key: cord-0888667-tdlcb9bf authors: GAUTRET, Philippe; LAGIER, Jean Christophe; PAROLA, Philippe; HOANG, Van Thuan; MEDDED, Line; MAILHE, Morgan; DOUDIER, Barbara; COURJON, Johan; GIORDANENGO, Valerie; ESTEVES VIEIRA, Vera; TISSOT DUPONT, Herve; HONORE, Stephane; COLSON, Philippe; CHABRIERE, Eric; LA SCOLA, Bernard; ROLAIN, Jean Marc; BROUQUI, Philippe; RAOULT, Didier title: Hydroxychloroquine and Azithromycin as a treatment of COVID-19: preliminary results of an open-label non-randomized clinical trial date: 2020-03-20 journal: nan DOI: 10.1101/2020.03.16.20037135 sha: 4da0f2ec885057de83d5eef38a770177f91d9f7e doc_id: 888667 cord_uid: tdlcb9bf Background Chloroquine and Hydroxychloroquine have been found to be efficient on COV-19, and reported to be efficient in Chinese patients infected by this virus. We evaluate the role of Hydroxychloroquine on respiratory viral loads. Patients and methods Patients were included in a single arm protocol to receive 600mg of hydroxychloroquine daily and their viral load in nasal swabs was tested daily. Depending on their clinical presentation azithromycin was added to the treatment. Untreated patients from another center and cases refusing the protocol were included as negative control. Presence and absence of virus at Day-6 was considered the end point. Results Twenty cases were treated in this study and showed a significant reduction of the viral carriage at D-6 compared to controls, and much lower than reported average carrying duration of untreated patients in the literature. Azithromycin added to Hydroxychloroquine was significantly more efficient for virus elimination. Conclusion : Hydroxychloroquine is significantly associated with viral load reduction/disappearance in patients with COVID-19 and its effect is reinforced by Azithromycin. In late December 2019, an outbreak of an emerging disease (COVID-19) due to a novel coronavirus (named SARS-CoV-2 latter) started in Wuhan, China and rapidly spread in China and outside [1, 2] . The WHO declared the epidemic of COVID-19 as a pandemic on March 12 th 2020 [3] . According to a recent Chinese stud, about 80% of patients present with mild disease and the overall case-fatality rate is about 2.3% but reaches 8.0% in patients aged 70 to 79 years and 14.8% in those aged >80 years [4] . However, there is probably an important number of asymptomatic carriers in the population, and thus the mortality rate is probably overestimated. France is now facing the COVID-19 wave with more than 4500 cases, as of March 14 th 2020 [5] . Thus, there is an urgent need for an effective treatment to treat symptomatic patients but also to decrease the duration of virus carriage in order to limit the transmission in the community. Among candidate drugs to treat COVID-19, repositioning of old drugs for use as antiviral treatment is an interesting strategy because knowledge on safety profile, side effects, posology and drug interactions are well known [6, 7] . A recent paper reported an inhibitor effect of remdesivir (a new antiviral drug) and chloroquine (an old antimalarial drug) on the growth of SARS-CoV-2 in vitro, [8] and an early clinical trial conducted in COVID-19 Chinese patients, showed that chloroquine had a significant effect, both in terms of clinical outcome and viral clearance, when comparing to controls groups [9,10]. Chinese experts recommend that patients diagnosed as mild, moderate and severe cases of COVID-19 pneumonia and without contraindications to chloroquine, be treated with 500 mg chloroquine twice a day for ten days [11] . Hydroxychloroquine (an analogue of chloroquine) has been demonstrated to have an anti-SARS-CoV activity in vitro [12] . Hydroxychloroquine clinical safety profile is better than that of chloroquine (during long-term use) and allows higher daily dose [13] and has fewer concerns about drug-drug interactions [14] . Our team has a very comprehensive experience in successfully treating patients with chronic diseases due to intracellular bacteria (Q fever due to Coxiella burnetii and Whipple's disease due to Tropheryma whipplei) with long-term hydroxychloroquine treatment (600 mg/day for 12 to 18 months) since more than 20 years. [15, 16] We therefore started to conduct a clinical trial aiming at assessing the effect of hydroxychloroquine on SARS-CoV-2-infected patients after approval by the French Ministry of Health. In this report we describe our early results, focusing on virological data in patients receiving hydroxychloroquine as compared to a control group. This ongoing study is coordinated by The Méditerranée Infection University Hospital Institute in Marseille. Patients who were proposed a treatment with hydroxychloroquine were recruited and managed in Marseille centre. Controls without hydroxychloroquine treatment were recruited in Marseille, Nice, Avignon and Briançon centers, all located in South France. Hospitalized patients with confirmed COVID-19 were included in this study if they fulfilled two primary criteria: i) age >12 years; ii) PCR documented SARS-CoV-2 carriage in nasopharyngeal sample at admission whatever their clinical status. Patients were excluded if they had a known allergy to hydroxychloroquine or chloroquine or had another known contraindication to treatment with the study drug, including retinopathy, G6PD deficiency and QT prolongation. Breastfeeding and pregnant patients were excluded based on their declaration and pregnancy test results when required. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Patients were seen at baseline for enrolment, initial data collection and treatment at day-0, and again for daily follow-up during 14 days. Each day, patients received a standardized clinical examination and when possible, a nasopharyngeal sample was collected. All clinical data were collected using standardized questionnaires. All patients in Marseille center were proposed oral hydroxychloroquine sulfate 200 mg, three times per day during ten days (in this preliminary phase ,we did not enrolled children in the treatment group based in data indicating that children develop mild symptoms of COVID-19 [4] ). Patients who refused the treatment All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 20, 2020. . https://doi.org/10.1101/2020.03. 16.20037135 doi: medRxiv preprint or had an exclusion criteria, served as controls in Marseille centre. Patients in other centers did not receive hydroxychloroquine and served as controls. Symptomatic treatment and antibiotics as a measure to prevent bacterial super-infection was provided by investigators based on clinical judgment. Hydroxychloroquine was provided by the National Pharmacy of France on nominative demand. Patients were grouped into three categories: asymptomatic, upper respiratory tract infection (URTI) when presenting with rhinitis, pharyngitis, or isolated low-grade fever and myalgia, and lower respiratory tract infections (LRTI) when presenting with symptoms of pneumonia or bronchitis. Native hydroxychloroquine has been dosed from patients' serum samples by UHPLC-UV using a previously described protocol [18] . The peak of the chromatogram at 1.05 min of retention corresponds to hydroxychloroquine metabolite. The serum concentration of this metabolite is deduced from UV absorption, as for hydroxychloroquine concentration. Considering both concentrations provides an estimation of initial serum hydroxychloroquine concentration. Culture All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 20, 2020. . https://doi.org/10.1101/2020.03. 16.20037135 doi: medRxiv preprint For all patients, 500 µL of the liquid collected from the nasopharyngeal swab were passed through 0.22-µm pore sized centrifugal filter (Merck millipore, Darmstadt, Germany), then were inoculated in wells of 96-well culture microplates, of which 4 wells contained Vero E6 cells (ATCC CRL-1586) in Minimum Essential Medium culture medium with 4% fetal calf serum and 1% glutamine. After centrifigation at 4,000 g, microplates were incubated at 37°C. Plates were observed daily for evidence of cytopathogenic effect. Presumptive detection of virus in supernatant was done using SU5000 SEM (Hitachi) then confirmed by specific RT-PCR. The primary endpoint was virological clearance at day-6 post-inclusion. Secondary outcomes were virological clearance overtime during the study period, clinical follow-up (body temperature, respiratory rate, long of stay at hospital and mortality), and occurrence of sideeffects. Assuming a 50% efficacy of hydroxychloroquine in reducing the viral load at day 7, a 85% power, a type I error rate of 5% and 10% loss to follow-up, we calculated that a total of 48 COVID-19 patients (ie, 24 cases in the hydroxychloroquine group and 24 in the control group) would be required for the analysis (Fleiss with CC). Statistical differences were evaluated by Pearson's chi-square or Fisher's exact tests as categorical variables, as appropriate. Means of quantitative data were compared using Student's t-test. Analyses were performed in OpenEpi version 3.0. (detailed results are available in supplementary Table 1 ) All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 20, 2020. . side-effects will be described in a further paper at the end of the trial. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 20, 2020. . Mean hydroxychloroquine serum concentration was 0.46 µg/ml+0.2 (N=20). The proportion of patients that had negative PCR results in nasopharyngeal samples significantly differed between treated patients and controls at days 3-4-5 and 6 post-inclusion (Table 2) . At day6 post-inclusion, 70% of hydroxychloroquine-treated patients were virologicaly cured comparing with 12.5% in the control group (p= 0.001). When comparing the effect of hydroxychloroquine treatment as a single drug and the effect of We could isolate SARS-CoV-2 in 19 out of 25 clinical samples from patients. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 20, 2020. . https://doi.org/10.1101/2020.03. 16.20037135 doi: medRxiv preprint For ethical reasons and because our first results are so significant and evident we decide to share our findings with the medical community, given the urgent need for an effective drug against SARS-CoV-2 in the current pandemic context. We show here that hydroxychloroquine is efficient in clearing viral nasopharyngeal carriage of SARS-CoV-2 in COVID-19 patients in only three to six days, in most patients. A significant difference was observed between hydroxychloroquine-treated patients and controls starting even on day3 post-inclusion. These results are of great importance because a recent paper has shown that the mean duration of viral shedding in patients suffering from COVID- Very recently, a Chinese team published results of a study demonstrating that chloroquine and hydroxychloroquine inhibit SARS-CoV-2 in vitro with hydroxychloroquine (EC50=0.72%µM) found to be more potent than chloroquine (EC50=5.47%µM) [14] . These in vitro results corroborate our clinical results. The target values indicated in this paper [14] were reached in our experiments. The safer dose-dependent toxicity profile of hydroxychloroquine in humans, compared to that of chloroquine [13] allows using clinical doses of hydroxychloroquine that will be over its EC50 observed in vitro [14] . Our preliminary results also suggest a synergistic effect of the combination of hydroxychloroquine and azithromycin. Azithromycin has been shown to be active in vitro against Zika and Ebola viruses [20-22] and to prevent severe respiratory tract infections when administrated to patients suffering viral infection [23] . This finding should be further explored to know whether a combination is more effective especially in severe cases. Speculated potential risk of severe QT prolongation induced by the association of the two drugs has not been established yet but should be considered. As for each treatment, the cost benefits of the risk should be evaluated individually. Further studies on this combination are needed, since All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 20, 2020. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 20, 2020. . (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 20, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. control patients from centers other than Marseille did not underwent daily sampling, but were sampled every other day, they were considered positive for PCR when actually positive the day before an the day after the day with missing data. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. A review of the 2019 Novel Coronavirus (COVID-19) based on current evidence WHO Director-General's opening remarks at the media briefing on COVID-19 -11 Characteristics of and important lessons from the coronavirus disease 2019 (COVID-19) outbreak in China: summary of a report of 72 314 cases from the Chinese Center for Disease Control and Prevention COVID-19, France et Monde Chloroquine for the 2019 novel coronavirus SARS-CoV Chloroquine and hydroxychloroquine as available weapons to fight COVID-19 No reuse allowed without permission. 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