/24 h, and anticoagulation: mg/kg body weight.

In the plasma group, the median survival at 21 days was 19.5 days [95% CI 17.5-21.5), and in the control group, it was 16 days (95% CI 12.136-19.864), with a median of 17.0 days [95% CI 12.3-21.65). There was an increased survival in patients receiving plasma (p=0.001), regardless of the outcome (HR 0.129 [95% CI 0.039-0.432]). Figure 2 shows the cumulative 21-day mortality curves.

A post hoc multivariate analysis was performed to determine the contribution of some clinically essential variables to mortality; three variables were associated with the model: plasma application (p<0.001), disease severity (p=0.006), and thromboprophylaxis (p=0.028). However, in the multivariate analysis, no variables were independently associated with mortality.

Convalescent plasma in early discharge

Early discharge was considered when patients were discharged up to 15 days after CP/albumin solution infusion; of the patients who received CP, 71.0% were discharged before 15 days, and 29.0% were discharged after 15 days. Of those who received albumin solution, 60.0% were discharged before 15 days and 40% after, which showed no significant difference in the number of days they remained in the hospital after either CP or albumin solution.

Convalescent plasma as a protector for invasive mechanical ventilation (IVM)

Of the 24 patients who were not treated with IVM at the time of CP or albumin application, only 4/19 who received CP received IVM, while in the group receiving albumin, only 3/5 received IVM, showing no significant protective effect against IVM for CP application over albumin (OR=0.178 [0.022-1.15], p= 0.09).

Safety

There were no adverse events related to the transfusion of convalescent plasma or in the control group in the trial participants.

We reported the use of convalescent plasma together with standard treatment versus albumin in the control group plus standard treatment. We found a significant decrease in mortality in the CP group compared to that in the control group (13.8% vs. 80.0%, p=<0.001), as well Abolghasemi H. et al, [11] who found low mortality in the CP group: 14.8 % versus 24.3 % in control group (p=0.09), with a lower hospital stay partial and total.

Rasheed et al reported in a small study of 49 critically ill patients, 21/49 received 400 mL of CP in addition to standard care. Patients who received PC reported shorter time to clinical improvement and low mortality (4.8% vs 28%, p=0.03). [12]

Libster et al. conducted a randomized, placebo-controlled double-blind trial in which the plasma applied contained high titers of antibodies. Their results showed a more significant effect of early infusion: RR=0.4, 95% CI: 0.2-0.81; however, these results were directly related to the number of antibodies in the transfused plasma. [13] In our study, the selection of plasmas for infuse was based on the specificity to neutralize SARS-COV2, which is reflected in the high PU concentration achieved at seven days in patients who received CP. In this study, we used a neutralization assay with a high degree of specificity, which also showed a significantly higher titer in the group that received CP at seven days than in those who received albumin (96 vs 512, p=0.006), a situation that may make a difference in the benefit of using convalescent plasma when it was chosen based on a neutralization assay such as ours. Conversely, several published studies did not perform neutralization assays, and this lack of standardization in the selection of donor plasmas could influence the difference in results between publications. [8,9,14]

It is worth noting that the change in the SOFA score value in the group that received plasma, which decreased by almost two units from baseline (SOFA baseline=5.1, SOFA 7 days=3.7, N=19, p=0.014), although a 2 unit increase in this scale was used to predict mortality risk, [15] we could consider the possible relationship with improvement when it decreased. Regarding the PAO2/FIO2 index, both groups presented clinically similar baseline values (Table 1). Seven days after the intervention, the group that received CP presented a significantly higher value than the control group. Our data are similar to those reported by Shen C et al., who in a series of five cases in which convalescent plasmas with titers equal to or higher than 1/1000, in patients with severe COVID-19, the Kirby index also showed a significant elevation in patients who evolved favorably. [16] As well Sarkar S. et al., [17] who in a systematic review and meta-analysis reported that convalescent plasma showed less mortality than traditional treatment OR=0.44, CI 95 %: 0.25-0.77. These results were related to increased viral clearance OR=11.3, CI %: 4.9-25.9, and a better clinical evolution even though the latter was not significant.

Our study found no significant differences in inflammation-related variables such as ferritin, CRP, and D-dimer levels, which may be due to the limited sample size.

During acute SARS-CoV-2 infection, pro-inflammatory cytokines may contribute to the pathology leading to acute respiratory distress syndrome, the life-threatening form of the infection. Several cytokines such as TNF-α, IL-6, IL-8, IL-18, amongst others, have been associated; these proteins contribute to tissue damage. [18] However, when evaluating the different cytokines in this study, we only found in the CP group an increase in IFN-γ and INF-α2 at day three post-transfusion and its maintenance at day 7, which are directly associated with viral elimination and the maintenance of cells of the innate immune response, promoting the activation of more cells and initiating a specific adaptive response. In previous studies, a good prognosis was observed in patients with increased type I interferon such as INF-α2, where patients treated with this cytosine improved and efficiently eliminated the virus in less time, demonstrating the direct effect on the clearance of the infection. [19] Cytokine levels have been reported as poor prognostic factors for patients with COVID-19 because they generate hyper-inflammation, including IL-2, IL-6, IL-7, IL-8, IL-10, G-SCF, IP10, MCP1, MCP1, MIP1A, and IL1-β. [20] In our study, we observed an increase in the cytokines as mentioned earlier in patients in the control group up to day 7, which indicates a state of continuous inflammation that can directly over activate the innate immune response, such as macrophages and polymorphonuclears amongst others for their chemotaxis, perpetuating the arrival of more cells and promoting tissue damage, which could lead to clinical deterioration in the patient. In contrast, the levels of cytokines IL-17a, IL-23, and IL-33 were increased in patients with CP. These cytokines play an essential role in inflammation, since previous studies have shown that patients recovered from COVID-19 have persistent circulating cells that produce IL-33 in response to virus-specific T-cell activation. These cells were correlated with increased amounts of specific antibodies. [21] In our study, we observed a correlation between CP-treated patients and increased IL-33 expression, which could be attributed to the patient's clinical improvement.

Therefore, it is necessary to thoroughly evaluate immunological findings in these patients, from cytosines in supernatants to cell populations, as the difference in some treatments may be associated with immunological responses such as in CP.

One of the most extensive meta-analyses published included 430 781 participants with 115 fatal events without conclusive results. In this study, Klassen et al. reported an association with reduced mortality based on 13 non-randomized studies (OR: 0.50, 95% CI: 0.37-0.67), and the authors allude to the lack of uniformity in the protocols for the use of CP, which allowed the application of plasmas in late stages or with very low antibody titers. Hence, they performed a sub-analysis excluding the results of the PLACID trial, where the donated plasmas showed very low titers of neutralizing antibodies 1:40 (interquartile range, 1:30-1:80) so the sub-analysis is in favor of the use of plasma, and there is still a need for standardization in the processes involved in the use of convalescent plasma to treat COVID-19. [22, 23]

The REMAP-CAP trial (NCT02735707), the RECOVERY trial (ISRCTN50189673) and the CONCOR-1 trial (NCT04348656), are large randomized controlled trials that issued the closure of recruitment for the PC intervention because their preliminary results did not show any benefit with its use and in the case of mortality, they found no significant difference at 28 days. However, we must wait for their final results and it is of great interest to know their experience with the use of PC. [24, 25, 26]

Limitations of the study.

We recognize the limitations in terms of the number of participants that did not allow us to find significant differences in the variables studied on factors that could be independently associated with the use of plasma, and therefore, with the increased survival of the patients who received it. However, the use of CP under the conditions of our study shows that it is safe and that it decreases mortality in severe and moderate stages.

In our experience, the convalescent plasma is safe and effective to treat COVID-19, as it decreases mortality compared to control group.

Arterial Hypertension: “AHT”

D-Dimer: “DD”

Diabetes Mellitus: “DM”

C Reactive Protein: “CRP”

Convalescent Plasma: “CP”

Invasive Mechanical Ventilation: “IVM”

Middle East respiratory syndrome: “MERS”

Oxygenation Index: “PAO₂/FIO₂“

Protective Units: “PU”

Reverse Transcriptase- Polymerase Chain Reaction: “RT-PCR”

Sequential Organ Failure Assessment: “SOFA”

Ethics approval and consent to participate.

The authors confirm that all methods were carried out in accordance with relevant guidelines and regulations.

This study was approved from the different committees of each hospital:

Hospital General de México. Ethics in Investigation Committee with registration number CE/168/20 (23/04/2020). Investigation Committee with registration number CI/140/20 (23/04/2020).
Centro Medico Naval: Biosecurity Committee with registration number CB/01/20 (13/04/2020). ANNEX 1

Informed consent was obtained from all the patients or responsible family member, as well as from convalescent plasma donors before their participation.Written informed consent was obtained from each patient or responsible family member, as well as from convalescent plasma donors. ANNEX 2

Clinical Trial Registration ClinicalTrials.gov: NCT04405310 (28/05/2020). The protocol was authorized by the Comisión Federal para la Protección contra Riesgos Sanitarios (Federal Commission for the Protection Against Health Risks, COFEPRIS) with the registration number: CE/168/20. ANNEX 3

Consent for publication.

Not applicable

Good practices.

The authors confirm that all methods were carried out in accordance with relevant guidelines and regulations.

Availability of data and materials

The data supporting the conclusions of this study are available in "related files" on the platform.

Declaration of conflict of interest and Funding.

The researchers declare that they have no conflict of interest in this research and there is no financial support from laboratories, pharmaceutical industry, or private initiative. This work was supported by CEMEVAV (Centro Medico Naval, Naval Medical Center).

Conflict of interest letter from participants. ANNEX 4

Authors contributions

AUTORES	Substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work.	Drafting the work or revising it critically for important intellectual content.	Final approval of the version to be published.	Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
A. Yanet Ventura-Enríquez [email protected] ORCID: 0000-0003-1691-0730	X	X	X	X
B. Carlos Cabello-Gutiérrez [email protected] ORCID: 0000-0002-3192-8915	X	X	X	X
C. Ángel Augusto Pérez-Calatayud [email protected] ORCID:0000-0003-1691-0730	X	X	X	X
D. Evelyn Cortina-De la Rosa [email protected] ORCID: 0000-0002-0604-2872	X	X	X	X
E. Javier Fareli-González Christian [email protected] ORCID: 0000-0001-8654-9077	X	X	X	X
F. Paola Castillo-Juárez [email protected] ORCID: 0000-0001-5086-3902		X	X	X
G. Carlos Alberto Peña-Pérez [email protected] ORCID: 0000-0003-2388-7823		X	X	X
H. Elí Omar Zavaleta-Martínez [email protected] ORCID: 0000-0002-5194-0508			X
I. Elizabeth Díaz-Padilla [email protected] ORCID: 0000-0002-2807-3418			X
J. SandraMurrieta [email protected] ORCID: 0000-0002-0138-2245			X
K. Violeta Deyanira Álvarez-Jiménez [email protected] 0000- 0002- 3873- 3109 0000- 0002- 3873- 3109 0000- 0002- 3873- 3109 ORCID: 0000-0002-3873-3109		X	X
L. Juan Alberto Díaz Ponce-Medrano [email protected] ORCID: 0000-0002-8206-7174			X
M. Catalina Casillas-Suárez [email protected] ORCID: 0000-0002-0508-478X			X
N. María Angelica Ocampo-Ocampo [email protected] ORCID: 000-0002-0199-1163			X
O. Verónica Fernández-Sánchez [email protected] ORCID: 0000-0001-9732-2265	X	X	X	X

Acknowledgments.

We would like to acknowledge the enthusiasm and participation of the medical, nurses and laboratory personal from both Medical Centers.

CONSORT 2010 checklist. ANNEX 5

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<li>&nbsp;REMAP-CAP Statement 12 January 2021. Available at:&nbsp;<a href="https://www.sciencemediacentre.org/expert-reaction-to-remap-cap-recruitment-of-severely-ill-covid-19-patients-into-convalescent-plasma-trial-being-paused-after-initial-analysis-suggested-it-did-not-improve-outcomes.%20Accessed%205%20February%202021">https://www.sciencemediacentre.org/expert-reaction-to-remap-cap-recruitment-of-severely-ill-covid-19-patients-into-convalescent-plasma-trial-being-paused-after-initial-analysis-suggested-it-did-not-improve-outcomes. Accessed 5 February 2021</a>.</li>

<li>&nbsp;RECOVERY Statement 15 Janurary 2021. Available at:&nbsp;<a href="https://www.recoverytrial.net/news/statement-from-the-recovery-trial-chief-investigators-15-january-2021-recovery-trial-closes-recruitment-to-convalescent-plasma-treatment-for-patients-hospitalised-with-covid-19" target="_blank">https://www.recoverytrial.net/news/statement-from-the-recovery-trial-chief-investigators-15-january-2021-recovery-trial-closes-recruitment-to-convalescent-plasma-treatment-for-patients-hospitalised-with-covid-19</a> Accessed 5 February 2021.</li>

<li>&nbsp;CONCOR-1: A Randomized, Open-Label Trial of Convalescents Plasma for Hospitalized Adults With Acute COVID-19 Respiratory Illness. Statement 3 February 2021. Available at:&nbsp;<a href="https://concor1.ca/" target="_blank">https://concor1.ca</a>. Accessed 5 February 2021.</li>

No competing interests reported.

Convalescent Plasma to Treat Covid-19: a Randomized Double Blind 2 Centers Trial

Status:

Version 1

Abstract

Figures

Highlights

Introduction

Methodology