key: cord-0842114-romff7hl authors: Gyselinck, Iwein; Liesenborghs, Laurens; Belmans, Ann; Engelen, Matthias M.; Betrains, Albrecht; Van Thillo, Quentin; Nguyen, Pham Anh Hong; Goeminne, Pieter; Soenen, Ann-Catherine; De Maeyer, Nikolaas; Pilette, Charles; Papleux, Emmanuelle; Vanderhelst, Eef; Derweduwen, Aurélie; Alexander, Patrick; Bouckaert, Bernard; Martinot, Jean-Benoît; Decoster, Lynn; Vandeurzen, Kurt; Schildermans, Rob; Verhamme, Peter; Janssens, Wim; Vos, Robin title: Azithromycin for Treatment of Hospitalised COVID-19 Patients: a randomised, multicentre, open-label clinical trial (DAWn-AZITHRO) date: 2022-01-27 journal: ERJ Open Res DOI: 10.1183/23120541.00610-2021 sha: f0cc3a69cdc8bf60d844e508f46fd4d4547d84dc doc_id: 842114 cord_uid: romff7hl BACKGROUND AND OBJECTIVES: Azithromycin was rapidly adopted as a repurposed drug to treat COVID-19 early in the pandemic. We aimed to evaluate its efficacy in patients hospitalised for COVID-19. METHODS: In a series of randomised, open-label, phase 2 proof-of-concept, multicenter clinical trials (Direct Antivirals Working against the novel Coronavirus [DAWn]), several treatments were compared with standard of care. In 15 Belgian hospitals, patients hospitalised with moderate to severe COVID-19 patients were allocated 2:1 to receive standard of care plus azithromycin or standard of care alone. The primary outcome was time to live discharge or sustained clinical improvement, defined as a two-point improvement on the WHO ordinal scale sustained for at least 3 days. RESULTS: Patients were included between April 22 and December 17, 2020. When 15-day follow-up data were available for 160 patients (56% of preset cohort), an interim analysis was performed at request of the independent Data Safety and Monitoring Board. Subsequently, DAWn-AZITHRO was stopped for futility. In total, 121 patients were allocated to the treatment arm and 64 patients to the standard of care arm. We found no effect of azithromycin on the primary outcome with Hazard ratio of 1.044 (95% confidence interval, 0.772–1.413; p=0.7798). None of the predefined subgroups showed significant interaction as covariates in the Fine-Gray regression analysis. No benefit of azithromycin was found on any of the short- and longer-term secondary outcomes. CONCLUSION: Time to clinical improvement is not influenced by azithromycin in patients hospitalised with moderate to severe COVID-19. The high disease burden and the rapid spread of the SARS-CoV-2 pandemic immediately directed the search for disease-modifying agents towards the repurposing of existing molecules. The Belgian Direct Antivirals Working against the novel Coronavirus studies were a parallel series of proof-of-concept trials, assessing several treatment strategies for hospitalized patients with COVID-19: itraconazole (1), azithromycin (2) , anakinra combined with intensified anticoagulation (3) , and convalescent plasma (4) . Azithromycin is a macrolide antibiotic of which the use in COVID-19 was encouraged by a documented in vitro antiviral activity against SARS-CoV-2 and other viruses (5, 6) , previous clinical experiences with influenza (7, 8) and data from uncontrolled studies early during the pandemic (9, 10) . Apart from its antibiotic and potential antiviral properties, it was hypothesized that azithromycin's broad range of immunomodulatory effects could temper COVID-19 induced hyperinflammation (11) . Until now, primary endpoints that have been assessed in randomised controlled trials with azithromycin in hospitalized COVID-19 patients are clinical status at day 15 (12, 13) and 28-day mortality (14, 15) . No benefit of azithromycin was shown for any of these outcomes. In this randomised controlled trial, we assessed the effect of azithromycin on time to discharge or sustained clinical improvement, based on the World Health Organization (WHO) issued ordinal scale. DAWn-Azithro was an open-label, randomised, adaptive clinical trial conducted in 15 Belgian hospitals and coordinated by University Hospitals Leuven, to assess if azithromycin added to standard of care could shorten time to discharge or clinical improvement in hospitalized COVID-19 patients. The study protocol and statistical analysis plan are available in the appendix, and have been published previously (2) . The study was approved by a central Ethics Committee (Comité d'Éthique Hospitalo- guidelines. An independent Data Safety and Monitoring Board (DSMB) assessed the trial progress and patient safety and wellbeing. The DSMB performed pre-planned safety reviews after 80 and 160 patients, and could perform ad-hoc analyses in case of substantial evidence of a safety issue. Adults (≥ 18 years) hospitalized on a dedicated COVID-19 ward were eligible if they had symptomatic illness of any duration with (1a) radiographic infiltrates or (1b) clinical signs of pneumonia with an oxygen saturation of ≤ 94% on room air or required respiratory support) and (2a) recent laboratory or (2b) radiographic confirmed diagnosis of COVID-19 (≤ 72h before randomisation). Exclusion criteria were elevated liver transaminases (AST or ALT > 5 times the upper limit of normal), pregnancy or breast-feeding, allergy to macrolides, any medical condition which would impose an unacceptable safety hazard by participation to the study, heart failure with severely reduced ejection fraction (≤ 30%), a prolonged corrected QT interval on electrocardiogram (> 470 ms for males and > 480 ms for females), the use of macrolides during the last week prior to admission. Informed consent was obtained prior to randomisation. When written informed consent was not obtainable due to restrictions for research staff to access the isolation ward, oral consent was documented in the electronic medical record, and completed with a signed consent as soon as possible. If patients were unable to provide consent, the legal representative was consulted instead. Eligible and consenting patients were randomly allocated to azithromycin on top of standard of care (intervention group) or standard of care alone (control group) according to a 2:1 allocation scheme stratified by study site, using randomly selected block sizes of 6 or 9. Randomisation was done using a centralized web-based randomisation application. The study was open label. Patients, clinicians, and study personnel were aware of the assigned treatment. The trial statistician was not given access to the full database and was not aware of the allocated treatments. The trial statistician remained blinded until database lock. In the intervention group, 500 mg of azithromycin was given on top of standard of care for the first 5 consecutive days, administered once daily as oral tablets or syrup. Standard-of-care treatment followed regularly updated national and international recommendations, as allowed by the adaptive design. ECG was monitored in patients at risk for long QT. When QTc exceeded 500ms and/or QTc increased with more than 60 ms compared to baseline, azithromycin was interrupted or discontinued. We collected patients' demographics and clinical data, including medical history, use of concomitant therapies, clinical investigation and National Early Warning Score (NEWS), laboratory results and radiographic investigations. Clinical status was assessed daily until discharge and on day 15, day 29 and the follow-up visit 5-7 weeks after discharge. The primary outcome was the time from randomisation to alive discharge or sustained clinical improvement, the latter defined as an improvement of > 2 points compared to the highest value of day 0 and 1 and sustained for at least 3 days. The clinical status was recorded on a 7-point ordinal scale: (1) not hospitalized, no limitations on activities; (2) not hospitalized, limitation on activities; (3) hospitalized, not requiring supplemental oxygen; (4) hospitalized, requiring supplemental oxygen; (5) hospitalized, on non-invasive ventilation or high flow oxygen devices; (6) hospitalized, on invasive mechanical ventilation or ECMO; and (7) death. Secondary outcomes were daily clinical status on the ordinal scale while hospitalized and on days 15 and 29, daily NEWS while hospitalized and on days 15 and 29, cumulative clinical status up to day 15 (i.e., sum of daily clinical status scores from days 1 to 15), mortality on day 15 and day 29, time to ICU admission, time to death, duration of supplemental oxygen, duration of mechanical ventilation, duration of hospitalization, duration of intensive care stay, adverse events graded as grade 4 or 5 or SAEs, QTc abnormalities and a combined cardiac endpoint (hs-TroponinT levels > 0.5 ng per mL and/or ventricular arrhythmia requiring intervention and/or sudden cardiac death). Based on the trial of Cao et al (16) , we assumed that 40% of patients would have reached a 2-point improvement on the ordinal scale with standard of care at day 15. Using a log-rank test, with a 2-sided significance level of 5% and a power of 80% and using a (2:1) randomisation ratio in favor of azithromycin, we estimated that a total sample size of 269 patients to detect an absolute improvement of 17.5%. Taking early dropouts into account, we decided on a total sample size of 282 patients. A detailed description of the analysis is provided in the Statistical Analysis Plan (SAP), which was finalised and filed before database lock (appendix). A brief summary is provided here. Analysis sets were finalised during a Blind Review Meeting prior to database lock. The Full Analysis Set (FAS) included all randomised patients, except patients that were confirmed to be SARS-CoV-2 negative, and patients who withdrew consent to use any data immediately after randomisation and before treatment administration. The Per Protocol Set (PPS) included all FAS patients, but patients randomised to azithromycin who missed 2 or more days of dosing were excluded. The primary analysis set of interest was the FAS. All efficacy analyses were repeated on the PPS as a sensitivity analysis. Missing clinical status data were accounted for by means of multiple imputation, using a total of 100 imputations (17) . Treatment effects for all endpoints were estimated by an appropriate measure and presented with 95% confidence intervals and were adjusted for study site (small versus large sites, according to median size) and period (1 st and 2 nd wave). The primary endpoint was compared using competing-risk methodology, using cumulative incidence functions (CIF) to estimate event rates and a Fine & Grey regression model to obtain cause-specific hazard ratios. Daily clinical status was analysed a proportional odds logistic regression to estimate the common odds ratio. All-cause mortality and survival without mechanical ventilation up to 30 days were assessed using a Cox regression to obtain hazard ratios. Incidence rates were estimated using Kaplan-Meier methodology. Time to hospital discharge, incidence and duration of supplemental oxygen, mechanical ventilation and ICU were analysed using the same methodology as for the primary endpoint. Cumulative clinical status scores were analysed using a general linear model on the log-transformed scores to obtain a treatment ratio of geometric means between the treatment groups. Pre-specified subgroup analyses were performed for the primary endpoint, considering the following subgroups: duration of symptoms prior to enrolment (according to observed median); age groups (according to observed median); study period; clinical status at baseline (3&4 vs 5&6) and ethnicity. All tests were two-sided and assessed at a significance level of 5%. No correction was made for multiple secondary endpoints. All analyses were performed using SAS software version 9.4 for Windows 10. No safety issues were raised during the pre-planned safety reviews. Online preprint publication of data regarding azithromycin from the RECOVERY-trial (14) , which failed to demonstrate a benefit of azithromycin, became available just prior to completion of our 15-day follow up of the first 160 included patients. Therefore, the trial steering committee and DSMB issued to additionally perform a futility analysis on these patients, included in the second safety review. Recruitment was halted on December 18 th awaiting the results; 185 patients were randomized at that time. Eventually, the trial was stopped for futility on January 25 th , as the conditional power to detect a significant difference for the primary outcome was 0.4%. (futility analysis and DSMB recommendations are included in the appendix). Between April 24 th , 2020 and December 17 th , 2021, 185 patients were allocated to azithromycin (n = 121) or standard of care (n = 64). Two patients in the intervention group were excluded from the full analysis set (FAS, n = 119, equals safety set [SS] ), as they withdrew consent and were not treated according to allocation (consort diagram: supplementary figure 1). Baseline data are presented in Table 1 , and were well matched between intervention and SOC group. Participants had a mean age of 62 years (standard deviation [SD] 15 years), 37.8% of them were women. 16 .9% had a history of diabetes and 44.8% a history of arterial hypertension. 40,0% were current or former smokers. During hospitalization, 14.9% of patients received hydroxychloroquine. There was a nonsignificant trend towards increased use in the SOC group. Use of other concomitant medication was similar between both groups, with over half of patients receiving systemic corticosteroids (supplementary table 1). One hundred forty patients (76.5%) presented at the ambulatory pulmonology clinic for a follow-up visit 5 to 7 weeks after discharge. Data on exploratory outcomes were collected at that timepoint, such as diffusion capacity for carbonic monoxide (n = 137), high resolution CT scan (n = 115) and 6-minute walking distance (n= 121). In the azithromycin group and the SOC group respectively, the primary outcome was met in 39.5% vs. 35 figure 1 ). No statistically significant interactions or treatment effects could be observed for any of the predefined subgroups (figure 2). Secondary endpoints are shown in In 63% of patients in both the azithromycin and SOC group, chest CT was performed after 5-7 weeks. There was a similar proportion of patients with normal CTs (25/ These findings remained consistent across all prespecified subgroups. A convincing lack of benefit of azithromycin on 28-day mortality in hospitalized patients with COVID-19 was already shown in the RECOVERY-trial (14) . Moreover, Cavalcanti et al (12) and Furtado et al (13) demonstrated no effect on clinical status at day 15. However, assessment of outcomes at a fixed timepoint, may risk missing the time of clinical benefit (18) , and the often used proportional odds model is difficult to clinically interpret. The primary outcome of DAWn-AZITHRO, a time-to-eventanalysis of the WHO-issued ordinal scale, was therefore still relevant and readily interpretable. A strength of our study, was the follow-up after hospital discharge. While the lack of effect of azithromycin on short-term outcomes like 15-and 28-day clinical status and mortality have been extensively published, few have reported on longer term and functional outcomes. It has been hypothesized that Azithromycin-treatment in COVID-19 may promote tissue repair and reduce post-COVID fibrosis by modulation of macrophage and myofibroblast function (19) . At our five-to-seven- week follow-up visit, the proportion of patients with complete resolution of COVID-related radiological abnormalities was similar in treated and untreated patients. Correspondingly, we also found no meaningful difference in diffusion capacity nor walking distance. We thus could not demonstrate that azithromycin promotes post-COVID recovery. There may be several reasons why azithromycin fails to benefit COVID-19 patients. First, the in vitro antiviral activity is weak, compared with direct-acting antivirals like remdesivir (1), and azithromycin has not shown to aid viral clearance in vivo(20). Second, the incidence of bacterial superinfection in COVID-19 is low (21) , and, contrary to what is seen in influenza (7, 8, 22) , a prophylactic effect against post-viral pneumococcal and atypical pneumonia is thus absent. Last, the broad immunomodulatory actions of azithromycin are well known and broadly exploited in day-to-day respiratory care, albeit mostly for long-term treatment of chronic respiratory conditions as COPD, cystic fibrosis (CF) and non-CF bronchiectasis, or chronic lung allograft rejection (23) . In the acute setting of COVID-19 however, azithromycin may miss the potency to benefit. This seems equally true even in mild and early stage disease, as has recently been shown in studies assessing outpatient treatment with azithromycin (24) . Our trial has some limitations. First, the trial was prematurely stopped after an interim analysis and is therefore underpowered to exclude for a type II error. However, given the complete overlap of survival curves, the absence of any significant or numerical benefit on secondary outcomes, and the negative evidence on Azithromycin for COVID-19 from other trials, the likelihood of wrongly accepting the null hypothesis is extremely low. Second, the trial was open label and not placebo-controlled. This might have led to performance bias in early management, and bias during follow-up and even assessment of the ordinal scale, which is subjective to some degree, both for caregivers (e.g. decision to stop oxygen, discharge) and patients (e.g. perceived degree of limitations at home). Third, the definition of standard of care was not strictly defined, could change according to updated national and international guidelines, and was ultimately left at the treating physician's discretion. This has however been corrected by including study period as a covariate in the Fine and Gray regression, showing no significant interaction. Last, no viral outcome was assessed, as other antiviral compounds like remdesivir, that showed much stronger in vitro SARS-CoV-2 antiviral activity than azithromycin (1) had already failed to significantly reduce viral load in a similar population of hospitalized patients (25) . Therefore, eventually, we did not expect the potential antiviral activity of azithromycin to contribute much to the effect size and -in this late disease stage-rather assessed its anti-inflammatory and immunomodulatory effect. In summary, we showed that in hospitalized patients with COVID-19, azithromycin did not reduce the time to sustained clinical improvement or discharge. We were also able to confirm a lack of efficacy on clinical status or mortality at the fixed timepoints that were previously assessed by large trials. The undersigned confirm that the above referenced protocol has been acknowledged and accepted, and agree to conduct the Trial in compliance with the approved protocol, and will adhere to: The undersigned agree not to disclose the confidential information contained in this document for any purpose other than the evaluation or conduct of the Trial, without prior written consent of the Sponsor. The undersigned also commit to making the findings of the Trial publicly available through publication and/or other dissemination tools, in accordance with this protocol and applicable regulations, without any unnecessary delay and to provide an honest, accurate and transparent account of the Trial; and to explain any discrepancies or deviations from the approved Trial protocol. No fault liability insurance has been taken out by UZ Leuven for treating and/or compensating Trial participants who are harmed as a consequence of participation in the Trial. The Principle Investigator (PI) is responsible for the conduct of the Trial at his/her Participating Site, and for protecting the rights, safety and well-being of the Trial participants. As such the PI must ensure adequate supervision of the Trial conduct at the Participating Site. If any tasks are delegated, the PI will maintain a log of appropriately qualified persons to whom he/she has delegated specified Trial-related duties. The PI will ensure that adequate training is provided and documented for all Trial staff, prior to conducting assigned Trial-related activities. It is the Coordinating Investigator's (CI's) responsibility to supervise the general conduct (e.g. Trial progress, communication, protocol training and support of the participating sites, annual reporting to the Ethics Committee (EC), end of Trial notification(s) and results reporting…) of the Trial. The CI fulfils both Investigator and Sponsor responsibilities, as outlined in International Conference on Harmonisation -Good Clinical Practice (ICH-GCP) E6(R2) and applicable regulations. PI and CI shall each be referred to as « Investigator(s)». TMP-CTC ALG-001-1.0_14022020 UZ Leuven -KUL Long QT (> 470 msec males and > 480 females) is an exclusion for participation. In patients with no long QT on ECG but at risk, a QT monitoring will be performed with intermittent ECG monitoring at d2-3 or continuous follow-up on ICUs. When QTc> 500 ms and/or delta QTc>60ms, IMP will be interrupted/discontinued at the discretion of the investigator. The patient's medication will be reviewed daily to evaluate DDIs including drugs prolonging the QTc interval according to what is listed in Appendix 2. TMP-CTC ALG-001-1.0_14022020 Page 13 of 50 Protocol template for non-commercial UZ Leuven -KUL Based on the current information, two strategies hold great promise for a successful reduction of COVID-19 disease burden. The first is of course the reduction of viral replication. In the Laboratory of Virology and Chemotherapy at the Rega Institute (KU Leuven), a library of existing drugs that were previously tested in clinical trials, of which some are available on the marked, was screened for activity against SARS-CoV-2 with the aim to repurposing drugs for COVID-19. It is expected that several compounds will be identified that show anti-viral activity in vitro, which subsequently need to be investigated in patients 2 . The second strategy is to modify the (in some cases excessive) host response. Widespread systemic inflammation and subsequent activation of the coagulation and complement system have repeatedly been described in severe COVID-19 3, 4 . Moreover, drugs influencing these pathways (e.g. anticoagulation) have suggested improved outcome in some small and non-randomized observational studies 5 . Additional strata that may thus be added to the DAWN study include new antiviral drugs (e.g. favipiravir), intensifying anticoagulation (e.g. with low molecumal weight heparin), adding anti-inflammatory molecules (e.g. interleukin receptor antagonists, or C1-esterase inhibitors) or reconvalescent plasma. One of the candidate drugs which may impact on COVID-19 is Azithromycin. Azithromycin is a macrolide molecule that exerts anti-inflammatory and immunomodulatory effects in a broad range of respiratory and infectious diseases through modulation of innate and adaptive immune responses, as previously extensively summarized by our group 6 and others 7, 8 . Different clinical trials have proven its efficacy in inflammatory respiratory diseases such as COPD, bronchiectasis, asthma and lung transplantation [9] [10] [11] [12] [13] . Azithromycin also has direct and indirect antiviral activity in bronchial epithelial cells 14, 15 and other host cells, as has been shown for influenza virus 16 27 . Also, azithromycin-use was associated with decreased 60-day mortality (HR 0.31; 95 % confidence interval, 0.11-082; P=0.02) and shorter time of ventilator-dependency in patients with sepsisassociated acute respiratory distress syndrome 28 . The mechanism of action of azithromycin in structural cells (such as epithelial and endothelial cells), monocytes and leukocytes is mainly related to 6 : 1/ altered intra-cellular signal transduction between cell surface and nucleus (modulation of MAPK pathway), 2/ lysosomotropic action (by lysosomal accumulation, leading to change of pH and modification of endocytosis and intracellular lysosome-trafficking), 3/ modulation of protein synthesis (by changed gene expression, inhibition of transcription factors (i.e. NFκ-B, AP-1) and altered translation trough ribosomal interaction) In virus-infected host cells, azithromycin broadly interferes at different levels with viral replication, i.e. by interfering with the initial virus internalization process, its endocytic activity, viral protein transcription/ translation and lysosome-trafficking of newly synthesized virus-particles to the cell surface. Furthermore, azithromycin has been shown to induce intracellular mRNA expression of antiviral genes, interferon (IFN)-stimulated genes and IFN production in infected host cells, mounting to an antiviral response mediated by the IFN pathway 14, 15, 18, 19, 22, 29 . Specific to SARS-CoV-2, recent quantum mechanical modeling suggests a potential role of AZ in interfering with viral entry via binding interaction between the SARS-CoV-2 spike protein and host receptor ACE2 (angiotensin converting enzyme-2) protein 30 ; however further experimental work on this is necessary to confirm the model. Apart from these antiviral effects, azithromycin reduces the production of several pro-inflammatory cytokines (e.g. interleukin (IL)-1, IL-2, IL-6, IL-8, IL-17, TNF-α) by (virus-infected) host cells, stimulated stuctural cells and/or activated monocytes/leukocytes, which in its way reduces proinflammatory macrophage activation (shift from M1 to M2 profile), increases macrophage phagocytosis, inhibits activation and proliferation of CD4+ and CD8+ T-lymphocytes (both Th1 and Th2), attenuates neutrophil chemotaxis and function (degranulation, active oxygen generation, release of neutrophil extracellular traps) 6, 31 . Finally, azithromycin attenuates TGFB-induced myofibroblast differentiation 32 , epithelial to mesenchymal transition (EMT) 33 , fibroblast collagen secretion 34 and extracellular matrix (ECM) remodeling (via reduction of matrix metalloproteinase production) 35 , as well as fibroblast growth factors-induced vascular endothelial growth factor production 36 . Altogether, this results in a reduction of the damaging effects of inflammation, fibrosis formation and vascular remodeling. These non-antimicrobial, anti-inflammatory, immunomodulatory and antiviral properties, together with its excellent safety profile and well-known clinical pharmacology characteristics, make azithromycin a very promising drug to study in COVID-19, the respiratory disease caused by the novel coronavirus SARS-CoV-2, as recently also stated by other research groups 24, 37 . Our hypothesis is that azithromycin may have a beneficial disease-modying effect by attenuation of viral replication, the associated (hyper)inflammation (so-called "cytokine storm"), evolution to acute respiratory distress syndrome (ARDS) and post-ARDS fibrosis in patients with COVID-19. inhibitor all cells will die after a few replication cycles of the virus. In the presence of an inhibitor of viral replication, some cells will survive and this low number of surviving cells is then amplified by leaving the cells to grow for 5 days. This test was previously validated for SARS-CoV 2 and detects activity of strong antiviral drugs like remdesivir and chloroquine, but not that of lopininavir. In this semi-quantitative antiviral compound-screening model, the antiviral effects of azithromycin were independently demonstrated in 4 different experiments (date 24/03/20, 04/04/20, 07/04/20 and 08/04/20) (figure 1 in appendix) in a window between 11-100 µm, with the strongest antiviral effect at a concentration of 33 µm (and limited DMSO toxicity of the assay conditions with 100 µm) This is in the same range as was seen for hydroxy-chloroquine (11-33 µm, 24/0320, 04/04/20 and 08/04/20) in the same experiments, which strengthens this finding of a antiviral effect of azithromycin. In the same assays, a direct experimental antiviral agent (GS-441524) exhibited stronger antiviral effects in the same range (3.7-100 µm). Currently, these pilot findings are being validated in a quantitative assay in which viral RNA is measured using qPCR. These results will become available in the next weeks. These findings are corroborated by the antiviral effects of other macrolides demonstrated in the same range (10-30 µm, without significant cytotoxicity of Vero-FM or CaCo2 cells) on human coronaviruses SARS-CoV, HCoV-NL63 and HCoV-229E 38 . These findings corroborate other very recent investigations which reported in vitro antiviral activity of azithromycin against viral pathogens with 50% inhibitory concentrations ranging from approximately 1 µM to 6 µM, with the exception of H1N1-influenza. The in vitro EC50 (50% effective concentration) for azithromycin against SARS-CoV-2, the virus responsible for COVID-19, was 2.12 µM (EC90: 8.65 µM) following a 72-hour incubation period post-infection, with a ratio of infectious virions to cells in culture (multiplicity of infection; MOI) of 0.002. In the same study, under the same experimental conditions, the in vitro EC50 for hydroxychloroquine was 4.17 µM 24 . In a pre-print study, following a 60-hour incubation period, a synergistic effect with the combination hydroxychloroquine 2 µM + azithromycin 10 µM was observed in vitro on SARS-CoV-2 at concentrations expected in human lung, leading to total inhibition of viral replication 39 . Caution should be exercised in comparing EC50 values across these studies due to the differences in experimental conditions (eg, different cell lines, MOI, time of drug addition to culture, incubation times, and analytical methods), yet altogether these findings confirm an antiviral effect of azithromycin against SARS-CoV-2. Clinical pharmacology perspectives on the use of azithromycin (and hydroxychloroquine) in COVID-19 were recently summarized elsewhere 24 . The pharmacokinetics of azithromycin are well understood. Azithromycin is rapidly absorbed following oral administration, has a long serum half-life (68 hours) and large volume of distribution (31 L/kg). Azithromycin is taken up by leucocytes at concentrations that are about 300-fold higher than plasma. In infected tissues, azithromycin concentrations are higher than in plasma, due to recruitment of leucocytes at the site of infection. Numerous studies have shown excellent penetration of azithromycin in a variety of infected tissues, which are summarized in this review 24 . Moreover, azithromycin is known to strongly accumulate in immune cells, at concentrations exceeding those of the epithelium by factor 7 40, 41 . Lung tissue homogenates and alveolar macrophages have azithromycin concentrations well in excess of the EC50 for SAR-CoV-2, as well as for other respiratory viruses, following approved doses of azithromycin. One limitation of these data is that concentrations in lung homogenates may not represent concentrations in infected cells. These data are extensively summarized elsewhere 24 . Once in the lung, concentrations of azithromycin persist for several days after plasma concentrations become undetectable. The estimated terminal half-life in lung tissue and bronchial washings were 133 hours and 74 hours, respectively. It is plausible that due to this unique pharmacokinetic property of azithromycin, coupled with target tissue concentrations in excess of in vitro EC50 against several viruses, azithromycin could play a potential therapeutic role in respiratory viral infections, including SARS-CoV-2 24 . Pharmacokinetic studies show that administration of 500mg azithromycin OD for 3 days results in bronchial epithelial concentrations that are in the range of 15-20µm. Considering tissue accumulation with prolonged administration (5days of 500mg Azithromycin OD) and the massive migration of UZ Leuven -KUL polymorphonuclear cells and monocytes to the site of inflammation, one can reasonably assume local tissue concentrations reaching the range of a direct antiviral effects 41, 42 . Increasing the uploading dose (1 gram) or the total cumulative dose of azithromycin by prolonged intake will likely increase local tissue concentrations but also the risk for side effects and cardiac toxicity. Azithromycin once daily for 5 consecutive days is deemed safe for treatment of hospitalized patients with community-acquired pneumonia according to the clinical practice guidelines of the American Thoracic Society and Infectious Diseases Society of America 43 . The slightly higher dosing than the standard regimen complies with aiming for the therapeutic concentration, while still keeping the gastrointestinal side effects tolerable and avoiding a high incidence of acquired long QT due to concomitant use of hydroxychloroquine as part of standard care. Moreover, one cannot underestimate the anti-inflammatory effects of the treatment, particular with high neutrophilic influx 6 . However, dose adjustment is not considered to be required for geriatric patients with normal renal and hepatic function, or in subjects with mild-to-moderate renal or hepatic impairment 24 . Interestingly, several studies of azithromycin +chloroquine, at doses up to 2000 mg azithromycin and 600 mg chloroquine (base), was shown to be generally well-tolerated, safe in patients with uncomplicated malaria, and safe to be used in different age groups (age range from 18 to >75 years) including pediatric patients (age range from 6 months to 12 years) and pregnant women 24 . In conclusion, the moderate antiviral effect, together with the more potent intrapulmonary antiinflammatory and immunomodulatory effects of azithromycin are the basis of a solid rationale for adjunctive azithromycin treatment in COVID-19. Confirmatory evidence with randomized controlled trials is essential to understand the role of azithromycin in the treatment of the current COVID-19 pandemic. Azithromycin is a marketed drug since many years, and many generic preparations are available. Today, many of the indications and clinical uses extend the approved marketing license. Typically, maintenance treatments with azithromycin 250 mg once daily, or 500 mg 3 weekly are prescribed for the prevention of acute infectious events in chronic respiratory diseases 38, 39 . According to clinicaltrials.gov Azithromycin is currently considered as therapeutic intervention for SARS-CoV-2 in 6 different study protocols worldwide with similar dosing regimens. Moreover, some countries in Europe (eg Turkey) have implemented Azithromycin as standard care and even a few centers in Belgium are adopting the proposed IMP strategy in clinical routine, without any good clinical evidence. We therefore need a Belgian study that urgently and rapidly explores the question in a Belgian setting before off label use with potential risks and no benefits is broadly implemented. It is obvious that by using the WHO scale, our study results can and will be merged with other results of ongoing international trials to provide strong type A evidence for or against the use of Azithromycin for SARS-CoV-2. Eligible adult patients who tested positive for SARS-CoV-2 and are admitted to the hospital will be randomized and assessed daily during hospitalization. Discharged patients will be contacted by telephone at days 15, 29 and 90. All subjects will undergo efficacy and safety assessments, including laboratory assays, which are aligned with clinical care. Also blood samples and nasopharyngeal swabs will be done according to clinical need (standard of care). If feasible an additional nasopharyngeal swab will be taken on day 6. The study should not put an extra burden on healthcare workers and on the hospital's resources. All recommendations of Sciensano are applied on COVID units to minimize risk and superinfections. UZ Leuven -KUL The study outcomes are based on the WHO master protocol. All outcomes will be presented overall as well as separately for patients with mild/moderate vs severe disease at baseline. This DAWN study is an adaptive, randomized, open-label clinical trial to evaluate the safety and efficacy of promising antiviral agents in hospitalized adult patients diagnosed with COVID-19. The outcomes of the study protocol are in part based on the draft master protocol of the WHO for trials that evaluate safety and efficacy of investigational therapeutics for the treatment of COVID-19 in hospitalized patients. The study is a phase 2 proof-of-concept multicenter trial. The DAWN study will compare standard of care vs. standard of care with the investigational therapeutic agent. Since there are no current approved treatment options for COVID-19, the standard of care is mostly supportive. However, the standard of care will reflect the guidance by (inter)national guidelines and hence may change during the course of the study. The clinical outcomes of this study have been choosen based on the outcomes of the WHO master template for clinical studies to allow pooling of the data with other ongoing studies. The adaptive study design of DAWN allows for the addition of new treatment arms and or strata during the study. The DAWN-AZITHRO will operate as a multicenter trial with a first wave and second wave of participating centres in complete alignment with the DAWN master study protocol. The first wave of 15 centres will be started after study initiation and appropriate training according to GCP. In case lower recruitment is noticed than expected with changing epidemiology, additional centres will be added by amendment to EC and FAGG will be notified. The DAWN-AZITHRO will randomize with a 2:1 allocation to SOC + Azithromycine versus SOC. Block randomisation per groups of 6 or 9 patients in every participating center will be implemented. DAWN -AZITHRO will also add and exploratory study visit 5-7 weeks post discharge for functional and radiological assessment. All tests in DAWN-AZITHRO protocol are part of standard clinical practice and good clinical follow-up The trial is expected to start April 2020, with a duration of 3 years. UZ Leuven -KUL Participants eligible for inclusion in this Trial must meet all of the following criteria: 1. Subject (≥18 years old) or legally authorized representative provides informed consent prior to initiation of any study procedures. When signed informed consent is not possible (e.g. due to restrictions to prevent viral transmission), verbal informed consent in the presence of a witness will be obtained and documented in the medical files. Signed informed consent will be obtained as soon as the safety concerns are mitigated. 2. Subject (or legally authorized representative) understands and agrees to comply with planned study procedures. All participants that are considered for Trial participation, per the above criteria will be documented on the Screening Log, including Screen Failures. Participants eligible for this Trial must not meet any of the following criteria: 1. ALT/AST > 5 times the upper limit of normal. 2. Pregnancy or breast feeding. 3. Allergy to any study medication. 4. Any medical condition which would impose an unacceptable safety hazard by participation to the study. 5. Study drug specific exclusion criteria:  for Azithromycin: o heart failure with severely reduced ejection fraction (  30%) o known prolonged long QT interval on ECG (> 470 msec males and > 480 females with Fridericia criteria; for patients with ventricular conduction delay the use of Rautaharju formula is also allowed) o patients on Macrolides during the last week before admission  For other treatment strata, see arm-specific protocols. Participants who meet one or more of the above exclusion criteria must not proceed to be enrolled/randomized in the Trial and will be identified on the Screening Log as Screen Failure. The Trial will be conducted only on the basis of prior informed consent by the Trial participants and/or their legally authorized representative(s). As such, no Trial-related procedures will be conducted prior to obtaining written informed consent from potential Trial participants. When signed informed consent is not permitted because of safety regulations related to the prevention of the transmission of SARS-CoV-2, verbal informed consent shall be documented in the medical records. Signed informed consent shall then be obtained as soon as permitted based on safety regulations to prevent the transmission of SARS-CoV-2. The process for obtaining and documenting initial and continued informed consent from potential Trial participants will be conducted in accordance with ICH-GCP E6(R2) guidelines, applicable regulatory requirements and internal Standard Operating Procedures (SOPs). All originally signed obtained Informed Consent Forms (ICFs) must be retained/archived in the Investigator Site File (ISF) at the Participating Site and must not be destroyed (even when a scanned copy is available) before expiration of the legal archiving term as defined in the protocol section entitled "Archiving". Participants may voluntarily withdraw consent to participate in the Trial for any reason at any time. The participant's request to withdraw from the Trial must always be respected without prejudice or consequence to further treatment. Consent withdrawal will be documented in the participant's medical record. The PI must take into account the consequences of such withdrawal: (1) further use of personal data/Trial data, (2) use of human biological materials already collected, (3) safe transition to alternative treatment options, etc. as applicable. Only adult hospitalized patients diagnosed with COVID-19 will be included. To ensure the integrity of the Trial, a randomization procedure through a computerized system has been established, generated by the data management unit of the clinical trial center leuven. For the multicentre DAWN-AZITHRO study a 2 Azithromycin versus 1 usual care will be allocated. Block randomisation (groups of 6 or 9) in every participating center will be implemented. Patients with documented COVID-19 who require hospitalization will be screened for eligibility. Informed consent will be obtained. When written informed consent is not possible due to restrictions to prevent the transmission of SARS-CoV-2, verbal informed consent will be documented in the medical files, and completed with written informed consent as soon as the restrictions do no longer apply. After consent has been given, following data will be obtained from the patient's file: Demographic parameters will be obtained. Medical history will be obtained as part of routine clinical care. Parameters and values of assessments from the moment of admission will be obtained retrospectively from the patient's file (vital signs, DNR-code, clinical assessments, historytaking, respiratory support, ECG, lab-UZ Leuven -KUL . When study-related procedures impose an additional ²burden on the clinical care of patients, they can be waived. Parameters should be obtained as part of routine clinical care. When study related procedures impose an additional burden on the clinical care of patients, they can be waived. The assessment closest in time or most relevant to the situation at baseline wil then be used instead. Study drug will be administered when randomized to the investigational drug arm. Medication will be reviewed using the electronic medical files. Medication of special interest is specified in appendix 2. Serious adverse events and adverse events grade 4 and 5 will be collected when these are not outcomes of the study. -Administration of study drug -Vital signs including SpO2 -Clinical data collection for assessment of study outcomes -Targeted medication review (see appendix 2) -Adverse event evaluation Serious adverse events and adverse events grade 4 and 5 will be collected when these are not outcomes of the study. When study-related procedures impose an additional burden on the clinical care of patients, they can be waived. These visits can be phone visits when patients are no longer hospitalized or when safety issues don't permit physical contact. To avoid burden on clinical care in a time of a strained health care system, laboratory tests are part or routine clinical care and are not mandatory, but when available will be collected (CRP, white cell count, haemoglobin, platelets, creatinine, hsTroponinT, glucose, total bilirubin, ALT, and AST on days 1; 3, 5, 8, 11, 15 and 29). In the exploratory visit 5-7 weeks post discharge, laboratory is part of clinical routine and will be collected.  The study includes two optional measurements on Day 6 (+-2). on the condition that this does not hinder routine clinical care: An additional assessment (e.g. nasopharyngeal swab) for SARS-CoV-2 quantitative and qualitative PCR.  The study includes two optional blood samples: One additional serum tube will be obtained within the first week after diagnosis, and one at the ambulatory visit of 5 to 7 weeks after discharge. The time-window for the first sampletaking is deliberately wide, to easily combine this with a blood drawing performed for the clinical routine, and thus minimize the burden for caregivers and patients, and avoid the waste of protectional gear.  The study includes a QTc assessment on ECG (day 2-3) or continuous ECG monitoring during administration of the study drug in patients on ICU, taking into account potential co-treatment with QTc prolonging drugs as listed in Appendix 2. UZ Leuven -KUL A telephone call on D90 (+/-5days) will check for hospital admission or survival status The study includes the collection of data on a clinical follow-up visit at 5-7 weeks post discharge, on the condition that the patient is able to visit ambulatory practice and to perform the functional and radiological evaluation which is part of good clinical follow-up. In case patient's physical condition permits no ambulatory monitoring visit, an additional call will be organized by the studyteam for follow-up.  Clinical examination  Medication and adverse event review  Questionnaire (mMRC, CAT, Cough Hypersensitivity)  Spirometry with reversibility  Longvolumes and diffusing capacity  Low dose CT scan  6 minutes walk (at the physicians discretion) Trial termination is defined as the date of the last visit of the last patient undergoing the trial. Participants may voluntarily discontinue Trial treatment and/or prematurely end their participation in the Trial for any reason at any time. In such case the Investigator must make a reasonable effort to contact the participant (e.g. via telephone, e-mail, letter) in order to document the primary reason for this decision. The Investigator may also decide at any time during the course of the Trial, to temporarily interrupt or permanently discontinue the Trial treatment if it is deemed that continuation would be detrimental to, or not in the best interest of the participant. In particular, when QTc> 500 ms and/or delta QTc>60ms, IMP will be interrupted/discontinued at the discretion of the investigator and PI and sponsor will be informed. Similarly, the Sponsor, Ethics Committee or authorized regulatory authority can decide to halt or prematurely terminate the Trial when new information becomes available whereby the rights, safety and well-being of Trial participants can no longer be assured, when the integrity of the Trial has been compromised, or when the scientific value of the Trial has become obsolete and/or unjustifiable. Circumstances requiring premature treatment interruption or discontinuation of the Trial, include but are not limited to:  Safety concerns related to IMP or unacceptable intolerability In any such case of early Trial termination and/or treatment interruption/discontinuation, the Investigator will continue to closely monitor the participant's condition and ensure adequate medical care and followup. For participants whose status is unclear because they fail to appear for Trial visits without stating an intention to discontinue or withdraw, the Investigator must make every effort to demonstrate "due diligence" by documenting in the source documents which steps have been taken to contact the participant to clarify their willingness and ability to continue their participation in the Trial (e.g. dates of telephone calls, registered letters, etc.). A participant should not be considered lost to follow-up until due diligence has been complet UZ Leuven -KUL The study design is adaptive, to allow the adjustment of a treatment arm and or stratum, the addition of new treatment arms/strata or the removal of treatment arms/strata based on the most updated information in a rapidly evolving field, based on the continuous assessment of the existing evidence available for the IMP and other potential drug candidates. The DAWN-AZITHRO will randomize participants 2:1 to standard of care in combination with the investigational product or to standard of care alone. Investigational drug will be administered to hospitalized patients. The hospital electronic medical prescription will be monitored to assess drug accountability. On the first 5 days, azithromycin 500mg will be administered as oral tablets, once daily, with or without a meal. In patients with a nasogastric tube or enteral feeding, syrup (suspension) 200 mg/5 mL can be given or tablets can be crushed, suspended in water and administered via the tube. Before and after administration, the tube will be rinsed with 20ml of water. The study design allows standard care or best supportive care to be changed in function of the Belgian Sciensano recommendations for treating COVID-19. Standards of care may rapidly change in pandemic situations, even during the enrolment of study participants. For differences between centres, or new recommendations for standard care, statistical adjustments will be made in the analysis. There are currently no approved treatments for COVID-19. Patients will receive the standard of care as continuously updated by national and international guidance. There are no restrictions for supportive care and we recommend to follow standard of care for Belgium according to the Scienesano website, which is regularly updated. https://epidemio.wiv-isp.be/ID/Pages/2019-nCoV.aspx UZ Leuven -KUL The only safety concern with azithromycin during acute hospital admission is longQT syndrome and torsade des pointes. Of the macrolide antibiotics, Azithromycine has the least QT prolonging potential. A recent large study in COPD acute exacerbations demonstrated that in patients at risk with no longQT prolongation at baseline, similar doses of Azithromycin did not induce longQT during the admission period. ECG will be mandatory for patients hospitalized and at risk for prolonged QTc and fatal arrhythmias. Long QT (> 470 msec males and > 480 females) will be an exclusion for participation. In patients with no long QT on ECG but at risk, a QT monitoring will be performed with intermittent ECG monitoring at d2-3 or continuous follow-up on ICUs. When QTc> 500 ms and/or delta QTc>60ms, IMP will be interrupted/discontinued at the discretion of the investigator. The patient's medication will be reviewed daily to evaluate DDIs including drugs prolonging the QTc interval according to what is listed in Appendix 2. An AE is any untoward medical occurrence in a patient or subject during an experiment, and which does not necessarily have a causal relationship with the study treatment. An AE can therefore be any unfavourable and unintended sign (including an abnormal laboratory finding), symptom or disease temporally associated with the use of a product, whether or not considered related to the product. Any worsening (i.e., any clinically significant adverse change in the frequency or intensity of a pre-existing condition) should be considered an AE. An AR is any untoward and unintended responses to an investigational medicinal product or to an experiment and, when an investigational product is concerned, related to any dose administered. An SAE is untoward medical occurrence that results in any of the following:  Death  A life-threatening a experience  In-patient hospitalisation or prolongation of existing hospitalisation  A persistent or significant disability or incapacity  A congenital anomaly or birth defect UZ Leuven -KUL A SUSAR is an adverse reaction, the nature or severity of which is not consistent with the information on the experiment, and, when a clinical trial is concerned, with the applicable product information (e.g. investigator's brochure for an unauthorised investigational product or the patient leaflet joined to the summary of product characteristics for an authorised product). In general, the following should not be reported as AEs:  Pre-existing conditions, including those found as a result of screening (these should be reported as medical history or concomitant illness).  Pre-planned procedures, unless the condition for which the procedure was planned has worsened from the first trial-related activity after the subject has signed the informed consent. The following events not to be considered as SAEs are:  Pre-planned hospitalisations unless the condition for which the hospitalisation was planned has worsened from the first trial-related activity after the subject has signed the informed consent.  Hospitalisation as part of a standard procedure for protocol therapy administration. However, hospitalisation or prolonged hospitalisation for a complication of therapy administration will be reported as an SAE.  Hospitalisation or prolongation of hospitalisation for technical, practical, or social reasons, in absence of an AE. For this trial, only Serious Adverse events and Adverse Events graded as grade 4 or 5 shall be collected, i.e. adverse events that are life-threatening and/or require an urgent intervention. Adverse events that are also outcomes of the trial, are also exempt from reporting. Investigators will seek information on AEs during each patient contact. All events, whether reported by the patient or noted by trial staff, will be recorded in the patient's medical record within a reasonable time after becoming aware, as will SAE's/grade 4/5 AE in the eCRF. If available, the diagnosis should be reported on the AE form, rather than the individual signs or symptoms. If no diagnosis is available, the Investigator should record each sign and symptom as individual AEs. The following minimum information should be recorded for each AE:  None -An AE which is not related to the IMP or experiment  Unlikely -An AE for which an alternative explanation is more likely (e.g. concomitant medication(s), concomitant disease(s)), and/or the relationship in time suggests that a causal relationship is unlikely  Possible -An AE which might be due to the use of the IMP or the experiment. An alternative explanation is inconclusive. The relationship in time is reasonable; therefore the causal relationship cannot be ruled out.  Probable -An AE which might be due to the use of the IMP or the experiment. The relationship in time is suggestive (e.g. confirmed by dechallenge). An alternative explanation is less likely.  Definitely -An AE which is listed as a possible adverse reaction and cannot be reasonably explained by an alternative explanation. The relationship in time is very suggestive (e.g. it is confirmed by dechallenge and rechallenge). For this trial, only Adverse Events grade 4 or 5 shall be collected, i.e. adverse event that are life-threatening and/or require an urgent intervention. Adverse events that are also collected as outcomes will not be separately reported. All SAEs must be reported to the Sponsor within 24 hours of the trial staff becoming aware of the event. The immediate report shall be followed by detailed, written reports. The immediate and follow-up reports shall identify subjects by Trial identification. SAE details will be reported by the Investigator to the Sponsor:  By completing the SAE form in the (e)CRF The Investigator must record follow-up information by updating the patient's medical records and the appropriate forms in the (e)CRF. The worst-case severity and seriousness of an event must be kept throughout the trial. SAE follow-up information should only include new (e.g. corrections or additional) information and must be reported within 24 hours of the Investigator's first knowledge of the information. This is also the case for previously non-serious AEs which subsequently become SAEs.  All SAEs must be followed up until the outcome of the event is 'recovered', 'recovered with sequelae', 'not recovered' (in case of death due to another cause) or 'death' (due to the SAE) and until all related queries have been resolved, or until end of trial (whichever occurs first).  Non-serious AEs must be followed up until the patient's last study visit, and until all related queries have been resolved. SAEs after the end of the trial: If the Investigator becomes aware of an SAE with suspected causal relationship to the IMP or experiment after the subject has ended the trial, the Investigator should report this SAE within the same timelines as for SAEs during the trial. All deaths will be reported without delay to the sponsor (irrespective of whether the death is related to disease progression, the IMP, study procedure or is an unrelated event). The sponsor will notify all deaths, as soon as possible after becoming aware, to the Central EC and the EC of the concerned site and provide additional information if requested. The Investigator is responsible for ensuring that all safety events are recorded in the (e)CRF and reported to the Sponsor in accordance with instructions provided below. The Sponsor will promptly evaluate all SAEs against medical experience to identify and expeditiously communicate possible new safety findings to Investigators, EC's and applicable CA's based on applicable legislation. After receiving the SAE report form from the Investigator, the Sponsor has to make a causality (relationship) assessment. The term SADR (Serious Adverse Drug Reaction) is to be used whenever either the Investigator or the Sponsor deems the SAE as possibly or probably related to the IMP. The Sponsor must evaluate (and document the evaluation of) the expectedness for each SADR against the Reference Safety Information, e.g. in the Investigator's Brochure or applicable product information. In case the event is Unexpected (= a SUSAR) it must be reported by the Sponsor to the EC's, CA's (through the EudraVigilance database) and other participating Investigators within the following timelines: The Sponsor has the obligation to, once a year throughout the clinical trial (or on request), submit a progress report to the EC's and CA's containing an overview of all SARs occurred during the reporting period and taking into account all new available safety information received during the reporting period. Due to the exceptional circumstances, namely a pandemic of the SARS-CoV-2 virus, and the urgency with which this DSMB has been assembled, DSMB members are not independent from the Sponsor. For further information see separate document DSMB Charter for "DAWN-azithromycine" COVID-19 trial (S63935) (v1 15 April 2020) Given the severity of illness in COVID-19, there are no pre-specified study stopping rules for safety. The protocol team will review AE / SAE data on an ongoing basis. If there are a concerning number of unexpected AEs, the DSMB will be asked to review safety data in an ad hoc meeting. The DSMB will review safety data after 80 subjects are entered into the trial and ad hoc reviews will be undertaken if there are other specific safety concerns. The study will not stop enrolment awaiting these DSMB reviews, though the DSMB may recommend temporary or permanent cessation of enrolment based on their safety reviews. There are no pre-specified treatment stopping rules. There interim monitoring will allow early stopping for safety, or if new effective therapies are identified through these trials and should become standard of care immediately, in an attempt to control the COVID-19 pandemic as quickly as possible. Findings of the DSMB: immediate communication by the Coordinating Investigator or his delegate to all study investigators and relevant study personnel Ongoing review of safety information will be performed by the Coordinating Investigator, in case of unexpected trends this information will be reviewed by the DSMB All unexpected events which affect the benefit-risk balance of the clinical trial, but are not suspected unexpected serious adverse reactions, will be reported by the Coordinating Investigator to the FAHMP, ethics committees and study investigators as soon as possible, but no later than 15 days from the date the sponsor became aware of this event. Urgent safety measures: Where an unexpected event is likely to seriously affect the benefit-risk balance, the sponsor shall take appropriate urgent safety measures to protect the subjects. These will be reported by the Coordinating Investigator or his delegate as soon as possible to the FAHMP and ethics committees but no later than seven calendar days from the date the measures have been taken. Relevant new safety information will be communicated to the trial subjects by means of an update to the informed consent form or any other communication pre-approved by the ethics committee. SUSAR: if an SAE is reported in the eCRF, an automatic email notification is sent to the Coordinating Investigator and Safety reviewer of the CTC of UZ Leuven. In case the reporting Investigator assesses the event to be possibly, probably or definitely causally related to the study medication, the Coordinating Investigator will evaluate the expectedness of the event based on the Reference Safety Information. This information will be recorded in the eCRF as soon as possible, preferably within the same working day. In case of a SUSAR, the reporting Investigator will be contacted by the Coordinating Investigator and asked to provide all relevant information related to the event to the Coordinating Investigator and CTC, using the CIOMS template, within 3 working days. The CTC will report the event to the FAHMP (via EudraVigilance) and ethics committees within 3 working days. The Coordinating Investigator will report the event to all study investigators and relevant study personnel. Statistical analysis will be performed in accordance with ICH E9; a detailed description of the analysis is provided in the separate Trial-specific Statistical Analysis Plan (SAP). ICH E3 and E8 will guide the structure and content of the clinical trial report. A brief summary is provided here. Details will be described in the SAP. The general statistical approach of the DAWN study can still be revisioned, as it is subject to the development of future treatment strata. General considerations: This study is intended to allow for the ability to add a new experimental arm/stratum if one becomes available. If additional data become available to add an experimental therapy, analyses of experimental arms or strata will be performed comparing concurrently enrolled control subjects. The primary outcome is based on an ordinal severity scale with 7 categories. This scale has been proposed by the WHO for COVID-19 related research and has been previously used in trials of patients with influenza. Previously reported studies and ongoing studies record the same primary outcome, which allows cross-study data pooling. Primary outcome will be time from Day 0 to sustained clinical improvement or life discharge, whichever comes first, whereby a sustained clinical improvement is defined as an improvement of > 2 points vs the highest value of Day 0 and 1 and sustained for at least 3 days. The null hypothesis being tested is that the primary outcome is the same for the standard of care and experimental treatment arms. Despite rapid dissemination of data from clinical case series and some early stage clinical trials, detailed information about the course of the disease is limited in this stage of the COVID-19 pandemia. The samples sizes presented here are only illustrative. The larger the number randomised the more accurate the results will be, but the numbers that can be randomised will critically depend on how large the epidemic becomes. Furthermore, in the absence of treatments with a known benefit, rapid changes in standard of care are to be expected and important signs of a benefit or a harm of a treatment under investigations will require rapid reporting. If good external evidence emerges while the trial is continuing that some other treatment(s) should also be being evaluated then it can be decided that one or more extra arms/strata will be added while the trial is in progress. Therefore, for our sample size calculations, we assume that a 40% improvement rate will be observed at Day 15 in the control group. Based on the log-rank test, with a 2-sided significance level of 5% and 80% statistical power and using a (2:1) randomization ratio in favour of azithromycin, we estimate that a total sample size of 354 patients will suffice to detect an absolute improvement of 15% (i.e. 55% in intervention group). To detect an absolute improvement of 20% (60% in intervention group), a total sample of 196 patients will suffice. We propose a pragmatic sample size of 282 patients taking into account early dropouts. 258 patients will be sufficient to detect an absolute improvement of 17.5% with a statistical power of 80% at a 2-sided significance level of 5%. The following analysis sets will be defined: The FAS will include all randomised patients according to their randomised treatment. Patients randomised to the interventional group will be excluded if they did not receive any dose of study medication. The FAS will be used for the evaluation of all efficacy endpoints. The SS will include all patients who were randomised according to their actual treatment. Patients randomised to the interventional group who did not receive any study treatment will be included in the Standard Of Care group, The SS will be used for the evaluation of all safety parameters. This is an open label controlled randomized trial testing a superiority hypothesis with a two-sided type I error rate of 0.05. In this explorarory study, secondary hypotheses will be tested in a non-hierachical way. These will be described according to the appropriate summary statistics (e.g., proportions for categorical data, means with 95% confidence intervals for continuous data, median for time-to-event data). A statistical analysis plan (SAP) will be developed and filed with the study sponsor prior to database lock. The primary endpoint will be analysed by means of competing risk analyses whereby death without any improvement will be considered as a competing risk. Event rates will be estimated using cumulative incidence functions (CIF). Median times to improvement will be calculated by treatment group. The effect of treatment will be assessed by performing a Fine&Gray competing risk regression model that includes the baseline value on Day 0 as a covariate and randomised treatment as a factor. From the Fine&Gray model, the treatment effect and associated 95% confidence interval will be estimated 1. Cumulative clinical status up to Day 15 will be analysed using a general linear model adjusted for clinical status on Day 0. The treatment effect will be estimated by the difference of mean values between the groups. 2. Cumulative clinical status recorded daily during hospital stay and on Days 15 and 19 will be analysed by means of a proportional odds logistic regression model, adjusted for clinical status on Day 0. The treatment effect will be estimated by the common odds ratio. 3. All-Cause mortality rates will be estimated by treatment group using the Kaplan-Meier method. The resulting Kaplan-Meier curves will be compared using a log-rank test. The treatment effect will be estimated by the hazard ratio using a Cox regression. 4. Other Time-to-event parameters with competing risk: event rates will be estimated using cumulative incidence functions (CIF), the resulting CIF curves will be compared using Gray's test. The treatment effect will be estimated by the subdistribution hazard ratio. 5. Duration of hospital and ICU stay: both parameters will be analysed as time-to-event parameters with competing risk, whereby the event of interest is discharge from hospital/ICU and the competing risk is hospital/ICU death. 6. Continuous normally distributed variables (e.g. QTc) will be analysed using a 2-sample t-test. Treatment effects will be estimated by the difference in mean values between the groups. If applicable, changes from baseline will be calculated. Comparisons between treatment groups will be done by performing an analysis of covariance (ANCOVA) on the post-baseline value, using the baseline value as a covariate. 7. Contnuous non-normally distributed variables (clinical status, NEWS score, duration of supplemental oxygen, duration of mechanical ventilation) will be analysed using a Wilcoxon ranksum test. Change in ordinal scale at specific time points will be compared using Wilcoxon ranksum tests. Missing data procedures will be described in the SAP. Safety endpoints are described above. These events will be analysed univariately and as a composite endpoint. Time-to-event methods will be used for death and the composite endpoint. Each AE will be counted once for a given participant and graded by severity and relationship to COVID-19 or study intervention. Adverse events leading to premature discontinuation from the study intervention and serious treatmentemergent AEs will be described as part of the primary publication of the study results. Baseline characteristics will be summarized by treatment arm/stratum. For continuous measures the mean and standard deviation will be summarized. Categorical variables will be described by the proportion in each category (with the corresponding sample size numbers). Early analysis UZ Leuven -KUL An initial blinded endpoint-evaluation phase will be enrolled prior to specification of the primary endpoint as described above. Analysis and decision making will be restricted to a blinded endpoint evaluation committee (DSMB). DSMB membership will be defined elsewhere and will consist only of individuals who are blinded to treatment assignment. Principles of blinded endpoint-evaluation will be defined in a separate document. Additional early analyses include monitoring enrolment, baseline characteristics, and follow-up rates throughout the course of the study by the study team. Analyses will be conducted blinded to treatment assignment. An data and monitoring safety board (DSMB) will monitor ongoing results to ensure patient well-being and safety as well as study integrity. The DSMB will be asked to recommend early termination or modification only when there is clear and substantial evidence of a safety issue. Subgroup analyses for the primary and selected secondary outcomes will evaluate the treatment effect across the following subgroups: duration of symptoms prior to enrolment, age groups, disease severity at baseline and co-morbidities. A forest plot will display confidence intervals across subgroups. Interaction tests will be conducted to determine whether the effect of treatment varies by subgroup. The DSMB will review safety data after 80 subjects are entered into the trial and ad hoc reviews will be undertaken if there are other specific safety concerns. The study will not stop enrolment awaiting these DSMB reviews, though the DSMB may recommend temporary or permanent cessation of enrolment based on their safety reviews. Given the severity of illness in COVID-19, there are no pre-specified study stopping rules for safety. The protocol team will review AE / SAE data on an ongoing basis. If there are a concerning number of unexpected AEs, the DSMB will be asked to review safety data in an ad hoc meeting. Their interim monitoring will allow to recommend early stopping for reasons of safety. If new effective therapies are identified through these trials, these snould become standard of care immediately, in an attempt to control the COVID-19 pandemic as quickly as possible. Because of the exceptional circumstances, the DSMB is part of UZ Leuven and cannot be considered as fully independent. A charter and terms of reference have been provided (see separate document DSMB Charter for "DAWN-azithromycine" COVID-19 trial (S63935) (v1 15 April 2020)) to make sure scientific independency of the DSMB members has been sufficiently assured. Data collection, handling, processing and transfer for the purpose of this Trial will be performed in compliance with applicable regulations, guidelines for clinical trials and internal procedures, as follows: Source Data will be collected and recorded in the Trial participant's files/medical records. Worksheets may be used for capturing some specific data in order to facilitate completion of the eCRF. Any such worksheets will become part of the Trial participant's source documentation and will be filed together with or as part of the medical records (during but also following completion of the Trial). It remains the responsibility of the Investigator to check that all data relating to the Trial, as specified in the Trial protocol, are entered into the eCRF in accordance with the instructions provided and that the forms are filled out accurately, completely and in a timely manner. eCRFs are provided by the Sponsor for each participant. The Trial data will be transcribed from the source records (i.e. participant's medical file or Trial-specific source data worksheets) into an eCRF by Trial Staff. Transcription to the eCRF will be done as soon as possible after a participant visit and in a pseudonymized manner using a unique identifier assigned by the Sponsor. The eCRFs will be available for review at the next scheduled monitoring visit (as applicable). All data relating to the Trial must be prepared and validated by the Investigator. Any eCRF entries, corrections and alterations must be made by the Investigator or other authorized Trial staff. Proper audit trails are available in REDCap to demonstrate the validity of the Trial data collected. This includes historical records of original data entries, by whom and when the data was entered, as well as detailed records of any corrections or additions made to the original data entry (i.e. who made the correction/addition, when and why), without obliterating the original data entry information. The Trial Data Manager will perform extensive consistency checks on the received data. Queries will be issued in case of inconsistencies in accordance with internal procedures. A Data Management Plan will be developed to map data flows, data validation measures that will be taken, how (interim) database lock(s) will be managed and, as applicable, the role and responsibilities of the Data and Safety Monitoring Board (DSMB) Any participant records or datasets that are transferred to the Sponsor or any partners of the Sponsor will contain the Trial-specific participant identifier only; participant names or any information which would make the participant identifiable will not be transferred. All pseudonymized data relating to the Trial must be transmitted in a secure manner to the Sponsor (see 8.1.2. legal requirements). All source data will be kept at a secured location with restricted access at all times. These data must be collected and processed with adequate precautions to ensure confidentiality and compliance with applicable data protection laws and regulations and more in particular the EU General Data Protection Regulation 2016/679 (GDPR) and relevant national laws implementing the GDPR. Appropriate technical and organizational measures to protect the data against unauthorized disclosure or access, accidental or unlawful destruction, or accidental loss or alteration must be established. Trial staff whose responsibilities require access to personal data agree to keep the data confidential. The Investigator and the Participating Site(s) (as applicable) shall treat all information and data relating to the Trial disclosed to them as confidential and shall not disclose such information to any third parties or UZ Leuven -KUL Confidential Protocol: Direct antivirals working against nCoV -Azithromycin treatment stratum (DAWN-AZITHRO) Version: 06_15Mei2020 Internal Ref.: 63935 TMP-CTC ALG-001-1.0_14022020 Page 36 of 50 Protocol template for non-commercial interventional clinical Trial with IMP use such information for any purpose other than the objectives of the Trial as described in this protocol. The collection, processing and disclosure of personal data, such as participant health and medical information is subject to compliance with applicable laws and regulations regarding personal data protection and the processing of personal data. The Investigator will maintain all source documents and completed eCRF that support the data collected from each Trial participant, and will maintain a Trial Master File (TMF) containing all Trial documents as specified in ICH-GCP E6(R2) Chapter 8 entitled "Essential Documents for the Conduct of a Clinical Trial", and as specified by applicable regulatory requirement(s). The Investigator will take appropriate measures to prevent accidental or premature destruction of these documents. Transfer of the pseudonymized data will be performed via a secured method of transfer taking into account all applicable security arrangements and regulations (such as the European General Data Protection Regulation). The receiving party will be bound by contractual agreement to keep the transferred data confidential at all times and to only process the data for the purpose of the Trial. To this end, appropriate Data Transfer Agreements (DTAs) will be established. The Investigator will permit direct access to Trial data and documents for the purpose of monitoring, audits and/or inspections by authorized entities such as but not limited to: the Sponsor or its designees and competent regulatory or health authorities. As such eCRFs, source records and other Trial related documentation (e.g. the Trial Master File, pharmacy records, etc.) must be kept current, complete and accurate at all times. In accordance with ICH-GCP E6(R2) the Sponsor is responsible for monitoring the Trial to ensure compliance with GCP and current legislation, and to verify, among other requirements, that proper written informed consent has been obtained and documented, that the Trial procedures have been followed as shown in the approved protocol, and that relevant Trial data have been collected and reported in a manner that assures data integrity. To this end Source Data will be compared with the data recorded in the eCRF. Monitoring of the Trial will be performed by qualified individuals (independent from the site Trial staff) according to the monitoring plan. The Sponsor and Investigator/Participating Site will permit direct access to the Trial data and corresponding Source Data and to any other Trial related documents or materials to verify the accuracy and completeness of the data collected. More details about the monitoring strategy are described in the Trial specific Monitoring Plan (MP). As specified in ICH-GCP E6(R2) section 8.1 Addendum the Sponsor and Investigator/Participating Site will maintain a record of the location(s) of all respective Essential Trial Documents (including but not limited to Source Documents, completed and final eCRF and ISF(s)/TMF). The Sponsor should ensure that the Investigator has control of and continuous access to the eCRF data reported to the Sponsor during the Trial. The Investigator/Participating Site should have control of all Essential Documents and records generated by the Investigator/Participating Site before, during and following termination of the Trial. The Sponsor is responsible for archiving Trial specific documentation (such as but not limited to the Trial protocol, any amendments thereto, the final Clinical Study Report (CSR) and the Trial database) according to ICH-GCP E6(R2). Source data and site-specific Trial documents (such as but not limited to the original signed ICFs) will be archived by the participating site(s) according to local practice, and for at least 25 years following termination of the Trial. Archived data may be held on electronic record, provided that media back-up exists, hard copies can be obtained, if required and measures are taken to prevent accidental or premature loss or destruction of data. Destruction of Essential Documents will require written authorisation from the Sponsor. Before the start of the Trial, this protocol and other related documents (e.g. ICF, advertisements, IB, etc.) will be submitted for review to the EC and to the relevant CA for Trial authorization. The Trial shall not commence until such approvals have been obtained. It is the responsibility of the CI to produce the Annual Progress Report (APR) and submit to the EC/CA within 30 days of the anniversary date on which favourable opinion to start the Trial was given, and annually until the Trial is declared ended. The CI shall notify the EC/CA of the end of the Trial. Should the Trial be ended prematurely, the CI will notify the EC/CA and include the reasons for premature termination within 15 days of the decision. The CI will submit a final report with the results, including any publications/abstracts, to the EC/CA within 1 year or within 6 months for paediatric Trials. The The Trial must be performed in accordance with the protocol, current ICH-GCP guidelines, and applicable regulatory and country-specific requirements. GCP is an international ethical and scientific quality standard for designing, conducting, recording, and reporting studies that involve the participation of human participants. Compliance with this standard provides public assurance that the rights, safety, and well-being of Trial participants are protected, consistent with the principles that originated in the most recent version of the Declaration of Helsinki, and that the Trial data are credible, reliable and reproducible. The Investigator and Trial team acknowledge and agree that prospective, planned deviations or waivers to the protocol are not permitted under applicable regulations on clinical studies. However, should there be an accidental protocol deviation, such deviation shall be adequately documented in the source documents and on the relevant forms and reported to the CI and Sponsor. Deviations should also be reported to the EC as part of the EC's continued review of the Trial (e.g. through the ASR, APR, etc.). Protocol deviations which are found to frequently recur, will require (immediate) action. Investigator acknowledges that such recurring protocol deviations could potentially be classified as a serious violation. It is understood that "a serious violation" is likely to affect to a significant degree: the safety or physical or mental integrity of the Trial participants; or the scientific validity of the Trial The Investigator is expected to take any immediate action required to protect the safety of any participant included in the Trial, even if this action represents a deviation from the protocol. In such cases, the Sponsor should be notified of this action and the EC at the Trial site should be informed according to local procedures and regulations. The Trial will be conducted in compliance with the requirements of the EU General Data Protection Regulation 2016/679 (GDPR), the relevant Belgian laws implementing the GDPR including the Belgian Privacy Act of 30 July 2018 on the protection of privacy in relation to the processing of personal data. Any collection, processing and disclosure of personal data, such as participant health and medical information is subject to compliance with the aforementioned personal data protection laws (cfr. Data UZ Leuven -KUL Processing Annex (DPA) in Appendix). In case personal data is transferred outside the European Economic Area, safeguards will be taken to ensure that appropriate protection travels with the data in accordance with the GDPR. (https://ec.europa.eu/info/law/law-topic/data-protection/internationaldimension-data-protection/rules-international-data-transfers_en#documents) Any personal data shall be treated as confidential at all times including during collection, handling and use or processing, and the personal data (including in any electronic format) shall be stored securely at all times and with all technical and organizational security measures that would be necessary for compliance with EU and national data protection legislation (whichever is more stringent). The Sponsor shall take appropriate measures to ensure the security of all personal data and guard against unauthorized access thereto or disclosure thereof or loss or destruction while in its custody. The Participating Site, the Investigator and Sponsor shall have and maintain in full force and effect during the term of this Trial, and for a reasonable period following termination of the Trial, adequate insurance coverage for: (i) medical professional and/or medical malpractice liability, and (ii) general liability. Art 29 of the Belgian Law relating to experiments on human persons dated May 7 th , 2004 applies. Prior to the start of the Trial, the Sponsor shall enter into an insurance contract in order to adequately cover Trial participants from Belgian sites in accordance with art. 29 of the said law. The Participating Site shall have and maintain in full force and effect during the term of this Trial (and for a reasonable period following termination of the Trial, adequate insurance coverage for other possible damages resulting from the Trial at the Participating Site, as required by local law. Each such insurance coverage shall be in amounts appropriate to the conduct of the services of the Participating Site under this Trial. The Participating Site and Sponsor shall be solely responsible for any deductible or self-insured retention under any such policies. Unless for urgent reasons as specified in ICH-GCP E6(R2) section 4.5.4, amendments must not be implemented prior to EC and/or CA review and/or approval, as applicable. In accordance with the Belgian law of May 7 th 2004 regarding experiments on humans, the Sponsor may develop a non-substantial amendment at any time during the Trial. If a substantial amendment to the clinical Trial agreement or the documents that supported the original application for the clinical Trial authorisation is needed, the Sponsor must submit a valid substantial amendment to the Competent Authority (CA) for consideration, and to the EC for review and approval. The CA and/or EC will provide a response in accordance with timelines defined by applicable regulations. It is the Sponsor's responsibility to assess whether an amendment is substantial or non-substantial for the purpose of submission to the CA and/or EC. Amendments to the Trial are regarded as 'substantial' when they are likely to have a significant impact on the safety or physical or mental integrity of the clinical Trial participants, or the scientific value of the Trial. https://ec.europa.eu/health/sites/health/files/files/eudralex/vol-10/2010_c82_01/2010_c82_01_en.pdf Not applicable. The scientific hypothesis and the sponsor has adequate oversight of the safety and integrity of the entire clinical trial. When initiating and conducting complex clinical trials in the EU/EEA, sponsors should identify potential risks associated with the IMPs, trial populations and operational complexity. The following key recommendations have been taken into account by the sponsors. Clear description and justification of the design. Maintenance of scientific integrity. 3 . Ensuring the quality of trial conduct and optimise clinical feasibility 4. Ensuring the safety of trial subjects 5. Maintenance of data integrity 6. Reassessment of benefit-risk balance throughout clinical trial 7. Full data transparency As risk mitigation strategies to accommodate these EU/EEA recommendations, independent review by FAGG/EC, a statistical team independent of the clinical study team and a DSMB operating along a predefined charter have been implemented. Individual patient safety monitoring during the trial, and an interim safety analysis after 100 participants is planned. Central support from sponsor, with a 24hours medical helpline, centrally appointed clinical research associates for local study team support, eCRF, electronic randomisation and an independent monitoring team has been foreseen. Remote video and onsite initiation and training visits are imposed according to standard GCP rules, to ensure quality of trial conduct and integrity of the data acquisition. Full transparency of the data will provide as specified in the data handling section. The Declaration of Helsinki (latest version) and European and Belgian regulations require that every research Trial involving human participants be registered in a publicly accessible database before recruitment of the first participant. The CI is responsible for registering the Trial. In addition, the CI will fulfil their ethical obligation to disseminate and make the research results publicly available. As such the CI is accountable for the timeliness, completeness and accuracy of the reports. Researchers, authors, Sponsors, editors and publishers must adhere to accepted guidelines for ethical reporting. Negative and inconclusive, as well as positive results must be published or otherwise made publicly available. Sources of funding, institutional affiliations and conflicts of interest must be declared in publication. Publications will be coordinated by the CI. Authorship to publications will be determined in accordance with the requirements published by the International Committee of Medical Journal Editors and in accordance with the requirements of the respective medical journal. For multi-centric Trials, it is anticipated that the primary results of the overall Trial shall be published in a multi-centre publication. Participating Sites are not allowed to publish any subset data or results from the Trial prior to such multicentre publication. Any publication by a Participating Site must be submitted to the Sponsor for review at least thirty (30) calendar days prior to submission or disclosure. Sponsor shall have the right to delay the projected publication for a period of up to three (3) months from the date of first submission to the Sponsor in order to enable the Sponsor to take steps to protect its intellectual property rights and know-how. Any know-how, inventions, methods, developments, innovations, discoveries and therapies, whether patentable or not, arising from the Trial or made in the performance of the Trial protocol ("Inventions") shall vest in the Sponsor. The Participating Site, its employees and Investigator(s) shall promptly disclose UZ Leuven -KUL to the Sponsor any such Inventions. Parties have expressly agreed that any and all Trial data as collected and prepared in the performance of the Trial protocol shall be the sole property of Sponsor. Publication policy guidelines will be created. In order to ensure the same quality and safety standards in patient care for clinical research as commonly applied by the Sponsor in its regular activities, and in accordance with JCI standards, the Sponsor shall comply with the following obligations: (a) the Sponsor will use trained and qualified employees or contractors to manage and coordinate the Trial; (b) the Sponsor will ensure that multi-center Trial reporting is reliable and valid, statistically accurate, ethical, and unbiased. (c) the Sponsor will not grant incentives, other than standard compensations and reimbursement of costs, to Trial participants or to participating site's staff that would compromise the integrity of the research; (d) the Sponsor is responsible for monitoring and evaluating the quality, safety, and ethics of the Trial and will respect the participating site's policies and processes when performing such monitoring and evaluation activities; (e) the Sponsor will protect the privacy and confidentiality of the Trial participants in accordance with all applicable laws. UZ Leuven -KUL Page 45 of 50 Protocol template for non-commercial interventional clinical Trial with IMP 6. Taking into account the nature of the processing, the Data Processor shall assist the Data Controller, by means of appropriate technical and organizational measures, insofar as this is possible, in fulfilling its obligation to respond to requests from Data Participants pursuant to laws and regulations in the area of privacy and data protection (such as, the right of access, the right to rectification, the right to erasure, the right to restrict the processing, the right to data portability and the right to object) The DAWN-AZYTHRO sub-protocol sees 240 patients randomised in a 2:1 ratio to either azithromycin or Standard Of Care (SOC) at multiple sites. Due to the fast-changing addition of novel treatments and hypotheses of interest, the Master statistical analysis plan (SAP) will describe the general statistical methods to be used for statistical evaluation of the primary, secondary, exploratory and safety endpoints for all compounds under investigation. This AZYTHRO-Specific SAP provides additional and specific details about the statistical methods for the evaluation of the outcomes for Azythromycin. The objective of the DAWN study is to evaluate the efficacy of various compounds of interest in the treatment of hospital-admitted COVID-19 patients. The primary outcome for the evaluation of Azythromycin is defined as follows: A. The following secondary outcomes will be of interest for the evaluation of Azythromycin: Adult patients who tested positive for SARS-CoV-2 and are admitted to the hospital are screened for eligibility. Once eligibility is confirmed, informed consent is sought from the patient to participate in the study. Patients who consented are randomised to either study treatment or standard of care. Patients are randomised in a 2:1 ratio to either Azythromycin or SOC, stratified by study site. This is an open-label study, meaning both patients and study personnel were aware of the assigned treatment. No formal interim analyses to check for early efficacy or futility are foreseen. A regular review of the safety data will be performed by a Data Safety Monitoring Board. Upon final database lock for Azythromycin, statistical analyses of the data will be performed according to the methods described in this document and the Master SAP. Any deviations will be documented. The analysis populations and analysis plan will be finalised at a Blinded Review Meeting prior to database lock where all attendees will be kept blind from randomised study treatment. All decisions taken at this meeting will be fully documented in a Blind Review Document. The agenda of the Blind Review Meeting will include (but not necessarily be limited to) the following:  Definition of Per Protocol Set (see Section 9.3);  Definition of Study Periods (see Section 10.5);  Pooling of Study Sites (see Section 10.6). In their study comparing clinical improvement rates for Lopinavir-Ritonavir in hospitalized patients with severe Covid-19, Cao et al. reported a clinical improvement rate in the control group of 37.7% on Day 14. Therefore, for our sample size calculations, it was assumed that a 40% improvement rate will be observed at Day 15 in the control group. Based on the log-rank test, with a 2-sided significance level of 5% and 80% statistical power and using a (2:1) randomization ratio in favour of azithromycin, it was estimated that a total sample size of 354 patients will suffice to detect an absolute improvement of 15% (i.e. 55% in intervention group). To detect an absolute improvement of 20% (60% in intervention group), a total sample of 196 patients will suffice. A pragmatic sample size of 282 patients was used, taking into account early dropouts. 258 patients will be sufficient to detect an absolute improvement of 17.5% with a statistical power of 80% at a 2-sided significance level of 5%. The following analysis sets will be of interest: The FAS will include all randomised patients according to their randomised treatment. However, the following patients will be excluded from the FAS: a) Covid-negative patients: inclusion criterion 4 The FAS will be used for the evaluation of all efficacy endpoints. The SS will include all patients from FAS according to their actual treatment received. Patients randomised to the interventional group who did not receive any study treatment will be included in the SOC group. Patients randomised to SOC but who received Azythromycin, will be included in the Azythromycin group. The SS will be used for the evaluation of all safety parameters. Patients from the FAS with major protocol deviations will be excluded from the perprotocol set (PPS). Major protocol deviations will include, but are not limited to, the following:  Failure to satisfy inclusion or exclusion criteria,  Taking any not permitted concomitant medication during the study,  Serious non-compliance to dose regimen and visit schedule. The Per Protocol Set will be reviewed and finalized at a Blind Review Meeting, which will take place after all data have been collected and cleaned and prior to the database lock and unblinding of the study treatment for the final analysis of the study. All major protocol deviations that lead to exclusion from the PPS will be fully documented in Analysis Sets Specification Document that will be dated and signed prior to final database lock. A summary by treatment group will be provided for the following: Important protocol violations and deviations that can impact the results of the statistical analyses will be fully documented prior to database lock in the Analysis Sets Specification Document. All data recorded at baseline will be summarized by treatment group and compared using the methods described in Section 10.3. Summaries will be presented for FAS, SS and PPS separately. The following baseline information will be presented:  Demographic characteristics  Medical history  Prior medications  Hospital admission: symptoms  Imaging data at hospital admission  Vital signs at hospital admission  Laboratory data at hospital admission  ECG at hospital admission 13 Primary, Secondary and Exploratory Endpoints The analysis of the primary outcome will be done on the FAS and, if defined, the PPS. Time to Sustained Clinical Improvement or Discharge will be derived from the imputed Clinical Status data. Time to clinical improvement will be analysed using methods for the analysis of competing risk data: the event of interest is clinical improvement, death without improvement will be considered to be the competing risk, patients for whom follow-up ended without clinical improvement will be censored on Day 15. Comparisons of the CIF curves will be done using Gray's test. Event rates over time will be estimated as cumulative incidence functions (CIF) and presented along with their 95% point-wise confidence intervals. Median times (or other more suitable quantiles) will be presented by treatment group. The treatment effect will be estimated by the subdistribution hazard ratio obtained from a Fine&Gray model. The Fine&Gray regression will include a factor for randomised treatment, study site and period and clinical status on Day 0 as covariate. The analysis of the secondary outcomes will be done on the FAS and, if defined, the PPS, unless specified otherwise. Daily Clinical Status (imputed) will be analysed by means of a proportional odds logistic regression model, performed on each day. The treatment effect will be estimated by the common odds ratio. The proportional odds logistic regression model will include a factor for randomised treatment, period and study site. Cumulative Clinical Status will be calculated from the imputed Clinical Status data. Cumulative Clinical Status up to Day 15 will be analysed by means of a general linear model. The treatment effect will be estimated by the difference between groups and presented together with its 95% confidence interval. The general linear model will include a factor for randomised treatment, period, study site and clinical status at baseline (Day 0). In addition to what was described in the protocol, the following will be done: At each study day between Day 1 and Day 15, the cumulative status from Day 1 up to that day will be calculated The obtained cumulative scores will be summarised by treatment group time and plotted versus time using boxplots. Mortality rates over time will be estimated by Kaplan-Meier methodology. 95% Confidence intervals will be calculated using the log-log transformation. Comparisons of the curves will be done using a log-rank test. Median times (or other more suitable quantiles) will be presented by treatment group. The treatment effect will be estimated as a hazard ratio, obtained using a Cox proportional hazards regression model. The Cox regression will include a factor for randomised treatment, period and study site. For the analysis of mortality on Day 15, all data beyond Day 15 will be censored at Day 15. Likewise, for the analysis of all-cause mortality on Day 29, all data beyond Day 29 will be censored at Day 29. In addition to duration, the incidence of supplemental oxygen will also be assessed. Incidence and duration of supplemental oxygen will be calculated from the imputed Clinical Status data. Incidence of supplemental oxygen will be analysed using competing risk methodology as described in Section 13.1.1 above, whereby death without the administration of supplement oxygen is considered a competing risk. Competing risk methodology will also be used to evaluate the duration of the supplemental oxygen. The event of interest will be the end of supplemental oxygen when alive, competing risk is the end of supplemental oxygen because of death. Two separate analyses will be done for the duration: first, patients who did not have any supplemental oxygen will be excluded from the analysis. Second, patients who did not have any supplemental oxygen will be included with a duration of zero days. In addition to duration, the incidence of mechanical ventilation will also be assessed Incidence and duration of mechanical ventilation will be calculated from the imputed Clinical Status data. The statistical methodology for the evaluation of this endpoint will be the same as for incidence and duration of supplemental oxygen (see Section 13.2.4). Duration of hospital stay with be analysed by means of competing risk methodology as described in Section 13.1.1 with in-hospital death the competing risk. Incidence of ICU stay will be analysed using the competing risk methodology described in Section 13.2.4, considering death without ICU stay as competing risk. Duration of ICU stay with be analysed by means of competing risk methodology as described in Section 13.2.4 with in-ICU death the competing risk. For patients who have multiple ICU stays, the durations will be added up. Daily NEWS scores will be summarised by treatment group and study day. The combined cardiac endpoint during hospital stay will be analysed using a logistic regression from which the treatment effect will be estimated as an odds ratio. The logistic regression will include a factor for randomised treatment, period and study site. The combined cardiac endpoint will be assessed in the Safety Set. QTc data and their changes from baseline will be summarised by study day and treatment group using appropriate summary statistics. The QTc data will be analysed using a general estimating equation (GEE) model that models all data over time, using an identity link-function and normally distributed errors. A GEE analysis is quite robust against deviations from the distributional assumptions. However, when serious deviations are observed, a log-transformation of the data can be considered. The GEE model will include all measurements and will include factors for time, treatment, their interaction, study site, period and the baseline value as a covariate. An appropriate variance-covariance matrix will be used to account for interdependencies between the timepoints. From the GEE model, treatment differences will be calculated at all timepoints and presented along with their 95% confidence interval. In addition, an overall 'average' treatment effect will be estimated by removing the interaction from the model. In addition, the number and proportion of patients will be calculated who meet either of the following criteria: 1. Change in QTc > 60 msec; OR 2. QTc > 500 msec. QTc data will be analysed using the SS. All exploratory outcomes will be analysed using the FAS, unless specified otherwise. Viral load will be summarised by treatment and timepoint. In addition, the number (%) of patients testing positive will be summarised by treatment and timepoint. Questionnaire data will be analysed by means of a general linear model that includes factors for treatment group, period and study site. If necessary, a log-transformation will be applied to the data. The treatment effect will be estimated as a treatment difference (or treatment ratio, for log-transformed data). Spirometry data will be analysed using the same methods as those described in Section 13.3.2. If available, the baseline value will be included in the model. Lung volumes will be analysed using the same methods as those described in Section 13.3.2. If available, the baseline value will be included in the model. CT scan parameters of interest will be summarised by treatment group. The 6 minute walking distance will be analysed using the same methods as those described in Section 13.3.2. If available, the baseline value will be included in the model. 14 Safety Data The number of events and the number of patients experiencing adverse events will be summarized by treatment group Summaries of adverse events will be presented for the SS. All laboratory data will be summarized using appropriate summary statistics by treatment group and study day. Summaries of the laboratory data will be presented for the SS. All other data will be summarized treatment group and, if applicable, by day, using the FAS. Table 7 Daily Clinical Status Table 8 All-Cause Mortality on Day 15 and 29 Table 9 Secondary Endpoint: Incidence and Duration of Supplemental Oxygen Figure 11 .2 Cumulative Incidence Curves Figure 11 .2.1 Cumulative Incidence Curves (FAS) Figure 11 .2.2 Cumulative Incidence Curves (PPS) Table 12 Secondary Endpoint: Incidence and Duration of ICU Stay Table 16 Exploratory Outcome: PCR for SARS-Cov-2 (FAS) Table 17 Exploratory Outcome: COPD Assessment Test -CAT (FAS) Table 24 Concomitant Medications Table 25 Other Data Itraconazole for COVID-19: preclinical studies and a proof-of-concept randomized clinical trial Direct antivirals working against the novel coronavirus: azithromycin (DAWn-AZITHRO), a randomized, multicenter, open-label, adaptive, proof-of-concept clinical trial of new antivirals working against SARS-CoV-2-azithromycin trial A randomized, open-label, adaptive, proof-of-concept clinical trial of modulation of host thromboinflammatory response in patients with COVID-19: the DAWn-Antico study Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study In vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine Correspondence COVID-19 : consider cytokine storm syndromes and Abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia Anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy Azithromycin: Mechanisms of action and their relevance for clinical applications Macrolides: From in vitro anti-inflammatory and immunomodulatory properties to clinical practice in respiratory diseases Azithromycin for Prevention of Exacerbations of COPD Azithromycin for prevention of exacerbations in severe asthma (AZISAST): A multicentre randomised double-blind placebo-controlled trial Azithromycin during Acute Chronic Obstructive Pulmonary Disease Exacerbations Requiring Hospitalization (BACE). A Multicenter, Randomized, Double-Blind, Placebo-controlled Trial A randomised controlled trial of azithromycin to prevent chronic rejection after lung transplantation Azithromycin for prevention of exacerbations in non-cystic fi brosis bronchiectasis (EMBRACE): A randomised, double-blind, placebo-controlled trial Azithromycin induces anti-viral effects in cultured bronchial epithelial cells from COPD patients Azithromycin induces anti-viral responses in bronchial epithelial cells Azithromycin, a 15-membered macrolide antibiotic, inhibits influenza A(H1N1)pdm09 virus infection by interfering with virus internalization process Macrolide therapy in respiratory viral infections Identification of novel macrolides with antibacterial, antiinflammatory and type I and III IFN-augmenting activity in airway epithelium Novel antiviral properties of azithromycin in cystic fibrosis airway epithelial cells Spiramycin and azithromycin, safe for administration to children, exert antiviral activity against enterovirus A71 in vitro and in vivo Azithromycin Protects against Zika Virus Infection by upregulating Virus-UZ Leuven -KUL Confidential Protocol: Direct antivirals working against nCoV -Azithromycin treatment stratum (DAWN-AZITHRO) Version: 06_15Mei2020 Internal Ref Induced Type I and III Interferon Responses Evaluation of Ebola Virus Inhibitors for Drug Repurposing Clinical Pharmacology Perspectives on the Antiviral Activity of Azithromycin and Use in COVID-19 Anti-inflammatory effects of adjunctive macrolide treatment in adults hospitalized with influenza: A randomized controlled trial Macrolides in critically ill patients with Middle East Respiratory Syndrome Adjunctive therapy with azithromycin for moderate and severe acute respiratory distress syndrome: a retrospective, propensity scorematching analysis of prospectively collected data at a single center Efficacy of azithromycin in sepsis-associated acute respiratory distress syndrome: a retrospective study and propensity score analysis Role of prophylactic azithromycin to reduce airway inflammation and mortality in a RSV mouse infection model Energetics Based Modeling of Hydroxychloroquine and Azithromycin Binding to the SARS-CoV-2 Spike (S)Protein -ACE2 Complex The immunomodulatory effects of macrolides-A systematic review of the underlying mechanisms Azithromycin attenuates myofibroblast differentiation and lung fibrosis development through proteasomal degradation of NOX4 Regional Differences in Susceptibiity of Bronchial Epithelium to Mesenchymal Transition and Inhibition by the Macrolide Antibiotic Azithromycin Azithromycin has enhanced effects on lung fibroblasts from idiopathic pulmonary fibrosis (IPF) patients compared to controls (Respiratory Azithromycin increases in vitro fibronectin production through interactions between macrophages and fibroblasts stimulated with Pseudomonas aeruginosa Macrolide treatment for COVID-19: Will this be the way forward? Replication of human coronaviruses SARS-CoV, HCoV-NL63 and HCoV-229E is inhibited by the drug FK506 In vitro testing of Hydroxychloroquine and Azithromycin on SARS-CoV-2 shows synergistic effect Julien Uptake of azithromycin by various cells and its intracellular activity under in vivo conditions Pharmacokinetics of azithromycin in lung tissue, bronchial washing, and plasma in patients given multiple oral doses of 500 and 1000 mg daily Comparative distribution of azithromycin in lung tissue of patients given oral daily doses of 500 and 1000 mg 6 6.2.3 (Long-Term) Exploratory Outcomes 6 7.2 Selection of Study Population Full Analysis Set Per Protocol Set 10 10.9 Methods for Withdrawals, Missing Data and 2.1 Clinical Status During Hospitalisation Cumulative Clinical Status from Day 1-15 14 13.2.5 Incidence and Duration of Mechanical Ventilation 15 13.3.1 PCR for SARS-CoV-2 Not hospitalized, limitations on activities Hospitalized, requiring supplement oxygen Hospitalized, on non-invasive ventilation or high flow oxygen devices Hospitalized, on invasive mechanical ventilation or ECMO Daily clinical status during hospitalization and on Days 15 and 29 Cumulative clinical status from Day 1 to Day 15, i.e. sum of the scores between Day 1 and 15 All-cause mortality on Days 15 and 29 Duration of supplemental oxygen Duration of mechanical ventilation Time to Discharge Time to and duration of ICU stay Time to Death NEWS assessed daily during hospitalization and on Days 15 and 29 Combined cardiac endpoint during in-hospital stay: any of the following: hsTrop>0.5 ng/mL, ventricular arrhythmia requiring intervention, reanimation, sudden cardiac death Long-Term) Exploratory Outcomes Qualitative and quantitative PCR for SARS-CoV-2 in (naopharyngeal) swab on day 1 and 6 for lung functional, functional and radiological evaluation if possible a. Questionnaire (mMRC, CAT In the AZYTHRO sub-protocol, patients are randomised in a 2:1 ratio to either azithromycin or standard of care (SOC) 4 of the SAS System for Windows. Copyright © 2002 SAS Institute Inc. SAS and all other SAS Institute Inc. product or service names are registered trademarks or trademarks of SAS Institute Inc Longitudinal data analysis of continuous and discrete responses for pre-post designs RV and WJ have received specific funding for DAWn-AZITHRO from the COVID-19 fund of the KU and UZ Leuven. 1. The first group of drugs that deserve special attention in the DAWN-Azithro study is the group causing drug-drug interacions. Some of them form contra-indications and are reason for exclusion. Others require close monitoring. Patients with a normal QTc, but at risk for QTc prolongation due to concomitant use of QTc prolonging drugs, will be monitored with the ECG monitoring schedule (see above