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ARTICLE IN PRESSJID: ANTAGE[m5G;June 30, 2020;0:48]International Journal of Antimicrobial Agents xxx (xxxx) xxxContents lists available at ScienceDirectInternational Journal of Antimicrobial Agentsjournal homepage: ine and azithromycin as a treatment of COVID-19:results of an open-label non-randomized clinical trial Philippe Gautret a,b, , Jean-Christophe Lagier a,c, , Philippe Parola a,b, Van Thuan Hoang a,b,d,Line Meddeb a, Morgane Mailhe a, Barbara Doudier a, Johan Courjon e,f,g,Valérie Giordanengo h, Vera Esteves Vieira a, Hervé Tissot Dupont a,c, Stéphane Honoré i,j,Philippe Colson a,c, Eric Chabrière a,c, Bernard La Scola a,c, Jean-Marc Rolain a,c,Philippe Brouqui a,c, Didier Raoult a,c, aIHU-Méditerranée Infection, Marseille, FranceAix Marseille Univ, IRD, AP-HM, SSA, VITROME, Marseille, FrancecAix Marseille Univ, IRD, APHM, MEPHI, Marseille, FrancedThai Binh University of Medicine and Pharmacy, Thai Binh, Viet NameInfectiologie, Hôpital de l’Archet, Centre Hospitalier Universitaire de Nice, Nice, FrancefUniversité Côte d’Azur, Nice, FrancegU1065, Centre Méditerranéen de Médecine Moléculaire, C3M, Virulence Microbienne et Signalisation Inflammatoire, INSERM, Nice, FrancehDepartment of Virology, Biological and Pathological Center, Centre Hospitalier Universitaire de Nice, 06200 Nice, FranceiService Pharmacie, Hôpital Timone, AP-HM, Marseille, FrancejLaboratoire de Pharmacie Clinique, Aix Marseille Université, Marseille, Franceba r t i c l ei n f oArticle history:Available online xxxEditor: Dr. Po-Ren HsuehKey eAzithromycinClinical triala b s t r a c tBackground: Chloroquine and hydroxychloroquine have been found to be efficient on SARS-CoV-2, andreported to be efficient in Chinese COV-19 patients. We evaluate the effect of hydroxychloroquine onrespiratory viral loads.Patients and methods: French Confirmed COVID-19 patients were included in a single arm protocolfrom early March to March 16th , to receive 600mg of hydroxychloroquine daily and their viral load innasopharyngeal swabs was tested daily in a hospital setting. Depending on their clinical presentation,azithromycin was added to the treatment. Untreated patients from another center and cases refusing theprotocol were included as negative controls. Presence and absence of virus at Day6-post inclusion wasconsidered the end point.Results: Six patients were asymptomatic, 22 had upper respiratory tract infection symptoms and eighthad lower respiratory tract infection symptoms.Twenty cases were treated in this study and showed a significant reduction of the viral carriage atD6-post inclusion compared to controls, and much lower average carrying duration than reported in thelitterature for untreated patients. Azithromycin added to hydroxychloroquine was significantly more efficient for virus elimination.Conclusion: Despite its small sample size, our survey shows that hydroxychloroquine treatment is significantly associated with viral load reduction/disappearance in COVID-19 patients and its effect is reinforcedby azithromycin. 2020 Published by Elsevier B.V.1. Introduction Given his role as Editor in Chief of this journal, Jean Marc Rolain had no involvement in the peer-review of this article and has no access to information regarding its peer-review. Full responsibility for the peer-review process for this article was delegated to P.R. Hsueh. Corresponding author.E-mail address: [email protected] (D. Raoult). equal workIn late December 2019, an outbreak of an emerging disease(COVID-19) due to a novel coronavirus (later named SARS-CoV2) 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 12th 2020 [3]. According to a recent Chinese 059490924-8579/ 2020 Published by Elsevier B.V.Please cite this article as: P. Gautret, J.-C. Lagier and P. Parola et al., Hydroxychloroquine and azithromycin as a treatment of COVID-19:results of an open-label non-randomized clinical trial, International Journal of Antimicrobial Agents, https://doi.org/10.1016/j.ijantimicag.2020.105949

ARTICLE IN PRESSJID: ANTAGE2[m5G;June 30, 2020;0:48]P. Gautret, J.-C. Lagier and P. Parola et al. / International Journal of Antimicrobial Agents xxx (xxxx) xxxabout 80% of patients present with mild disease and the overallcase-fatality rate is about 2.3% but reaches 8.0% in patients aged70 to 79 years and 14.8% in those aged 80 years [4]. However,there is probably an important number of asymptomatic carriersin the population, and thus the mortality rate is probably overestimated. France is now facing the COVID-19 wave with morethan 4500 cases, as of March 14th 2020 [5]. Thus, there is anurgent 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 candidatedrugs to treat COVID-19, repositioning of old drugs for use as antiviral treatment is an interesting strategy because knowledge onsafety profile, side effects, posology and drug interactions are wellknown [6,7].A recent paper reported an inhibitor effect of remdesivir (anew antiviral drug) and chloroquine (an old antimalarial drug) onthe growth of SARS-CoV-2 in vitro, [8] and an early clinical trialconducted in Chinese COVID-19 patients, showed that chloroquinehad a significant effect, both in terms of clinical outcome and viral clearance, when compared to controls groups [9,10]. Chineseexperts recommend that patients diagnosed with mild, moderateand severe cases of COVID-19 pneumonia and without contraindications to chloroquine, be treated with 500 mg chloroquine twicea day for ten days [11].Hydroxychloroquine (an analogue of chloroquine) has beendemonstrated to have an anti-SARS-CoV activity in vitro [12].Hydroxychloroquine clinical safety profile is better than that ofchloroquine (during long-term use) and allows a higher daily dose[13] and has fewer concerns regarding drug-drug interactions [14].Our team has a very comprehensive experience (over 20 years) insuccessfully 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) [15,16]. Wetherefore started to conduct a clinical trial aiming at assessing theeffect of hydroxychloroquine on SARS-CoV-2-infected patients after approval by the French Ministry of Health. In this report wedescribe our early results, focusing on virological data in patientsreceiving hydroxychloroquine as compared to a control group.2. Study population and Methods2.1. Setting2.3. Informed consentBefore being included in the study, patients meeting inclusioncriteria had to give their consent to participate to the study. Written informed signed consent was obtained from adult participants( 18 years) or from parents or legal guardians for minors ( 18years). An information document that clearly indicates the risksand benefits associated with the participation to the study wasgiven to each patient. Patients received information about theirclinical status during care regardless of whether they participatein the study or not. Regarding patient identification, a study number was assigned sequentially to included participants, accordingto the range of patient numbers allocated to each study centre. Thestudy was conducted in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines of good clinical practice, theHelsinki Declaration, and applicable standard operating procedures.The protocol, appendices and any other relevant documentation were submitted to the French National Agency for Drug Safety(ANSM) (2020-0 0 0890-25) and to the French Ethic Committee (CPPIle de France) (20.02.28.99113) for reviewing and were approved on5th and 6th March, 2020, respectively. This trial is registered in EUClinical Trials Register with number 2020-0 0 0890-25.2.4. ProcedurePatients were seen at baseline for enrollment, initial datacollection and treatment at day-0, and again for daily followup during a 14 day period. 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 the Marseillecenter were proposed oral hydroxychloroquine sulfate 200 mg,three times per day for ten days (in this preliminary phase,we did not enroll children in the treatment group based ondata indicating that children develop mild symptoms of COVID19 [4]). Patients who refused the treatment or had an exclusion criteria, served as controls in the Marseille centre. Patientsin other centers did not receive hydroxychloroquine and servedas controls. Symptomatic treatment and antibiotics as a measure to prevent bacterial super-infection was provided by investigators based on clinical judgment. Hydroxychloroquine wasprovided by the National Pharmacy of France on nominativedemand.This ongoing study is coordinated by The Méditerranée Infection University Hospital Institute in Marseille. Patients who wereproposed a treatment with hydroxychloroquine were recruited andmanaged in the Marseille centre. Controls without hydroxychloroquine treatment were recruited in Marseille, Nice, Avignon and Briançon centers, all located in Southern France.2.5. Clinical classification2.2. Patients2.6. PCR assayHospitalized patients with confirmed COVID-19 were includedin this study if they fulfilled two primary criteria: i) age 12 years;ii) PCR documented SARS-CoV-2 carriage in nasopharyngeal sample at admission regardless of 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, G6PDdeficiency and QT prolongation. Breastfeeding and pregnant patients were excluded based on their declaration and pregnancy testresults when required.SARS-CoV-2 RNAtranscription-PCR [17].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 lowerrespiratory tract infections (LRTI) when presenting with symptomsof pneumonia or bronchitis.wasassessedbyreal-timereverse2.7. Hydroxychloroquine dosageNative hydroxychloroquine has been dosed from patients’serum samples by UHPLC-UV using a previously described protocol[18]. The peak of the chromatogram at a retention time of 1.05 mincorresponds to hydroxychloroquine metabolite. The serum concentration of this metabolite is deduced from UV absorption, asPlease cite this article as: P. Gautret, J.-C. Lagier and P. Parola et al., Hydroxychloroquine and azithromycin as a treatment of COVID-19:results of an open-label non-randomized clinical trial, International Journal of Antimicrobial Agents, https://doi.org/10.1016/j.ijantimicag.2020.105949

ARTICLE IN PRESSJID: ANTAGE[m5G;June 30, 2020;0:48]P. Gautret, J.-C. Lagier and P. Parola et al. / International Journal of Antimicrobial Agents xxx (xxxx) xxx3Table 1Characteristics of the study population.Age (years)Male genderClinical statusTime between onset ofsymptoms and inclusion (days)Mean SDtp-valuen (%)p-valueAsymptomaticURTILRTIMean SDtp-valueHydroxychloroquinetreated patients(N 20)51.2 18.7-1.950.069 (45.0)0.652 (10.0)12 (60.0)6 (30.0) 0.304.1 2.6-0.150.88Control patients(N 16)37.3 24.06 (37.5)4 (25.0)10 (62.5)2 (12.5)3.9 2.8All patients (36)45.1 22.015 (41.7)6 (16.7)22 (61.1)8 (22.2)4.0 2.6p-valueURTI: upper tract respiratory infection, LRTI: lower tract respiratory infectionfor hydroxychloroquine concentration. Considering both concentrations provides an estimate of initial serum hydroxychloroquineconcentration.2.8. CultureFor all patients, 500 μL of the liquid collected fromthe nasopharyngeal swab were passed through a 0.22-μmpore 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 (ATCCCRL-1586) in Minimum Essential Medium culture medium with4% fetal calf serum and 1% glutamine. After centrifigationat 4,0 0 0 g, the microplates were incubated at 37 C. Plateswere observed daily for evidence of cytopathogenic effect.Presumptive detection of the virus in the supernatant wasdone using SU50 0 0 SEM (Hitachi) then confirmed by specificRT-PCR.2.9. OutcomeThe primary endpoint was virological clearance at day-6 postinclusion. Secondary outcomes were virological clearance over thetime of the study period and clinical follow-up (body temperature,respiratory rate, lenght of stay in hospital and mortality), and occurrence of side-effects.one transferred on day2 post-inclusion who was PCR-positive onday1, one transferred on day3 post-inclusion who was PCR-positiveon days1-2 and one transferred on day4 post-inclusion who wasPCR-positive on day1 and day3; one patient died on day3 postinclusion and was PCR-negative on day2; one patient decided toleave the hospital on day3 post-inclusion and was PCR-negativeon days1-2; finally, one patient stopped the treatment on day3post-inclusion because of nausea and was PCR-positive on days12-3. The results presented here are therefore those of 36 patients(20 hydroxychloroquine-treated patients and 16 control patients).None of the control patients were lost during follow-up. Basicdemographics and clinical status are presented in Table 1. Overall,15 patients were male (41.7%), with a mean age of 45.1 years. Theproportion of asymptomatic patients was 16.7%, that of patientswith URTI symptoms was 61.1% and that of patients with LRTIsymptoms was 22.2%. All patients with LRTI symptoms had confirmed pneumonia by CTScan. Hydroxychloroquine-treated patientswere older than control patients (51.2 years vs. 37.3 years). Nosignificant difference was observed between hydroxychloroquinetreated patients and control patients with regard to gender,clinical status and duration of symptoms prior to inclusion(Table 1). Among hydroxychloroquine-treated patients six patientsreceived azithromycin (500mg on day1 followed by 250mg perday for the next four days) to prevent bacterial super-infection andwere kept under daily electrocardiogram control. Clinical follow-upand occurrence of side-effects will be described in a further paperat the end of the trial.2.10. StatisticsAssuming a 50% efficacy of hydroxychloroquine in reducing theviral load at day 7, an 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 thecontrol group) would be required for the analysis (Fleiss with CC).Statistical differences were evaluated by Pearson’s chi-square orFisher’s exact tests as categorical variables, as appropriate. Meansof quantitative data were compared using Student’s t-test. Analyseswere performed in Stata version 14.2.3. Results (detailed results are available in supplementaryTable 1)3.1. Demographics and clinical presentationWe enrolled 36 out of 42 patients meeting the inclusion criteriain this study that had at least six days of follow-up at the timeof the present analysis. A total of 26 patients received hydroxychloroquine and 16 were control patients. Six hydroxychloroquinetreated patients were lost during the follow-up of the surveybecause of early cessation of treatment. Reasons are as follows:three patients were transferred to intensive care unit, including3.2. Hydroxychloroquine dosageMean hydroxychloroquineμg/ml 0.2 (N 20).serumconcentrationwas0.463.3. Effect of hydroxychloroquine on viral loadThe 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 patientswere virologically cured compared to 12.5% in the control group(p 0.001).When comparing the effect of hydroxychloroquine treatmentas a single drug and the effect of hydroxychloroquine andazithromycin in combination, the proportion of patients that hadnegative PCR results in nasopharyngeal samples was significantlydifferent between the two groups at days 3-4-5 and 6 postinclusion (Table 3). At day6 post-inclusion, 100% of patients treatedwith a combination of hydroxychloroquine and azithromycin werevirologically cured compared to 57.1% of patients treated with hydroxychloroquine only, and 12.5% in the control group (p 0.001).Please cite this article as: P. Gautret, J.-C. Lagier and P. Parola et al., Hydroxychloroquine and azithromycin as a treatment of COVID-19:results of an open-label non-randomized clinical trial, International Journal of Antimicrobial Agents, https://doi.org/10.1016/j.ijantimicag.2020.105949

ARTICLE IN PRESSJID: ANTAGE4[m5G;June 30, 2020;0:48]P. Gautret, J.-C. Lagier and P. Parola et al. / International Journal of Antimicrobial Agents xxx (xxxx) xxxTable 2Proportion of patients with virological cure (negative nasopharyngeal PCR) by day, in COVID-19 patients treated with hydroxychloroquine and in COVID-19 control patients.Day3 post inclusionDay4 post inclusionDay5 post inclusionDay6 post inclusionNumber ofnegativepatients/totalnumber ofpatients%p-valueNumber ofnegativepatients/totalnumber ofpatients%p-valueNumber ofnegativepatients/totalnumber ofpatients%p-valueNumber ofnegativepatients/totalnumber ofpatients%Hydroxychloroquinetreated patients(N 0.0Control patients(N trol patients from centers other than Marseille did not undergo daily sampling, but were sampled every other day in most cases, they were considered positive forPCR when actually positive the day(s) before and the day(s) after the day(s) with missing data.Table 3Proportion of patients with virological cure (negative nasopharyngeal PCR) by day, in COVID-19 patients treated with hydroxychloroquine only, in COVID-19 patients treatedwith hydroxychloroquine and azithromycin combination, and in COVID-19 control patients.Day3 post inclusionDay4 post inclusionDay5 post inclusionDay6 post inclusionNumber ofnegativepatients/totalnumber ofpatients%p-valueNumber ofnegativepatients/totalnumber ofpatients%p-valueNumber ofnegativepatients/totalnumber ofpatients%p-valueNumber ofnegativepatients/totalnumber ofpatients%Control 2.5Hydroxychloroquinetreatment uineand azithromycincombined treatment5/683.35/683.36/61006/6100p-value 0.001Fig. 1. Percentage of patients with PCR-positive nasopharyngeal samples from inclusion to day6 post-inclusion in COVID-19 patients treated with hydroxychloroquine and inCOVID-19 control patients.These results are summarized in Figs. 1 and 2. Drug effect was significantly higher in patients with symptoms of URTI and LRTI, ascompared to asymptomatic patients with p 0.05 (data not shown).Of note, one patient who was still PCR-positive at day6post inclusion under hydroxychloroquine treatment only, receivedazithromycin in addition to hydroxychloroquine at day8-post inclusion and cured her infection at day-9 post inclusion. In contrast,one of the patients under hydroxychloroquine and azithromycincombination who tested negative at day6 post-inclusion was testedpositive at low titer at day8 post-inclusion.Please cite this article as: P. Gautret, J.-C. Lagier and P. Parola et al., Hydroxychloroquine and azithromycin as a treatment of COVID-19:results of an open-label non-randomized clinical trial, International Journal of Antimicrobial Agents, https://doi.org/1