Test design and supervision
We conducted a randomized, double-blind, placebo-controlled trial between June 4, 2020 and October 25, 2020 (when the last patient completed follow-up), in clinical centers and geriatric units in Argentina. The trial was approved by the institutional review boards of participating institutions and the state of Buenos Aires and was supervised by an independent data and security monitoring board. The authors who designed the study and wrote the manuscript are listed in Table S15 in the Supplementary Appendix, available with the full text of this article at NEJM.org. All authors compiled the data and attest to the accuracy and integrity of the data and the study’s adherence to the protocol, available at NEJM.org. Three of the authors analyzed the data. The last author wrote the first draft of the manuscript. No one who is not an author contributed to the writing of the manuscript. No confidentiality agreement related to the data is in effect between the sponsors and the authors or their institutions.
Trial patients
Patients 75 years of age or older, regardless of current coexisting diseases, or between 65 and 74 years of age with at least one coexisting disease, were identified and assessed for eligibility. The coexisting conditions, which are defined in Table S1, included hypertension or diabetes for which the patient was currently receiving pharmacological treatment, obesity, chronic renal failure, cardiovascular disease and COPD. At the time of screening for SARS-CoV-2 by polymerase reverse transcriptase chain reaction (RT-PCR), eligible patients had at least one of each sign or symptom in the following two categories for less than 48 hours: temperature of at least 37.5 ° C, unexplained sweating or chills; and dry cough, dyspnoea, fatigue, myalgia, anorexia, sore throat, dysgeusia, anosmia or rhinorrhea. Exclusion criteria included severe respiratory disease (the primary outcome), any disease listed in Table S5, or both.
Patients who gave consent for the screening received home visits, and samples of nasopharyngeal and oropharyngeal secretions were obtained for testing with an RT-PCR assay (iAMP COVID-19, Atila BioSystems) for the detection of SARS-CoV-2. Patients with SARS-CoV-2 detectable RNA were transported to the study hospitals and asked to sign the informed consent form. After July 22, 2020, legal guardians provided consent for patients with cognitive disabilities. As of July 27, 2020, since several geriatric institutions with SARS-CoV-2 outbreaks have been transformed into low-complexity inpatient units for residents with mild SARS-CoV-2-infected diseases, we screen and invite residents that met the study criteria in the on-site test.
Randomization and Intervention
Eligible patients who provided written informed consent were randomly assigned to receive 250 ml of convalescent plasma with an IgG titer greater than 1: 1000 against the SARS-CoV-2 spike (S) protein (COVIDAR IgG, Instituto Leloir, Argentina) or 250 ml of placebo (normal 0.9% saline). Convalescent plasma was arbitrarily defined as “high titer” and included concentrations of antibodies in the upper 28th percentile. A computer-generated randomization sequence with a balanced swapped block design (block size 2) was prepared in the data center.
Convalescent plasma or placebo was administered less than 72 hours after the onset of symptoms, and the infusions were administered over a period of 1.5 to 2.0 hours. Both the convalescent plasma and the placebo were concealed with opaque bags and tape to cover the infusion catheter. Patients were monitored for adverse events up to 12 hours after the intervention.
A total of 479 potential plasma donors who had SARS-CoV-2 infection for at least 10 days and who remained asymptomatic for 3 days or more and had two negative RT-PCR tests17 were identified through hospital lists and an online campaign. Potential donors who provided written informed consent were visited at home and screened for SARS-CoV-2 S IgG at a titre greater than 1: 1000 in serum. Each of the 135 candidates (28%) with suitable titles donated 750 ml of plasma (see Fig. S6).
Clinical and Laboratory Monitoring
24 hours after the end of the infusion, a sample of venous blood (5 ml) was obtained from the patients. Serum samples were preserved at -20 ° C until the end of the assay. We tested the anti-S IgG SARS-CoV-2 using the COVIDAR IgG test. In addition, we analyzed the samples using the SARS-CoV-2 Spike S1-RBD IgG immunoenzyme assay detection kit (GenScript) and the SARS-CoV-2 surrogate virus neutralization test kit (GenScript).
The clinical status of the patients was monitored daily by the trial physicians until the 15th to assess the primary outcome events that occurred in the hospital, participating geriatric institutions or at home if the patients had been discharged (Figs. S7 and S8). Patients who had persistent symptoms for which medical care was required were followed up until symptom resolution or for a maximum of 25 days to assess secondary outcome events. The study doctors used predefined questionnaires to collect clinical information. None of the patients received any experimental therapy for Covid-19 other than convalescent plasma. Data were recorded on paper forms and entered twice in an electronic database.
Test end points
The primary endpoint of the study was the development of severe respiratory disease, defined as a respiratory rate of 30 breaths per minute or more, an oxygen saturation of less than 93% while the patient was breathing ambient air, or both. Patients were evaluated for this final event between 12 hours after the infusion of convalescent plasma or placebo and the 15th day of participation in the study.
The pre-specified secondary clinical outcomes were life-threatening respiratory disease (defined as oxygen supplementation in a fraction of inspired oxygen [Fio2] 100%, non-invasive or invasive ventilation, admission to an intensive care unit or any combination thereof, critical systemic disease (respiratory failure with a ratio of partial pressure of oxygen to Fiotwo ≤200 mm Hg, shock, multiple organ dysfunction syndrome or any combination thereof) and death associated with Covid-19. Patients in whom the disease was not resolved were evaluated for these endpoint events until the 25th day of participation in the study. On July 22, 2020, we changed the protocol to include a fourth secondary endpoint that included any of the three secondary endpoints described above, either alone or in combination.
Early test termination
The trial began when the number of Covid-19 cases in Buenos Aires was high. However, as the number of cases decreased, it became clear that it would take about 5 months to reach the enrollment goal. Consequently, after discussions with the data and security monitoring board and enrollment of 76% of the target population, we decided that it would be logistically impossible and ethically questionable, given the daily cost of the pandemic in lives and illnesses, to continue the study, and we stopped to examine the results.
Statistical analysis
Given the complexity of implementing this intervention, the minimal clinically important difference was defined as a relative reduction from 40% to 50% of patients in the placebo group and 30% of patients in the convalescent plasma group who would have a primary endpoint-event. We estimate that a total sample size of 210 patients (105 per trial group) would give the trial 80% power to detect a difference between groups, at a significance level of α = 0.05. We used a bilateral z test of proportions with continuity correction and a planned interim analysis with the O’Brien-Fleming spending function to determine the test limits.
In the intention to treat analysis, the end points were evaluated from the moment of randomization. Continuous variables are presented as means and standard deviations or medians and interquartile ranges, as appropriate, and categorical variables are presented as percentages.
In the primary analysis strategy, we used the Kaplan-Meier product limit estimates to compare the time to reach the primary outcome in the test groups. An estimate of the relative risk and 95% confidence interval was also reported. A modified intention-to-treat analysis excluded patients who became ineligible between randomization and administration of convalescent plasma or placebo.
The pre-specified protocol included an assessment of IgG protection correlates and a subgroup analysis that was suggested by the data and security monitoring board and approved by the institutional review boards on November 2, 2020. This analysis included an event assessment final in patients who were 75 years of age or older, regardless of coexisting diseases, and in those between 65 and 74 years of age who had at least one coexisting disease.