Test design and monitoring
We conducted this three-group trial in 55 hospitals in Brazil. The trial was designed by the executive committee (see Supplementary Appendix, available with the full text of this article at NEJM.org) and approved by the Brazilian National Research Ethics Commission, the Brazilian Health Regulatory Agency (ANVISA) and ethics committees. at participating sites The trial was funded by hospitals and research institutes participating in the Covid-19 Brazil Coalition (see Supplementary Appendix). EMS Pharma provided additional funding and logistical support for the trial and also donated and supplied the trial medications. EMS Pharma had no role in conducting the trial, the analysis, or the decision to submit the manuscript for publication. The trial was supervised by an independent international data and security monitoring committee. The executive committee guarantees the integrity and accuracy of the data and the fidelity of the trial to the protocol (available at NEJM.org).
Participants
The trial included consecutive patients who were 18 years of age or older and who had been hospitalized with suspected or confirmed Covid-19 14 or fewer days from the onset of symptoms. Among the reasons for exclusion from the trial were the use of supplemental oxygen at a rate of more than 4 liters per minute administered by a nasal cannula or at a level of at least 40% administered by a Venturi mask; the use of supplemental oxygen administered by a high-flow nasal cannula or invasive or non-invasive ventilation; Previous use of chloroquine, hydroxychloroquine, azithromycin, or any other macrolide for more than 24 hours before enrollment (and from the onset of symptoms); and a history of severe ventricular tachycardia or electrocardiographic findings with a corrected QT interval (QTc) of at least 480 msec. Full information on the inclusion and exclusion criteria is provided in the Supplementary Appendix. All patients gave written or electronic informed consent prior to randomization.
Randomization, interventions and follow-up
Patients were randomized in a 1: 1: 1 ratio to receive standard care (control group), standard care plus hydroxychloroquine at a dose of 400 mg twice daily for 7 days (hydroxychloroquine group alone), or standard care plus hydroxychloroquine at a dose of 400 mg twice a day plus azithromycin at a dose of 500 mg once a day for 7 days. Randomization was performed in blocks of six and stratified according to whether or not supplemental oxygen was used at the time of randomization. Randomization was performed centrally using an electronic case report form system (RedCap) as described in the Supplementary Appendix.12
Current standard care for Covid-19 was at the discretion of the treating physicians. The use of glucocorticoids, other immunomodulators, antibiotic agents, and antiviral agents was allowed (see Supplemental Appendix). Administration of hydroxychloroquine or chloroquine was not allowed in the control group, and the use of macrolides was not allowed in the control group or in the hydroxychloroquine group alone. Guidance was provided to researchers on how to adjust or discontinue treatment based on side effects and laboratory abnormalities.
The data was collected daily, from randomization to day 15, in electronic form as a case report. For patients who were discharged before day 15, an interviewer unaware of the assigned trial group made a structured phone call to the patient or the patient’s family to assess vital status and return to routine activities.
Results
The primary outcome was clinical status at 15 days, assessed using a seven-level ordinal scale. The scores on the scale were defined as follows: a score of 1 indicated that he was not hospitalized without limitations in activities; 2, not hospitalized but with limitations in activities; 3, hospitalized and not receiving supplemental oxygen; 4, hospitalized and receiving supplemental oxygen; 5, hospitalized and receiving oxygen supplements administered by a high-flow nasal cannula or non-invasive ventilation; 6, hospitalized and receiving mechanical ventilation; and 7, death.
Secondary outcomes included clinical status at 7 days, assessed using a six-level ordinal scale (see below and see Supplemental Appendix); an indication for intubation within 15 days; receiving supplemental oxygen administered by a high-flow nasal cannula or non-invasive ventilation between randomization and 15 days; length of hospital stay; death in hospital; thromboembolic complications; Acute kidney injury; and the number of days alive and without respiratory support up to 15 days. A live day without respiratory support was defined as any day on which the patient did not receive supplemental oxygen or invasive or non-invasive mechanical ventilation, from randomization to day 15. Patients who died during the 15-day period were given assigned a value of 0 days alive and without respiratory support in this evaluation. Safety results are listed in the Supplemental Appendix. All trial results were evaluated by site investigators, who were aware of trial group assignments (except as noted above for patients who had been discharged before day 15 and who were evaluated for outcome. primary through a blinded telephone interview). No formal adjudication of trial results was made.
Sample size calculation and protocol changes
Originally, we had planned for the trial to include 630 patients, using the intention-to-treat analysis population, with a six-level ordinal outcome as the primary outcome, as described in the Supplementary Appendix. However, before the first interim analysis was performed, we shifted the primary outcome assessment to the seven-level ordinal scale and the primary analysis population of the intent-to-treat population to a modified intent-to-treat population that included only patients with a diagnosis of Covid-19 that had been confirmed by the reverse transcriptase-polymerase chain reaction (RT-PCR) test (using the test available at each site).
The change to using the seven-level ordinal scale was adopted because on April 10, 2020 (before the first recruited patient reached 15 days of follow-up), we established the ability to obtain 15-day information on the limitations of the activities. with the use of blinded telephone interviews. Therefore, we added another level to the ordinal result of six levels, dividing the first level (not hospitalized) into two levels (level 1, not hospitalized and without limitations in activities; and level 2, not hospitalized but with limitations in activities ). The change to the modified intention-to-treat population was adopted because, under the hypothesis that treatment would have beneficial effects on the primary outcome only for patients who had a confirmed diagnosis, the inclusion of unconfirmed cases would decrease the size and power of the patient. estimated effect. As a related change, we added external adjudication of unconfirmed cases, which were classified as probable, possible, or probably not as Covid-19 (see Supplementary Appendix).
The sample size was revised using the general distribution of the seven-level ordinal result on day 15 observed among the first 120 patients, with levels 1-7 with the following proportions of patients: 60%, 19%, 7%, 1%, 1%, 5% and 7%, respectively. With 630 patients who had been randomized and 510 patients included in the modified intention-to-treat analysis, we calculated that the trial would have an 80% power to detect a probability ratio of 0.5 between groups (two-for-two comparisons two), at a significance level of 5% and with the Bonferroni adjustment for multiple comparisons (α = 5%, divided by 3 for each comparison).13
Statistic analysis
The primary outcome was analyzed using mixed ordinal logistic regression with random interception by site, assuming proportional probabilities. We report all comparisons two by two. Binary outcomes were assessed using a mixed logistic regression model, except for in-hospital mortality, which was assessed with a Cox proportional hazards model. Continuous results were evaluated using generalized linear regression or mixed models for repeated variables, as appropriate. All models were adjusted for age and the use of supplemental oxygen at admission.
We also performed sensitivity analyzes that included all patients who had undergone randomization (intention to treat population) and sensitivity analyzes for the primary outcome for the following groups: patients with definitive, probable, or possible Covid-19; and patients with definitive or probable Covid-19. Two additional populations were considered. An efficacy population included patients with a confirmed diagnosis who received at least one dose of the assigned test drug. The safety population included patients according to the medications received, regardless of the assigned test group or the Covid-19 test result.
We plan three interim analyzes, to be performed when 120 patients, 315 patients, and 504 patients have completed 15 days of follow-up. However, only the first interim analysis was performed. Due to faster enrollment than expected, primary outcome data for the second and third interim analyzes were available only after trial recruitment was completed. After discussion with the data and security monitoring committee, the second and third provisional analyzes were canceled. The data monitoring and security committee used Haybittle – Peto14 stop limits, with a P value threshold less than 0.001 to stop the test for safety and a P value threshold less than 0.0001 to stop the test for efficacy. We did not adjust the final values of the hypothesis test for sequential analyzes.
The analyzes were performed with the use of the R (R Core Team) software.fifteen The P values for the primary outcome were adjusted using the Bonferroni correction. P values are not reported for secondary outcomes; the widths of the confidence intervals for secondary outcomes have not been adjusted for multiple comparisons, so the intervals should not be used to infer the definitive effects of treatment. The P values for the safety analyzes were not adjusted given the importance of identifying possible signs of damage. Additional details on statistical analyzes are provided in the supplemental appendix.
