Patients

Patients were assessed for eligibility on the basis of a positive reverse-transcriptase–polymerase-chain-reaction (RT-PCR) assay (Shanghai ZJ Bio-Tec or Sansure Biotech) for SARS-CoV-2 in a respiratory tract sample tested by the local Center for Disease Control (CDC) or by a designated diagnostic laboratory. Male and nonpregnant female patients 18 years of age or older were eligible if they had a diagnostic specimen that was positive on RT-PCR, had pneumonia confirmed by chest imaging, and had an oxygen saturation (Sao 2 ) of 94% or less while they were breathing ambient air or a ratio of the partial pressure of oxygen (Pao 2 ) to the fraction of inspired oxygen (Fio 2 ) (Pao 2 :Fio 2 ) at or below 300 mg Hg. Exclusion criteria included a physician decision that involvement in the trial was not in the patient’s best interest, presence of any condition that would not allow the protocol to be followed safely, known allergy or hypersensitivity to lopinavir–ritonavir, known severe liver disease (e.g., cirrhosis, with an alanine aminotransferase level >5× the upper limit of the normal range or an aspartate aminotransferase level >5× the upper limit of the normal range), use of medications that are contraindicated with lopinavir–ritonavir and that could not be replaced or stopped during the trial period (see the Supplementary Appendix, available with the full text of this article at NEJM.org); pregnancy or breast-feeding, or known HIV infection, because of concerns about the development of resistance to lopinavir–ritonavir if used without combining with other antiretrovirals. Patients who were unable to swallow received lopinavir–ritonavir through a nasogastric tube.

Trial Design and Oversight

This was an open-label, individually randomized, controlled trial conducted from January 18, 2020, through February 3, 2020 (the date of enrollment of the last patient), at Jin Yin-Tan Hospital, Wuhan, Hubei Province, China. Because of the emergency nature of the trial, placebos of lopinavir–ritonavir were not prepared. Eligible patients were randomly assigned in a 1:1 ratio to receive either lopinavir–ritonavir (400 mg and 100 mg, orally; freely provided by the national health authority) twice daily, plus standard care, or standard care alone, for 14 days. Standard care comprised, as necessary, supplemental oxygen, noninvasive and invasive ventilation, antibiotic agents, vasopressor support, renal-replacement therapy, and extracorporeal membrane oxygenation (ECMO). To balance the distribution of oxygen support between the two groups as an indicator of severity of respiratory failure, randomization was stratified on the basis of respiratory support methods at the time of enrollment: no oxygen support or oxygen support with nasal duct or mask, or high-flow oxygen, noninvasive ventilation, or invasive ventilation including ECMO. The permuted block (four patients per block) randomization sequence, including stratification, was prepared by a statistician not involved in the trial, using SAS software, version 9.4 (SAS Institute). To minimize allocation bias, we performed allocation concealment with an interactive Web-based response system until randomization was finished on the system through a computer or phone.

The trial was approved by the institutional review board of Jin Yin-Tan Hospital. Written informed consent was obtained from all patients or from the patient’s legal representative if the patient was too unwell to provide consent. The trial was conducted in accordance with the principles of the Declaration of Helsinki and the Good Clinical Practice guidelines of the International Conference on Harmonisation. The authors were responsible for designing the trial and for compiling and analyzing the data. The authors vouch for the completeness and accuracy of the data and for the adherence of the trial to the protocol. Full details about the trial design are provided in the protocol, available at NEJM.org.

Clinical and Laboratory Monitoring

Patients were assessed once daily by trained nurses using diary cards that captured data on a seven-category ordinal scale and on safety from day 0 to day 28, hospital discharge, or death. Safety was monitored by the Good Clinical Practice office from Jin Yin-tan Hospital. Other clinical data were recorded using the WHO-ISARIC (World Health Organization–International Severe Acute Respiratory and Emerging Infections Consortium) case record form (https://isaric.tghn.org).16 Serial oropharyngeal swab samples were obtained on day 1 (before lopinavir–ritonavir was administered) and on days 5, 10, 14, 21, and 28 until discharge or death had occurred and were tested at Teddy Clinical Research Laboratory (Tigermed–DiAn Joint Venture), using quantitative real-time RT-PCR (see the Supplementary Appendix). RNA was extracted from clinical samples with the MagNA Pure 96 system, detected and quantified by Cobas z480 qPCR (Roche), with the use of LightMix Modular SARS-CoV-2 (COVID19) assays (TIB MOBIOL). These samples were obtained for all 199 patients who were still alive at every time point. Sampling did not stop when a swab at a given time point was negative. Baseline throat swabs were tested for detection of E gene, RdRp gene, and N gene, and samples on the subsequent visits were quantitatively and qualitatively detected for E gene. Clinical data were recorded on paper case record forms and then double-entered into an electronic database and validated by trial staff.

Outcome Measures

The primary end point was the time to clinical improvement, defined as the time from randomization to an improvement of two points (from the status at randomization) on a seven-category ordinal scale or live discharge from the hospital, whichever came first. The end point of clinical improvement was used in our previous influenza study17 and was also recommended by the WHO R&D Blueprint expert group.18 Ordinal scales have been used as end points in clinical trials in patients hospitalized with severe influenza.16-19 The seven-category ordinal scale consisted of the following categories: 1, not hospitalized with resumption of normal activities; 2, not hospitalized, but unable to resume normal activities; 3, hospitalized, not requiring supplemental oxygen; 4, hospitalized, requiring supplemental oxygen; 5, hospitalized, requiring nasal high-flow oxygen therapy, noninvasive mechanical ventilation, or both; 6, hospitalized, requiring ECMO, invasive mechanical ventilation, or both; and 7, death.

Other clinical outcomes included clinical status as assessed with the seven-category ordinal scale on days 7 and 14, mortality at day 28, the duration of mechanical ventilation, the duration of hospitalization in survivors, and the time (in days) from treatment initiation to death. Virologic measures included the proportions with viral RNA detection over time and viral RNA titer area-under-the-curve (AUC) measurements.

Safety outcomes included adverse events that occurred during treatment, serious adverse events, and premature discontinuation of treatment. Adverse events were classified according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.0.

Statistical Analysis

The trial was initiated in rapid response to the Covid-19 public health emergency, at which time there was very limited information about clinical outcomes in hospitalized patients with Covid-19. The original total sample size was set at 160, since it would provide the trial with 80% power to detect a difference, at a two-sided significance level of α=0.05, of 8 days in the median time to clinical improvement between the two groups, assuming that the median time in the standard-care group was 20 days and that 75% of the patients would reach clinical improvement. The planned enrollment of 160 patients in the trial occurred quickly, and the assessment at that point was that the trial was underpowered; thus, a decision was made to continue enrollment by investigators. Subsequently, when another agent (remdesivir) became available for clinical trials, we decided to suspend enrollment in this trial.

Primary efficacy analysis was on an intention-to-treat basis and included all the patients who had undergone randomization. The time to clinical improvement was assessed after all patients had reached day 28, with failure to reach clinical improvement or death before day 28 considered as right-censored at day 28 (right-censoring occurs when an event may have occurred after the last time a person was under observation, but the specific timing of the event is unknown). The time to clinical improvement was portrayed by Kaplan–Meier plot and compared with a log-rank test. Hazard ratios with 95% confidence intervals were calculated by means of the Cox proportional-hazards model. Five patients who had been assigned to the lopinavir–ritonavir group did not receive any doses (three of them died within 24 hours) but were included in the intention-to-treat analysis, since no reciprocal removals occurred in the standard-care group. A modified intention-to-treat analysis that excluded three early deaths was also performed. Post hoc analyses include subgroup analysis for National Early Warning Score 2 (NEWS2)19 of 5 or below or greater than 5 and those who underwent randomization up to 12 days or more than 12 days after the onset of illness.

Because the statistical analysis plan did not include a provision for correcting for multiplicity in tests for secondary or other outcomes, results are reported as point estimates and 95% confidence intervals. The widths of the confidence intervals have not been adjusted for multiplicity, so the intervals should not be used to infer definitive treatment effects for secondary outcomes. Safety analyses were based on the patients’ actual treatment exposure. Statistical analyses were conducted with SAS software, version 9.4 (SAS Institute).