key: cord-0719576-kjf63oug authors: Huang, Mingxing; Li, Man; Xiao, Fei; Pang, Pengfei; Liang, Jiabi; Tang, Tiantian; Liu, Shaoxuan; Chen, Binghui; Shu, Jingxian; You, Yingying; Li, Yang; Tang, Meiwen; Zhou, Jianhui; Jiang, Guanmin; Xiang, Jingfen; Hong, Wenxin; He, Songmei; Wang, Zhaoqin; Feng, Jianhua; Lin, Changqing; Ye, Yinong; Wu, Zhilong; Li, Yaocai; Zhong, Bei; Sun, Ruilin; Hong, Zhongsi; Liu, Jing; Chen, Huili; Wang, Xiaohua; Li, Zhonghe; Pei, Duanqing; Tian, Lin; Xia, Jinyu; Jiang, Shanping; Zhong, Nanshan; Shan, Hong title: Preliminary evidence from a multicenter prospective observational study of the safety and efficacy of chloroquine for the treatment of COVID-19 date: 2020-05-28 journal: Natl Sci Rev DOI: 10.1093/nsr/nwaa113 sha: 4ef068d9f3ccad01df9c187f9858915e38557bad doc_id: 719576 cord_uid: kjf63oug BACKGROUND: Effective therapies are urgently needed for the SARS-CoV-2 pandemic. Chloroquine has been proved to have antiviral effect against coronavirus in vitro. In this study, we aimed to assess the efficacy and safety of chloroquine with different doses in COVID-19. METHOD: In this multicenter prospective observational study, we enrolled patients older than 18 years old with confirmed SARS-CoV-2 infection excluding critical cases from 12 hospitals in Guangdong and Hubei Provinces. Eligible patients received chloroquine phosphate 500 mg, orally, once (half dose) or twice (full dose) daily. Patients treated with non-chloroquine therapy were included as historical controls. The primary endpoint is the time to undetectable viral RNA. Secondary outcomes include the proportion of patients with undetectable viral RNA by day 10 and 14, hospitalization time, duration of fever, and adverse events. RESULTS: A total of 197 patients completed chloroquine treatment, and 176 patients were included as historical controls. The median time to achieve an undetectable viral RNA was shorter in chloroquine than in non-chloroquine (absolute difference in medians -6.0 days; 95% CI -6.0 to -4.0). The duration of fever is shorter in chloroquine (geometric mean ratio 0.6; 95% CI 0.5 to 0.8). No serious adverse events were observed in the chloroquine group. Patients treated with half dose experienced lower rate of adverse events than with full dose. CONCLUSIONS: Although randomised trials are needed for further evaluation, this study provides evidence for safety and efficacy of chloroquine in COVID-19 and suggests that chloroquine can be a cost-effective therapy for combating the COVID-19 pandemic. The coronavirus disease 2019 (COVID-19) emerged in late 2019 1,2 . The responsible virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), belongs to a distinct clade from the human severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) 3 . It has become a global pandemic, affecting over 100 countries with more than 240,000 confirmed cases and over 10,000 deaths globally as of March 20, 2020, calling for an urgent demand of effective treatment. Chloroquine has been proved effective in vitro to inhibit the replication of SARS-CoV 4 , HCoV-229E 5 , and the newly discovered SARS-CoV-2 6 , 7 . To evaluate the efficacy and safety of chloroquine for COVID-19, we previously conducted a single-arm pilot clinical study with 10 patients (Huang et al. Journal of Molecular Cell Biology, in press). Encouragingly, all patients achieved undetectable level of viral RNA within 14 days without serious adverse events. These results led us to conduct a multicenter prospective observational study in adult patients with COVID-19 to assess the efficacy and safety of chloroquine for COVID-19. Of the 233 enrolled patients for chloroquine, 197 (84.5%) completed treatment and were included in the final analysis (Figure 1, study in the non-chloroquine group. Across the two treatment groups, the majority patients were classified as moderate cases (93.4% in chloroquine; 89.2% in nonchloroquine) 8 Guidelines of COVID-19 later than the other therapies used in the non-chloroquine group. Therefore, we observed longer interval time between symptom onset and treatment initiation in chloroquine versus non-chloroquine (absolute difference 4 days; 95% CI 2 to 6 days; P < 0.0001). In addition, due to the rapid rise of patients in Wuhan and established mobile hospital in early February, the interval time between symptom onset and treatment initiation in Wuhan (median 17 days, IQR 10.5 to 21 days) is longer than that in Guangdong Province (median 5 days, IQR 3 to 10 days; Table 1 ). In the subgroup of patients from the Fifth Affiliated Hospital of Sun Yat-sen University (SYSU5), we obtained and evaluated the viral load at baseline between chloroquine (N=21) and non-chloroquine (N=8) group and did not observe statistically significant difference (absolute difference in medians = 2.93, 95% CI -0.8 to 6.6, p = 0.09). In the analysis of the full study population, patients in the chloroquine group have an accelerated time to undetectable viral RNA from that of patients in the non-chloroquine group (absolute difference in medians -5.4 days; 95% CI -6 to -4; P < 0.0001; Figure 2) . Secondly, by day 10 and day 14 since treatment initiation, higher proportion of patients had undetectable viral RNA in the chloroquine group (91.4% and 95.9% respectively; Table 2 ) comparing to the non-chloroquine group (57.4% and 79.6% respectively; Table 2 ). In the aspect of clinical manifestations, we found that the duration of fevers is shorter in chloroquine versus non-chloroquine among patients experienced fever symptom (geometric mean ratio 0.6; 95% CI 0.5 to 0.8; P = 0.0029; Supplementary Figure S1 ). To note, the antipyretic effects of chloroquine may have also contributed to this result. We observed no difference in the length of hospital stay (Supplementary Figure S2) . No patient died or admitted to ICU either in the chloroquine group or in the non-chloroquine group. Among patients who had moderate clinical symptoms at baseline, seven patients experienced aggravated symptoms from moderate to severe level, one in the chloroquine group and six in the non-chloroquine group. The proportion of patients having aggravated symptoms is lower in the chloroquine group but not statistically significant (absolute difference in proportions 3.28; 95% CI -6.96 to 1.43). All of the seven patients eventually were tested negative for the viral RNA within the study period. Due to the significant difference observed in clinical classification between chloroquine and non-chloroquine group at baseline, we further analyzed the primary and secondary outcomes in patients with moderate symptoms only. The number of patients in mild or severe subgroup were too few to compare. The benefit of chloroquine in viral suppression is consistent with the full analysis, except for nonsignificant difference observed for the proportion of patients with undetectable viral RNA by day 14 (Supplementary Table 2 ). In post hoc analysis, we examined the effect of chloroquine on the time to undetectable viral RNA stratified by different doses, types of clinical manifestation, the interaction between province and time from symptom onset to treatment initiation, and a representative center (Figure 3) . Chloroquine showed beneficial effect in all stratum. However, the beneficial effect is not statistically significant in patients with severe COVID-19 symptoms, patients from Guangdong Province treated later than 14 days after symptom onset, or patients from SYSU5. In order to assess the effect of chloroquine in more detailed clinical improvement outcomes in post hoc analysis, we collected detailed clinical data in patients from SYSU5, including the improvement of chest CT, the monitoring of serum chloroquine concentration, and the reappearance of positive viral RNA detection after hospital discharge. In this subgroup of patients, the interval time between symptom onset and treatment initiation were comparable. The medians are 7 days in chloroquine group (N=50) and 6 days in non-chloroquine group (N=21) (absolute difference in medians 1 day; 95% CI -3 to 4 days; P = 0.99; Supplementary Table 3) . We did not find statistically significant difference in the time to undetectable viral RNA between the two groups (absolute difference in medians -3.5 days; 95% CI -6 to 1 days). The chloroquine group have higher percentage of patients with improved chest CT by day 10 (absolute difference in proportions 9.7; 95% CI -16.0 to 35.6) and day 14 (absolute difference in proportions 6.3; 95% CI -22.2 to 32.0) than the nonchloroquine group but the difference is not statistically significant (Supplementary Table 3 ). This could be due to the small sample size or the delayed chest CT absorption 9 . We did not observe beneficial effect of chloroquine in the length of hospital stay and the duration of oxygen support (Supplementary Table 3) . Unprecedently, we observed 3 cases of so called "re-positive" patients in the chloroquine group. They were identified with negative viral RNA test from respiratory tract samples but positive viral RNA test from fecal samples within 7 days following hospital discharge. No such observation in the non-chloroquine group. Investigation is underway to examine whether it is due to re-infection or other factors. Among the 12 hospitals, one hospital explored different dosage of chloroquine, as 500 mg once daily, which is half of the protocol dosage. We compared the primary and secondary outcomes in patients from this subgroup (N=29) with the nonchloroquine group in Guangdong Province. The results mainly showed that chloroquine has benefit effect on the time to undetectable viral RNA (absolute difference in medians -5 days; 95% CI -6.0 to -4.0 days) and the proportion of patients with undetectable viral RNA by day 10 is higher in chloroquine group (absolute difference in proportions 32.7; 95% CI 23.9 to 42.1). The duration of fever was also shorter than those in the non-chloroquine group (geometric mean ratio 0.8; 95% CI 0.5 to 0.9) (Supplementary Table 4 ). A total of 53 patients (26.9%) in the chloroquine group and 57 (32.4%) in the non-chloroquine group reported adverse events during study period ( Table 3) . Gastrointestinal events including vomiting, abdominal distension, nausea, decreased appetite, thirst were more common in chloroquine than in the non-chloroquine group. The percentage of patients with neurological adverse events, including dizziness and sleep order, were higher in the chloroquine than in the non-chloroquine group. In addition, anxiety was observed more frequently in chloroquine than in the nonchloroquine group. We observed fewer adverse events in patients with half dose of chloroquine than full dose (absolute difference in proportions -40; 95% CI -60 to -29). Chloroquine phosphate has a long half-life (20-60 days) 10-12 and its mean residence time is approximately 20 days 10 . It may have cumulative effect 13 . In order to determine whether chloroquine has a cumulative effect in the short-term treatment with COVID-19, we measured the serum concentration of chloroquine in patients from SYSU5 during and off the treatment. The results showed that the mean of serum concentration of chloroquine gradually rising, with the highest reaching 1.80(±0.49) μmol/L during medication and reduced to 0.13(±0.08) μmol/L within 28±1 days off chloroquine (Supplementary Figure 3) . We did not observe statistically significant difference in treatment effect of chloroquine when stratifying by tertiles of serum chloroquine concentrations (Supplementary Figure 4) . In this study, we found that patients in the chloroquine group experienced significantly faster and higher rate of viral suppression comparing to the nonchloroquine group in both the full analysis and the post hoc stratified analysis. Even when the dose reduced to half, the benefit of chloroquine still remained (Figure 3) . These findings indicate that chloroquine could be effective in treating patients with COVID-19. To our knowledge, this is the first and largest clinical study on chloroquine phosphate for treating COVID-19 to date. We recognize that our study has several limitations. This study was carried out under the COVID-19 public health emergency. Due to the limited medical capacity and urgent clinical situation, we were unable to conduct a standard randomised controlled study to formally evaluate efficacy and safety of chloroquine versus placebo. As an observational study, we have to note that several factors may influence the interpretation of the result. It is reasonable to suspect that the dramatic improvement in the primary outcome in chloroquine could be due to the later treatment initiation since symptom onset. Firstly, gaining experience in treatment management and attenuation of the virus during the course of the epidemic could contribute to the improved outcomes. Secondly, we cannot rule out the possibility that among those with longer interval time between symptom onset and treatment, some may already have been on the course of recovery. Thirdly, although it is impossible to dissect the influence from other antiviral therapies used before chloroquine, it is a plausible assumption that chloroquine is the first antiviral therapy Review 2020, in press). Chloroquine has a favorable effect on glucose and lipid metabolism 18 . Therefore, chloroquine may exert its antiviral effect against SARS-CoV-2 by inhibiting endocytosis and autophagy, and stabilizing glucose and lipid metabolism. The adverse reactions of chloroquine drugs are of great concern to the community. Although it is an old anti-malarial drug, its safety in treating COVID-19 patients is still unknown. In the present study, we did not observe serious adverse events in patients with chloroquine. All adverse events observed during the study period are known side-effects for chloroquine ( Table 3) . The main adverse events were symptoms in gastrointestinal and neuropsychiatric systems. Chloroquine is known for its side effects in cardiovascular system. In the chloroquine group, we did not find significantly higher rate of adverse events in patients older than 65 or with pre-existing conditions (Supplementary Table 5 Table 6 ). There is no strong evidence that these antiviral treatments were safe and effective in COVID-19 patients 22 In conclusion, our preliminary evidence showed that chloroquine has the potential to shorten the time to SARS-CoV-2 viral suppression and duration of fever in patients with moderate symptoms at earlier stage of the disease, even with reduced dose. Further randomised studies are needed to determine the optimal dose, to assess its benefit for both severe cases and to assess its benefit in settings other than secondary care. Considering that there is no better option at present, chloroquine could be a viable option to combat the coronavirus pandemic under proper management. This study was a multicenter prospective observational study conducted from Table 6 ). The primary outcome is the time from treatment initiation to undetectable viral The original plan was to compare the efficacy between three groups, chloroquine only, Lopinavir/Ritonavir only, and chloroquine plus Lopinavor/Ritonavir. At the beginning of the outbreak, different therapies were proposed and tested for the treatment of COVID-19. Therefore, it is challenging to find sufficient patients with unified treatment across all centers. The epidemic in Guangdong had been brought under control rapidly during the study making it difficult to recruit patients as planned. The history of changes to the protocol is listed in Supplementary Table 7 . Thus, a decision was made to focus on recruiting chloroquine only and compare the efficacy with historical controls. The current sample size was based on feasibility within the fixed trial recruitment window and was felt would provide sufficient precision for the estimation of plausible effects. With right-censoring in time-to-event variables, generalized Wilcoxon test was used to compare the difference in medians and the 95% confidence intervals were calculated by bootstrapping 24 . For binary outcomes, Wilson test was implemented to calculate the difference in proportions and 95% confidence intervals. As this was an observational study, imbalance in the baseline characteristics of the two groups was expected. To adjust for this imbalance, we The sponsor of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data and had final responsibility for the decision to submit for publication. All authors declare no competing interests. The data that support the findings of this study are available from the corresponding author on reasonable request. Participant data without names and identifiers will be made available after approval from the corresponding author and Ministry of science and technology and Health Committee in Guangdong province. After publication of study findings, the data will be available for others to request. The research team will provide an email address for communication once the data are approved to be shared with others. The proposal with detailed description of study objectives and statistical analysis plan will be needed for evaluation of the reasonability to request for our data. The corresponding author and Ministry of science and technology and Health Committee in Guangdong province will make a decision based on these materials. Additional materials may also be required during the process. oxygen saturation in fingertip < 93%, oxygenation index < 300, pulmonary imaging showed that the lesion progressed significantly more than 50% within 24-48 hours. Abbreviations: CI, confidence interval; IQR, inter-quartile range; CV, coefficient of variation. § Definitions of outcomes are listed in Supplementary Methods. † 95% CI for continuous variables are calculated by bootstrapping. 95% CI for binary variables are calculated with Wilson method. The difference for duration of fever is geometric mean ratio of chloroquine group to non-chloroquine group. The differences for all other variables are the absolute difference between chloroquine group and non-chloroquine group. * The number of patients had at least one day of fever is 42 and 51 in the chloroquine and non-chloroquine group respectively. 1 (0.5) 0 (0) § Adverse events that occurred in more than 1 patient after treatment initiation during study period are shown. Some patients had more than one adverse event. Abbreviations: GD, Guangdong; HB, Hubei. 95% CI are calculated by bootstrapping. The differences for all other variables are the absolute difference between chloroquine group and non-chloroquine group. 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We also thank all the health care workers on the front lines of the COVID-19 pandemic. This work technology tackling key issues. We thank all the participants in this study. We acknowledge all health-care workers who provided care for the patients at the 12 hospitals. We thank Jiaxing Taimei Medical Technology Co., Ltd for Electronic Data Capture service.