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      Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open-label non-randomized clinical trial

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          Abstract

          Background

          Chloroquine and hydroxychloroquine have been found to be efficient on SARS-CoV-2, and reported to be efficient in Chinese COV-19 patients. We evaluate the role of hydroxychloroquine on respiratory viral loads.

          Patients and methods

          French Confirmed COVID-19 patients were included in a single arm protocol from early March to March 16 th, to receive 600mg of hydroxychloroquine daily and their viral load in nasopharyngeal 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 the protocol were included as negative controls. Presence and absence of virus at Day6-post inclusion was considered the end point.

          Results

          Six patients were asymptomatic, 22 had upper respiratory tract infection symptoms and eight had lower respiratory tract infection symptoms.

          Twenty cases were treated in this study and showed a significant reduction of the viral carriage at D6-post inclusion compared to controls, and much lower average carrying duration than reported of untreated patients in the literature. 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 reinforced by azithromycin.

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          Most cited references 20

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          Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro

          Dear Editor, In December 2019, a novel pneumonia caused by a previously unknown pathogen emerged in Wuhan, a city of 11 million people in central China. The initial cases were linked to exposures in a seafood market in Wuhan. 1 As of January 27, 2020, the Chinese authorities reported 2835 confirmed cases in mainland China, including 81 deaths. Additionally, 19 confirmed cases were identified in Hong Kong, Macao and Taiwan, and 39 imported cases were identified in Thailand, Japan, South Korea, United States, Vietnam, Singapore, Nepal, France, Australia and Canada. The pathogen was soon identified as a novel coronavirus (2019-nCoV), which is closely related to sever acute respiratory syndrome CoV (SARS-CoV). 2 Currently, there is no specific treatment against the new virus. Therefore, identifying effective antiviral agents to combat the disease is urgently needed. An efficient approach to drug discovery is to test whether the existing antiviral drugs are effective in treating related viral infections. The 2019-nCoV belongs to Betacoronavirus which also contains SARS-CoV and Middle East respiratory syndrome CoV (MERS-CoV). Several drugs, such as ribavirin, interferon, lopinavir-ritonavir, corticosteroids, have been used in patients with SARS or MERS, although the efficacy of some drugs remains controversial. 3 In this study, we evaluated the antiviral efficiency of five FAD-approved drugs including ribavirin, penciclovir, nitazoxanide, nafamostat, chloroquine and two well-known broad-spectrum antiviral drugs remdesivir (GS-5734) and favipiravir (T-705) against a clinical isolate of 2019-nCoV in vitro. Standard assays were carried out to measure the effects of these compounds on the cytotoxicity, virus yield and infection rates of 2019-nCoVs. Firstly, the cytotoxicity of the candidate compounds in Vero E6 cells (ATCC-1586) was determined by the CCK8 assay. Then, Vero E6 cells were infected with nCoV-2019BetaCoV/Wuhan/WIV04/2019 2 at a multiplicity of infection (MOI) of 0.05 in the presence of varying concentrations of the test drugs. DMSO was used in the controls. Efficacies were evaluated by quantification of viral copy numbers in the cell supernatant via quantitative real-time RT-PCR (qRT-PCR) and confirmed with visualization of virus nucleoprotein (NP) expression through immunofluorescence microscopy at 48 h post infection (p.i.) (cytopathic effect was not obvious at this time point of infection). Among the seven tested drugs, high concentrations of three nucleoside analogs including ribavirin (half-maximal effective concentration (EC50) = 109.50 μM, half-cytotoxic concentration (CC50) > 400 μM, selectivity index (SI) > 3.65), penciclovir (EC50 = 95.96 μM, CC50 > 400 μM, SI > 4.17) and favipiravir (EC50 = 61.88 μM, CC50 > 400 μM, SI > 6.46) were required to reduce the viral infection (Fig. 1a and Supplementary information, Fig. S1). However, favipiravir has been shown to be 100% effective in protecting mice against Ebola virus challenge, although its EC50 value in Vero E6 cells was as high as 67 μM, 4 suggesting further in vivo studies are recommended to evaluate this antiviral nucleoside. Nafamostat, a potent inhibitor of MERS-CoV, which prevents membrane fusion, was inhibitive against the 2019-nCoV infection (EC50 = 22.50 μM, CC50 > 100 μM, SI > 4.44). Nitazoxanide, a commercial antiprotozoal agent with an antiviral potential against a broad range of viruses including human and animal coronaviruses, inhibited the 2019-nCoV at a low-micromolar concentration (EC50 = 2.12 μM; CC50 > 35.53 μM; SI > 16.76). Further in vivo evaluation of this drug against 2019-nCoV infection is recommended. Notably, two compounds remdesivir (EC50 = 0.77 μM; CC50 > 100 μM; SI > 129.87) and chloroquine (EC50 = 1.13 μM; CC50 > 100 μM, SI > 88.50) potently blocked virus infection at low-micromolar concentration and showed high SI (Fig. 1a, b). Fig. 1 The antiviral activities of the test drugs against 2019-nCoV in vitro. a Vero E6 cells were infected with 2019-nCoV at an MOI of 0.05 in the treatment of different doses of the indicated antivirals for 48 h. The viral yield in the cell supernatant was then quantified by qRT-PCR. Cytotoxicity of these drugs to Vero E6 cells was measured by CCK-8 assays. The left and right Y-axis of the graphs represent mean % inhibition of virus yield and cytotoxicity of the drugs, respectively. The experiments were done in triplicates. b Immunofluorescence microscopy of virus infection upon treatment of remdesivir and chloroquine. Virus infection and drug treatment were performed as mentioned above. At 48 h p.i., the infected cells were fixed, and then probed with rabbit sera against the NP of a bat SARS-related CoV 2 as the primary antibody and Alexa 488-labeled goat anti-rabbit IgG (1:500; Abcam) as the secondary antibody, respectively. The nuclei were stained with Hoechst dye. Bars, 100 μm. c and d Time-of-addition experiment of remdesivir and chloroquine. For “Full-time” treatment, Vero E6 cells were pre-treated with the drugs for 1 h, and virus was then added to allow attachment for 2 h. Afterwards, the virus–drug mixture was removed, and the cells were cultured with drug-containing medium until the end of the experiment. For “Entry” treatment, the drugs were added to the cells for 1 h before viral attachment, and at 2 h p.i., the virus–drug mixture was replaced with fresh culture medium and maintained till the end of the experiment. For “Post-entry” experiment, drugs were added at 2 h p.i., and maintained until the end of the experiment. For all the experimental groups, cells were infected with 2019-nCoV at an MOI of 0.05, and virus yield in the infected cell supernatants was quantified by qRT-PCR c and NP expression in infected cells was analyzed by Western blot d at 14 h p.i. Remdesivir has been recently recognized as a promising antiviral drug against a wide array of RNA viruses (including SARS/MERS-CoV 5 ) infection in cultured cells, mice and nonhuman primate (NHP) models. It is currently under clinical development for the treatment of Ebola virus infection. 6 Remdesivir is an adenosine analogue, which incorporates into nascent viral RNA chains and results in pre-mature termination. 7 Our time-of-addition assay showed remdesivir functioned at a stage post virus entry (Fig. 1c, d), which is in agreement with its putative anti-viral mechanism as a nucleotide analogue. Warren et al. showed that in NHP model, intravenous administration of 10 mg/kg dose of remdesivir resulted in concomitant persistent levels of its active form in the blood (10 μM) and conferred 100% protection against Ebola virus infection. 7 Our data showed that EC90 value of remdesivir against 2019-nCoV in Vero E6 cells was 1.76 μM, suggesting its working concentration is likely to be achieved in NHP. Our preliminary data (Supplementary information, Fig. S2) showed that remdesivir also inhibited virus infection efficiently in a human cell line (human liver cancer Huh-7 cells), which is sensitive to 2019-nCoV. 2 Chloroquine, a widely-used anti-malarial and autoimmune disease drug, has recently been reported as a potential broad-spectrum antiviral drug. 8,9 Chloroquine is known to block virus infection by increasing endosomal pH required for virus/cell fusion, as well as interfering with the glycosylation of cellular receptors of SARS-CoV. 10 Our time-of-addition assay demonstrated that chloroquine functioned at both entry, and at post-entry stages of the 2019-nCoV infection in Vero E6 cells (Fig. 1c, d). Besides its antiviral activity, chloroquine has an immune-modulating activity, which may synergistically enhance its antiviral effect in vivo. Chloroquine is widely distributed in the whole body, including lung, after oral administration. The EC90 value of chloroquine against the 2019-nCoV in Vero E6 cells was 6.90 μM, which can be clinically achievable as demonstrated in the plasma of rheumatoid arthritis patients who received 500 mg administration. 11 Chloroquine is a cheap and a safe drug that has been used for more than 70 years and, therefore, it is potentially clinically applicable against the 2019-nCoV. Our findings reveal that remdesivir and chloroquine are highly effective in the control of 2019-nCoV infection in vitro. Since these compounds have been used in human patients with a safety track record and shown to be effective against various ailments, we suggest that they should be assessed in human patients suffering from the novel coronavirus disease. Supplementary information Supplementary information, Materials and Figures
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            Chloroquine and hydroxychloroquine as available weapons to fight COVID-19

            Repositioning of drugs for use as antiviral treatments is a critical need [1]. It is commonly very badly perceived by virologists, as we experienced when reporting the effectiveness of azithromycin for Zika virus [2]. A response has come from China to the respiratory disease caused by the new coronavirus (SARS-CoV-2) that emerged in December 2019 in this country. Indeed, following the very recent publication of results showing the in vitro activity of chloroquine against SARS-CoV-2 [3], data have been reported on the efficacy of this drug in patients with SARS-CoV-2-related pneumonia (named COVID-19) at different levels of severity [4,5]. Thus, following the in vitro results, 20 clinical studies were launched in several Chinese hospitals. The first results obtained from more than 100 patients showed the superiority of chloroquine compared with treatment of the control group in terms of reduction of exacerbation of pneumonia, duration of symptoms and delay of viral clearance, all in the absence of severe side effects [4,5]. This has led in China to include chloroquine in the recommendations regarding the prevention and treatment of COVID-19 pneumonia [4,6]. There is a strong rationality for the use of chloroquine to treat infections with intracellular micro-organisms. Thus, malaria has been treated for several decades with this molecule [7]. In addition, our team has used hydroxychloroquine for the first time for intracellular bacterial infections since 30 years to treat the intracellular bacterium Coxiella burnetii, the agent of Q fever, for which we have shown in vitro and then in patients that this compound is the only one efficient for killing these intracellular pathogens [8,9]. Since then, we have also shown the activity of hydroxychloroquine on Tropheryma whipplei, the agent of Whipple's disease, which is another intracellular bacterium for which hydroxychloroquine has become a reference drug [10,11]. Altogether, one of us (DR) has treated ~4000 cases of C. burnetii or T. whipplei infections over 30 years (personal data). Regarding viruses, for reasons probably partly identical involving alkalinisation by chloroquine of the phagolysosome, several studies have shown the effectiveness of this molecule, including against coronaviruses among which is the severe acute respiratory syndrome (SARS)-associated coronavirus [1,12,13] (Table 1 ). We previously emphasised interest in chloroquine for the treatment of viral infections in this journal [1], predicting its use in viral infections lacking drugs. Following the discovery in China of the in vitro activity of chloroquine against SARS-CoV-2, discovered during culture tests on Vero E6 cells with 50% and 90% effective concentrations (EC50 and EC90 values) of 1.13 μM and 6.90 μM, respectively (antiviral activity being observed when addition of this drug was carried out before or after viral infection of the cells) [3], we awaited with great interest the clinical data [14]. The subsequent in vivo data were communicated following the first results of clinical trials by Chinese teams [4] and also aroused great enthusiasm among us. They showed that chloroquine could reduce the length of hospital stay and improve the evolution of COVID-19 pneumonia [4,6], leading to recommend the administration of 500 mg of chloroquine twice a day in patients with mild, moderate and severe forms of COVID-19 pneumonia. At such a dosage, a therapeutic concentration of chloroquine might be reached. With our experience on 2000 dosages of hydroxychloroquine during the past 5 years in patients with long-term treatment (>1 year), we know that with a dosage of 600 mg/day we reach a concentration of 1 μg/mL [15]. The optimal dosage for SARS-CoV-2 is an issue that will need to be assessed in the coming days. For us, the activity of hydroxychloroquine on viruses is probably the same as that of chloroquine since the mechanism of action of these two molecules is identical, and we are used to prescribe for long periods hydroxychloroquine, which would be therefore our first choice in the treatment of SARS-CoV-2. For optimal treatment, it may be necessary to administer a loading dose followed by a maintenance dose. Table 1 Main results of studies on the activity of chloroquine or hydroxychloroquine on coronavirusesa Table 1 Reference Compound(s) Targeted virus System used for antiviral activity screening Antiviral effect [12] Chloroquine SARS-CoV Vero (African green monkey kidney) E6 cells EC50 = 8.8 ± 1.2 μM [16] Chloroquine Vero E6 cells EC50 = 4.4 ± 1.0 μM [17] Chloroquine, chloroquine monophosphate, chloroquine diphosphate SARS-CoV (four strains) Vero 76 cells Chloroquine: EC50 = 1–4 μMChloroquine monophosphate: EC50 = 4–6 μMChloroquine diphosphate: EC50 = 3–4 μM BALB/c mice Intraperitoneal or intranasal chloroquine administration, beginning 4 h prior to virus exposure: 50 mg/kg but not 10 mg/kg or 1 mg/kg reduced for the intranasal route (but not the intraperitoneal route) viral lung titres from mean ± S.D. of 5.4 ± 0.5 to 4.4 ± 1.2 in log10 CCID50/g at Day 3 (considered as not significant) [18] Chloroquine, hydroxychloroquine SARS-CoV Vero cells Chloroquine: EC50 = 6.5 ± 3.2 μMHydroxychloroquine: EC50 = 34 ± 5 μM Feline coronavirus Crandell–Reese feline kidney (CRFK) cells Chloroquine: EC50 > 0.8 μMHydroxychloroquine: EC50 = 28 ± 27 μM [19] Chloroquine HCoV-229E Human epithelial lung cells (L132) Chloroquine at concentrations of 10 μM and 25 μM inhibited HCoV-229E release into the culture supernatant [20] Chloroquine HCoV-OC43 HRT-18 cells EC50 = 0.306 ± 0.0091 μM Newborn C57BL/6 mice; chloroquine administration transplacentally and via maternal milk 100%, 93%, 33% and 0% survival rate of pups when mother mice were treated per day with 15, 5, 1 and 0 mg/kg body weight, respectively [21] Chloroquine Feline infectious peritonitis virus (FIPV) Felis catus whole fetus-4 cells FIPV replication was inhibited in a chloroquine concentration-dependent manner [22] Chloroquine SARS-CoV Vero E6 cells EC50 = 4.1 ± 1.0 μM MERS-CoV Huh7 cells (human liver cell line) EC50 = 3.0 ± 1.1 μM HCoV-229E-GFP (GFP-expressing recombinant HCoV-229E) Huh7 cells (human liver cell line) EC50 = 3.3 ± 1.2 μM [3] Chloroquine SARS-CoV-2 Vero E6 cells EC50 = 1.13 μM CCID50, 50% cell culture infectious dose; CoV, coronavirus; EC50, 50% effective concentration (mean ± S.D.); GFP, green fluorescent protein; HCoV, human coronavirus; MERS, Middle East respiratory syndrome; SARS, severe acute respiratory syndrome; S.D., standard deviation. a See also [1] (Table 1) for additional references.
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              Zika virus cell tropism in the developing human brain and inhibition by azithromycin.

              The rapid spread of Zika virus (ZIKV) and its association with abnormal brain development constitute a global health emergency. Congenital ZIKV infection produces a range of mild to severe pathologies, including microcephaly. To understand the pathophysiology of ZIKV infection, we used models of the developing brain that faithfully recapitulate the tissue architecture in early to midgestation. We identify the brain cell populations that are most susceptible to ZIKV infection in primary human tissue, provide evidence for a mechanism of viral entry, and show that a commonly used antibiotic protects cultured brain cells by reducing viral proliferation. In the brain, ZIKV preferentially infected neural stem cells, astrocytes, oligodendrocyte precursor cells, and microglia, whereas neurons were less susceptible to infection. These findings suggest mechanisms for microcephaly and other pathologic features of infants with congenital ZIKV infection that are not explained by neural stem cell infection alone, such as calcifications in the cortical plate. Furthermore, we find that blocking the glia-enriched putative viral entry receptor AXL reduced ZIKV infection of astrocytes in vitro, and genetic knockdown of AXL in a glial cell line nearly abolished infection. Finally, we evaluate 2,177 compounds, focusing on drugs safe in pregnancy. We show that the macrolide antibiotic azithromycin reduced viral proliferation and virus-induced cytopathic effects in glial cell lines and human astrocytes. Our characterization of infection in the developing human brain clarifies the pathogenesis of congenital ZIKV infection and provides the basis for investigating possible therapeutic strategies to safely alleviate or prevent the most severe consequences of the epidemic.
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                Author and article information

                Contributors
                Journal
                Int J Antimicrob Agents
                Int. J. Antimicrob. Agents
                International Journal of Antimicrobial Agents
                Elsevier Science Publishers
                0924-8579
                1872-7913
                20 March 2020
                20 March 2020
                Affiliations
                [a ]IHU-Méditerranée Infection, Marseille, France
                [b ]Aix Marseille Univ, IRD, AP-HM, SSA, VITROME, Marseille, France
                [c ]Aix Marseille Univ, IRD, APHM, MEPHI, Marseille, France
                [d ]Thai Binh University of Medicine and Pharmacy, Thai Binh, Viet Nam
                [e ]Infectiologie, Hôpital de l'Archet, Centre Hospitalier Universitaire de Nice, Nice, France
                [f ]Université Côte d'Azur, Nice, France
                [g ]U1065, Centre Méditerranéen de Médecine Moléculaire, C3M, Virulence Microbienne et Signalisation Inflammatoire, INSERM, Nice, France
                [h ]Department of Virology, Biological and Pathological Center, Centre Hospitalier Universitaire de Nice, 06200 Nice, France
                [i ]Service Pharmacie, Hôpital Timone, AP-HM, Marseille, France
                [j ]Laboratoire de Pharmacie Clinique, Aix Marseille Université, Marseille, France
                Author notes
                [* ]Corresponding author. Didier.raoult@ 123456gmail.com
                [$]

                equal work

                Article
                S0924-8579(20)30099-6 105949
                10.1016/j.ijantimicag.2020.105949
                7102549
                32205204
                © 2020 Published by Elsevier B.V.

                Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

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