key: cord-0888058-zatr4cjd authors: Zhaori, Getu; Lu, Lu; Liu, Chunyan; Guo, Yongli title: Progresses in clinical studies on antiviral therapies for COVID‐19—Experience and lessons in design of clinical trials date: 2020-12-28 journal: Pediatr Investig DOI: 10.1002/ped4.12227 sha: 6953c040ce7b7d36d6a8ddb8ae98153f9f247d30 doc_id: 888058 cord_uid: zatr4cjd Antiviral therapy with antiviral agents is a very important component of treatment for the 2019 novel coronavirus disease (COVID‐19) caused by the severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2). It is important to clarify how to evaluate efficacy and safety of antiviral agents in treatment of COVID‐19 during the pandemic of this disease. We need to answer the following questions: do we still need to use rigorously designed randomized controlled clinical trials (RCTs)? Or, will it be enough if we use loosened criteria, observational studies or even retrospective case series and case reports? The answer is “No, we still need to use the strictly designed preferably blinded multicenter RCTs to evaluate the antiviral agents.” In this article, we reviewed almost all the RCT reports on monotherapies and combined therapies with antiviral agents for COVID‐19, and found that among the reports on monotherapies, only remdesivir, and among combined antiviral agents, only the combined regimen with interferon‐β1b, lopinavir‐ritonavir and ribavirin were effective and safe based on evidences from RCTs. The results of five RCTs for chloroquine or hydroxychloroquine consistently showed that they were ineffective and unsafe in the treatment of COVID‐19, especially at larger doses. Many aspects in the design of the clinical trials may be related to success or failure of a trial and the relevant factors need to be analyzed, discussed and emphasized from the specific requirements and considerations of antiviral therapies. We hope such discussions be of certain use in designing clinical trials for pediatric antiviral therapies. The outbreak and global pandemic of 2019 novel coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) formed unprecedented threatening and challenges to the global populations. According to the World Health an extremely hazardous pandemic of infectious disease became the most prioritized tasks of medical care givers, including medical researchers. Unlike the previous outbreaks and epidemics of severe acute respiratory syndrome (SARS) in 2003 (during the whole process and thereafter), there was almost no randomized controlled well-designed clinical trials provided evidences for any therapeutic approach. 2 During this pandemic of COVID-19, at least one RCT presented evidences demonstrating that remdesivir was safe and effective against COVID-19. Based on the evidences, the Food and Drug Administration (FDA) of the United States issued and publicized an Emergency-Use Authorization for the treatment of adults and children patients with severe COVID-19 disease. 3 Antiviral treatment is an extremely important component of the treatment, control and prevention of COVID-19, especially during the early stage of the disease. However, dozens of drugs, including chloroquine, hydroxychloroquine, azithromycin and even famotidine were claimed to have antiviral effects against SARS-CoV-2. Of the total number of articles, up to 23 949, that can be retrieved at PubMed published since December 1, 2019, 2836 articles are related to or on antiviral therapies. We need to consider, which drugs are truly antiviral agents, which are not, and we should clarify how to evaluate antiviral agents, and reach a clear conclusion about which drug(s) are effective and safe in treatment and prevention of this pandemic disease and worthy of recommending for clinical application. In addition, it is also important for pediatricians to learn from experience and absorb lessons from the clinical trials that are already reported although most or all the clinical trials for antiviral treatments were conducted in adults, and make good use of them for future pediatric clinical studies for COVID-19 as well as for any other viral diseases in children. In this review article we discuss issues concerning antiviral therapies for COVID-19. However, we need to recall the following issues closely related to antiviral drugs/ therapies. What is antiviral therapy? Are azithromycin, chloroquine and hydroxychloroquine antiviral drugs? We could not find a definition for antiviral drugs/therapies. But we found requirements for antiviral agents, 4 they must have inhibitory effects on virus-specific events, including attachment to the cell or fusing with the cell membrane, uncoating of the genome of the virus, assembly of progeny virions, or on virus-directed synthesis of the viral nucleic acids and/or proteins. These characteristics are usually confirmed via laboratory experiments in cell culture of the viruses or in vivo in animals during the preclinical development stages. If drugs that have never been tested in laboratory for antiviral effects, in principle, those drugs should not be regarded as antivirals. Therefore, drugs such as azithromycin and chloroquine/hydroxychloroquine (unless they were studied for in vitro antiviral effects) should not be listed among antiviral agents. How to evaluate the efficacy and safety of antiviral agents in the setting of a pandemic? All the medical care givers follow guidelines in their normal daily practice. There are more than 6000 clinical practice guideline documents from more than 70 countries, 5 and most of the guidelines are evidencebased. The high quality of evidences are from multiple RCTs or meta-analyses and single RCTs, and large scale well designed RCTs are believed to provide high quality evidences. 6 Currently, the whole world is in an extremely unusual situation, in a rare global emergency health crisis. During such a pandemic, should we give up the routine ways of evaluating treatments with RCTs? Do we need to "loosen" or lower our criteria for the evidences, and to use case series, uncontrolled or not randomized trials? The answers are "No!" although there are many challenges and difficulties. We need to overcome the challenges and difficulties and to use rigorously designed, sufficiently large-scale RCTs. Designing and conducting RCTs are possible and feasible during the pandemic. The fact is that the total number of published articles on "COVID-19 treatment" searched at PubMed only was as high as 24 082, which included 122 clinical trials, and 67 RCTs, and for "antiviral treatment for COVID-19", the number of articles was 2853, which included 33 clinical trials and 19 RCT reports. RCT reports were also published during the pandemic of influenza in 2009. 7 These facts indicate that it is possible to conduct well-designed clinical trials, RCTs during a sudden public health crisis like SARS and COVID-19 pandemics. The numbers of published articles, ongoing and planned clinical trials are increasing rapidly, which clearly reflect the need for high-quality evidences for treatments of COVID-19. 8 As a panel of 29 pediatric experts of the US pointed out: "Antivirals should be tested in clinical trials, as this is the only way to establish efficacy and safety of these therapies for COVID-19." 9 The clinical trial is regarded as the most definitive tool for evaluation of the applicability of clinical research. Clinical trial has been called "the gold standard" on many occasions for measuring and evaluating all the clinical research. 10 For designing RCTs, there should be no many difficulties, however, there are challenges in conducting the trials and collecting data. In some studies, the sample size could not be fulfilled because in the setting of study the disease was soon controlled well and subsequent enrollment was impossible. 11 To obtain high quality evidences for antiviral treatments of COVID-19, it is important to rigorously design the studies. Specific considerations for clinical trials of antiviral agents should include the following. 1) Early beginning of antiviral medication. 2) There must be primary or secondary end points that reflect virological status, e.g., viral load in nasopharyngeal secretion. 3) Specimens for viral nucleic acid determination should be taken from each patient at least once a day or even more frequently, so that the virological parameters can be analyzed and compared accurately. 4) Stratification of the participants for various factors, including severity of diseases, age, and time between onset of symptoms and randomization, etc. Only one RCT had started antiviral therapy within 5 days after onset of symptoms, and collected specimens for virological testing at least once a day. 12 Remdesivir is an RNA-dependent RNA polymerase inhibitor, a nucleoside (adenosine) analog which was originally developed for treatment of Ebola virus infection. The mechanism of action is similar to that of the other well-established nucleoside/nucleotide analog antivirals. Its use 24 hours before an animal model infected with Middle East respiratory syndrome (MERS) coronavirus (CoV) showed that it could completely prevent development of symptoms caused by the virus, strong inhibition of viral replication in the respiratory tract and it prevented the formation of pulmonary lesions. However, a phase 2 clinical trial showed that the group treated with remdesivir could not lower the fatality rate. 13, 14 Recently, in vitro and animal model studies demonstrated that remdesivir had potent activity against SARS-CoV-2 and might become a more promising treatment for COVID- 19. 15 For evaluation of efficacy and safety of antiviral drugs, we will focus mainly on RCTs. Before WHO SOLIDARITY trial interim report became available on October 15, 2020, only three RCTs on remdesivir were published (Table 1) . Of them, only the ACTT-1 study 16 provided confirmed evidence (in shortening the time to recovery) to support the efficacy of remdesivir in treatment of patients with COVID-19 whose disease was moderate in severity. The study was a well-designed randomized, double-blind placebocontrolled phase 3 trial, which was conducted at 60 sites and 13 subsites in 10 countries. The results of the study showed that the time to recovery (primary outcome) in patients in remdesivir group was 4 days (in the final report that was published on October 8, 17 5 days) shorter than that of the placebo group (P < 0.001). The mortality by day 14 on the Kaplan-Meier estimate was 7.1% vs. 11.9% (in the final report, 6.7% vs. 11.9%); while the rate of serious adverse events was similar. These results are the evidences, although not very strong, that support the establishment of efficacy and safety of remdesivir in treatment of COVID-19 adult cases. Just because of the results described in the preliminary report, the US FDA had issued an emergency-use authorization of remdesivir for severe COVID-19 adult and children patients. While the other two RCTs failed to provide evidences due to different reasons (See below). An important concern about this study is that the primary outcome (time to recovery) was not directly related to the virus. Previous studies on other viral diseases and COVID-19 have used outcomes like time to first negative viral testing, 18 viral load reflected by viral nucleic acid determination, and daily changes of viral load etc. 12 Despite the challenges from different aspects, some studies applied virological parameters as primary outcomes and obtained significant results. 12 The importance of virological outcomes should be emphasized for clinical studies evaluating the efficacy and safety of an antiviral agent. 19 Another issue we must consider is the importance of early starting of antiviral therapy. If the therapy starts too late, the viral infection may have caused serious or irreversible lesions and therefore antiviral treatment will not help much for recovery. The other 2 RCTs 20,21 failed to provide any evidence for the efficacy and safety of remdesivir in treatment of COVID-19. The reasons are different. The major reason of Wang et al's study was obviously because the sample size could not reach the planned target, in fact the sample was almost only the half of what was planned. On the other hand, there seemed to be two major limitations in that study. 1) The patients were allowed to enter in the study as late as on the 12th day after the onset of the symptoms (in fact in some other studies, treatment with antiviral agents started as late as 17th days after the onset of the symptoms). Starting the antiviral treatment too late may affect the results of treatment very much. In the RCT by Hung et al, 12 the antiviral therapy was started within 5 days after the onset of symptoms, which is a very good practice. 2) No virological outcome was used in that study. Setting up of proper main outcome or and secondary outcomes are very important for a clinical trial. For clinical trials for antiviral agents, there should be virological or virus-related outcome or end point, so that the researchers can understand the effect of the antiviral therapy on the virus status. In addition, the patients in either group were allowed to use other drugs that might have antiviral effect against SARS-CoV-2, such as interferon, lopinavir-ritonavir, Arbidol etc., although none of these agents as a monotherapy has been demonstrated by RCTs to have antiviral effects against the SARS-CoV-2. If any of them really has an antiviral effect against the virus, and if the proportion of patients who were treated with such "other drugs", and the dosage applied are not balanced between groups and the length of time of the use of those drugs were different, bias may occur unless there is predefined stratification and well-planned subgroup analyses. The RCT reported by Spinner et al 21 experienced certain major amendments on their protocol during the process of the study. One of the notable amendments is the change of the lower limit of the participants' age: from 18 to 12 years. If the randomization is not stratified against the age, and if distribution of children between the ages of 12 and 18 years is not balanced between the two groups, since there is a tendency that children have less severe disease, the result may produce bias. Mainly based on the preliminary report of the ACTT-1 study and meta-analyses, Rochwerg et al (an international guideline panel composed of 27 persons) issued and published a clinical practice guideline for use of remdesivir for severe COVID-19. 22 The US National Institute of Health and Australian National Guideline made weak recommendation in favor of remdesivir for severe COVID-19. Important issues related to the use of remdesivir include the following: optimal time to administer the drug, the length of course of treatment, the possible problems with concomitant use of other medicines, contraindicated in patients with liver and renal dysfunction. The uncertainties in effects on mortality, length of hospitalization, and longterm safety and complications, and generalized use in other regions and populations will also need to be studied. In contrast to the RCT of Beigel et al 16, 17 that provided evidences for efficacy and safety of remdesivir in treatment of COVID-19, the interim report of WHO's SOLIDARITY trial 23 declared that remdesivir as well as three other monotherapies for COVID-19 were ineffective in this largest scale rigorously designed multinational RCT. However, this large-scale well designed clinical trial should not be regarded as the final trial, since some arguments concerning the study design should be considered, and further clinical studies that are ongoing and being planned may provide different results and evidences. 1) Inspiration from natural history of COVID-19. In the WHO trial, the design of the study seemed not to have considered and clearly defined early initiation of antiviral treatment. As an acute viral infection, the overall natural history of COVID-19 is about 3 to 4 weeks for most of the patients. The course of the disease can be divided into three stages as depicted by Dos Santos et al 24 : stage 1 is early infection; stage 2 is pulmonary phase; and stage 3 is hyperinflammatory phase, during which pneumonia may deteriorate, sepsis, respiratory failure, cytokine storm and even death may occur. Of the three stages, only the stage 1 and 2 are the best period for application of antiviral agents to stop replication of the virus or even clearing the virus from the body. On the other hand, regardless what the outcome of a patient is in this stage, which may not reflect the effectiveness of an antiviral agent. Therefore, from the point of antiviral therapy, early beginning of antiviral therapy is critically important. If we make a very preliminary rough estimation based on reports including that of Rees et al, 25 phase 1 may be around 5-7 days, phase 2 around 7-10 days and phase 3 about 3-8 days. A few previously reported RCTs enrolled patients as late as a mean of 16.6 days or 12 days after the onset of symptoms, that means that many of their patients have already missed the best time for antiviral therapy. Starting the antiviral therapy within 5 days after the onset of symptoms will be ideal. 2) Importance of virological outcomes. We have discussed about this earlier. 3) Importance of stratification of participants while they are randomized. Certain characteristics of participants are extraordinarily important for antiviral therapies, these include a) How long after onset of symptoms the antiviral therapy is started; if the study group and control group are not balanced in this regard, bias will occur in results; b) The severity of disease is another important factor. Imbalance in the severity of disease may also produce bias in the results. It will be better if participants are stratified against at least these two characteristics. 4) Choosing proper intervention for the control group. For antiviral and other drugs for treatment of human diseases, initial in-human trials ideally should use randomized placebo-controlled trials to confirm the efficacy of the drug. However, in such a pandemic of acute lifethreatening infectious disease, use of placebo is ethically not allowed and also not practical. Therefore, clinical trials have chosen standard of care, or another antiviral drug against COVID-19. Using standard care regimens for patients in each group, and administering the antiviral agent for patients in the test drug group generally is a good practice. However, the problem is that many of the standard of care protocols contain certain antiviral or potential antiviral agents, such as ribavirin, interferon, etc. It should be OK since there is a control group where no test antiviral is used. However, if the proportions of patients treated with the potential antiviral agents contained in standard of care are not balanced among the groups, which may still cause certain bias in the results. How to solve such problems? The only way is not to recommend listing potential antiviral medicines into standard regimens since the efficacy of them has not been established. The use of another antiviral medicine as control, although has been applied in clinical trials and reported in literature, it may raise the questions of uncertainty. For example, if the results show that there are no significant differences in the main and major secondary outcomes between the test drug group and control group, it will be hard to conclude if the test drug is effective or not since so far, except for remdesivir, none of the monotherapy with supposed antiviral drugs is confirmed to have antiviral effect on SARS-CoV-2. Therefore, for clinical trials for evaluation of antiviral drugs against SARS-CoV-2, the control group should not be treated with potential antiviral agent. In summary, based on the above-mentioned arguments, the WHO SOLIDARITY trial may not rule out the possibility that the tested drugs, especially remdesivir, may have antiviral effects on COVID-19. There has been only one RCT report on the efficacy and safety of monotherapy with lopinavir-ritonavir. 26 This study could not obtain any evidence for benefit. However, in the modified intention to treat analysis, after excluding 3 cases who died in the early stage of the disease, the median time to clinical improvement was 15 days vs. 16 days, and this difference was significant according to the authors. An important fact is that the patient population had relatively more severe disease with overall rate of death of this study was 22.1%, while in initial descriptive studies the death rates were between 11% and 14.5%. This study has a remarkable strength in design, which is virus-related outcome measures included in the secondary outcomes, one was viral RNA titer area-underthe-curve (AUC) (which may represent viral load) and proportions of patients positive for the viral RNA over time. Such outcome measures are essential for clinical trials evaluating efficacy of an antiviral agent, although more resources are required. The authors had to obtain the specimens from all the patients for detection of viral RNA on days 1, 5, 10, 14, 21, and 28. The viral RNA loads and the proportions of patients positive for the viral RNA were similar between groups. Use of lopinavir-ritonavir was judged by the researchers to be associated with gastrointestinal adverse and serious adverse events, no death was judged to be associated with the test drug. Respiratory failure, acute renal injury and secondary infection were more common in the control group. This RCT could not provide evidence for shortening the time to clinical improvement, and clearly showed that lopinavir-ritonavir had no antiviral efficacy against the SARS-CoV-2. Arbidol (umifenovir) was originally developed as an antiviral agent for treatment of influenza and was approved for treatment of influenza, SARS and Lassa viruses in Russia and China. 27 Its mechanism of action was presumed by Vankadari based on analyses on molecular dynamics and structure that SARS-CoV-2 spike glycoprotein is the target of the drug, and the drug can effectively block the trimerization of the SARS-CoV-2 spike glycoprotein, and thereby to prevent adherence to cell and entering into cell. 28 On the other hand, Arbidol efficiently inhibited virus infection in vitro in Vero E6 cell culture. 29 Literature search retrieved 4 original articles on treatment of COVID-19 with Arbidol, none of them was RCT, however. Two of the studies were retrospective case series and cohort study for treatment of COVID-19. 27, 30 In Zhu et al's study 50 patients were enrolled totally, and Arbidol group was better in viral load and in duration of positive viral RNA (P < 0.01). No apparent side effects were found in any group. 27 The other study enrolled 141 patients; those in the test group were treated with combined Arbidol and interferon (IFN)-α2b and the other group with the IFN alone. 30 Only one outcome, the absorption of pneumonia on CT was in favor of the test group. The other two studies were retrospective cohort studies and the authors concluded that Arbidol was effective in post-exposure prophylaxis of COVID-19. 31, 32 There was no evidence from RCT for Arbidol in either treatment or prophylaxis of COVID-19. Both chloroquine (CQ) and hydroxychloroquine (HCQ) are antimalarial and antirheumatic drugs; they had not been regarded as antiviral medicines before the outbreak of the COVID-19. Interestingly, however, as early as 40 years ago, there were studies showing that these drugs had some antiviral activity in vitro via different mechanisms, and acting on as many as 12 stages of viral life cycle and related processes although most of the mechanisms were presumed, not confirmed. 33 CQ analogues have been reported to have an in vitro nonspecific antiviral effect at high concentration against a number of viruses, and recently against SARS-CoV-2. 34, 35 Since the outbreak of the COVID-19 in late 2019, more than 1700 articles were published on CQ, HCQ and "COVID-19" (PubMed, from December 1, 2019 to October 18, 2020), among which 21 were reports of clinical trials and 15 were RCTs. However, only 5 were RCTs for evaluation of CQ or HCQ in treatment or prevention of COVID-19, and most of the remaining were study protocols. None of the five RCTs and a few retrospective cohort and case series studies could provide evidences for antiviral effects against COVID-19, instead, the studies showed severe adverse events, especially at higher dose and when they were used in combination with azithromycin or oseltamivir. Moreover, use of HCQ was even associated with more deaths of patients (Table 2) . [36] [37] [38] [39] With regard to antiviral effects on COVID-19 in relation to design, only two out of the 5 RCTs had virological end point. These two studies had a very clear result that the viral RNA negative rates had no significant difference between the test group and control group on day 7 40 and day 28 38 , respectively. The primary end point of the study reported by Borba et al 36 was mortality on day 13, which was a good choice, but the mortality on day 13 in high dose group was more than double of the low dose group. However, it is impossible to evaluate if the low dose therapy with CQ was effective or not because the study did not include a control group that was not treated with CQ. This lesson emphasizes the importance of a proper control group. The remaining two RCTs were well-designed, one was for treatment and the other was for post-exposure-prophylaxis, and both were ineffective. Based on the results of the 5 RCTs in terms of main outcome, it is clear that neither CQ nor HCQ was effective in either treatment or prophylaxis of COVID-19. We should also need to look at the secondary outcomes (clinical status, laboratory examinations, electrocardiogram, imaging, etc.) for efficacy and safety. In the study of Cavalcanti et al, 37 on day 15, clinical status was not improved either in HCQ (400 mg twice daily) + azithromycin (500 mg/d) or in HCQ alone group. There was no significant differences in any of the secondary outcomes. The primary and secondary outcomes were all similar between the 2 groups in the study of Tang et al. 38 In the study of Borba et al, 36 both groups had prolonged QTc interval (18.9% and 11.1% of cases). Creatine kinase increase was seen more frequently in high dose group (50% vs. 31.6%). Two of 37 (2.7%) in the high dose group had ventricular tachycardia before death without torsade de pointes. Overall death rate was 27.2%. Lethality was 39.0% (16/41) in high dose group and 15.0% (6/40) in low dose group. The authors say "No apparent differences despite more deaths in the high dosage group (log-rank, −2.183, P = 0.03). (It was very clearly written at the end of "Statistical Analysis" that: "… and a 2-tailed P < 0.05 was considered significant".) Side effects were more frequently seen in HCQ group. [37] [38] [39] Strength The design of the 5 RCTs was generally good, since some were double masked, all were randomized, some were multicenter, and sample size was based on calculation. Two of the RCTs had virological outcome. Primary outcome included virus negative conversion and clinical improvement, and secondary outcome included negative conversion of the virus at days 4, 7, 10, 14, or 21. a) The umber of enrolled cases was much smaller than calculated sample size (81 vs 440). b) There was no control group that was treated with a drug other than CQ, i.e., there was neither placebo nor standard care control, therefore, it is impossible to judge if the low dose chloroquine was effective or not. c) There was no virological outcome measure. 36 d) Single center. About 3/4 patients were virologically confirmed COVID-19 cases. e) Standard care included use of IFN, Arbidol, and or lopinavir-ritonavir, 38,40 which probably explains why most patients of both groups became viral RNA negative within 7 days. But it was not mentioned if the use of those was balanced. f) Mean duration from symptom onset to beginning of antiviral therapy was 16.6 days, too long. Ribavirin is an old antiviral drug, it is an analog of nucleoside (guanosine), and its main mechanism of antiviral action is similar to that of the other nucleosides/ nucleotides. Unlike remdesivir, there are no in vitro evidence showing that ribavirin has antiviral effects against the SARS-CoV-2, and its in vitro activity against the other SARS-CoV was limited and was observed to have dose-dependent hematologic toxicity, mainly hemolysis, which was seen in over 60% of patients and elevated liver transaminase was seen in 75% of patients. Combined treatment with ribavirin at lower doses and IFN-α2a, or -α2b seemed to be effective with less severe adverse events. 12, 41, 42 No evidences for the efficacy and safety of ribavirin monotherapy for COVID-19 from RCT or controlled clinical trials are available. Use of lopinavirritonavir with ribavirin was not recommended by a panel of experts. 9 Interferons (IFNs) are well-known for their antiviral effects. The mechanisms of antiviral action of IFNs is believed to involve activation of interferon-stimulated genes. 43 Since the outbreak of COVID-19, IFNs have been used in combination with a few other antiviral agents in treatment and clinical trials. However, there is only one randomized controlled clinical trial on evaluation of IFN-β1a monotherapy reported by researchers from Iran. 44 This was an open label RCT on 91 cases. Dose of the IFN was 44 mg subcutaneously injected 3 times a week for 2 weeks. No virologic outcome was applied. The primary outcome (time to clinical response) was not significantly different between the groups. However, significantly higher discharge rate and lower mortality were seen in IFN group. This study demonstrated that earlier start (before day 10 after symptom onset) of IFN therapy could lower the mortality as compared with later start. For antiviral drugs, the earlier the treatment starts, the less the virus causes lesions and severe lesions in the host. In an uncontrolled case series of 77 patients, 45 use of IFN-α2b with or without Arbidol significantly shortened the duration of virus persistence in the upper respiratory tract and reduced duration of elevated blood levels of inflammatory markers. Azithromycin, one of the macrolide antibiotics, should not be considered as an antiviral agent according to the requirements for antivirals mentioned earlier in this review, although it is believed to have certain immunomodulatory effects. 46, 47 There are no many clinical reports on the efficacy and safety of azithromycin as a monotherapy for COVID-19. Only 2 RCT reports are available, one was from Brazil; 48 397 patients constituted the intention-to-treat population; the primary outcome (clinical status at day 15 after randomization, assessed by an independent adjudication committee masked to treatment allocation) and the rate of adverse events were not significantly different between the two groups. This RCT demonstrated that azithromycin (combined in fact with HCQ) could not improve clinical outcome of patients with severe COVID-19. The other report was from Iran; 49 111 patients with COVID-19 were enrolled and the test group (n = 56) received azithromycin (oral, 500 mg/d) in addition to HCQ. Among the 7 components of the main outcomes, only the SpO 2 , respiratory rate and the "duration of admission" (hospitalization) were significantly better in azithromycin group. The mortality on day 15 was not significantly different. The patients received azithromycin had a better general status. Small case series studies showed opposite results for the combined use of HCQ and azithromycin. 50, 51 Therefore, there have been no strong evidences from RCTs to support the efficacy of either combined or monotherapy with azithromycin for treatment of COVID-19. Other drugs that are suggested by articles might have potential antiviral effects against SARS-CoV-2 include famotidine, chlorpromazine, and others. Rosa et al 52 pointed out that repositioning clinical trials possibly represent an attractive strategy because they facilitate the discovery of new classes of medicines at much lower costs and take less time to reach the market; and there are existing pharmaceutical supply chains for formulation and distribution, although such attempts may have certain limitations, and most importantly, these advantages can be utilized only if any of the drug(s) for repurposing is (are) confirmed to have solid clinical efficacy and safety. Computational analyses performed by Ortega et al 53 showed that famotidine should have inhibitory effect on the protease of SARS-CoV-2, but its affinity to the proteases is low, therefore, famotidine should be tested in combination with some other antiviral agent. According to a hypothesis based on recent in vitro studies on anti-MERS-CoV and anti-SARS-CoV activity (possibly via inhibition of clathrin-mediated endocytosis) of chloropromazine, an old psychotropic drug, and some clinical observation, a group of French researchers have planned to conduct a phase III randomized controlled therapeutic pilot clinical trial to evaluate repurposing for this drug. 54 There are many other potential candidate drugs and regimens that are either ongoing or have been planned for clinical studies. We wish the studies would be able to provide high quality strong evidences for their antiviral effects. For combined antiviral therapies against SARS-CoV-2 infection, there have been 3 RCT reports available at PubMed (as of November 15, 2020). The first is a phase 2 clinical trial conducted by Hung et al. 12 All the other available reports in this regard are on retrospective cohort study, case series or case reports. Table 3 shows the overview of the RCT and the retrospective cohort studies. The study reported by Hung et al provides the high quality evidence from the RCT that combination of IFN-β1b, lopinavir-ritonavir and ribavirin was effective in clearance of the viral RNA, clinical improvement and shortening hospital stay of patients with COVID-19. The other two RCTs are on sofosbuvir and daclatasvir compared with standard care 55 and sofosbuvir, daclatasvir and ribavirin compared with standard of care, 56 and both were conducted in Iran. In both trials the primary endpoints failed to show significant difference between the test group and the control group. However, some secondary endpoints provided evidences to support the efficacy, e.g. Sodeghi et al 55 showed hospital stay was significantly shorter and the rate of discharge was significantly higher in the combined therapy group, and Kasgari et al 56 showed the clinical recovery status was significantly better in the combined treatment group. Addition of ribavirin onto the sofosbuvir and daclatasvir seemed not to have shown any evidences suggesting increased benefit. The limitations of these two trials include: small sample size, no blinding was applied, and from the points of antiviral therapy, no virological outcomes were applied, no early beginning of antiviral treatment was specifically considered. For the particular combined regimens, we still need large scale, rigorously designed RCTs to confirm their efficacy and safety. Of the two retrospective cohort studies, Deng et al 57 evaluated combination treatment with Arbidol and lopinavir-ritonavir, and 2 outcomes supported the efficacy of the combined treatment (viral negative conversion at day 7 and day 14, chest CT improvement); safety profile was good in both groups. However, in the study of Xu et al, 30 the main outcome (viral RNA positive duration) failed to show significant difference between the groups, although CT images showed that the absorption of lung lesions was faster in the combined treatment group. After reading these articles on combined antiviral therapies against SARS-CoV-2, we have a general impression that combined antiviral therapies seem to be more effective as compared to the antiviral monotherapies, although such conclusions must be confirmed only based on high quality evidences from well-designed RCTs. In summary, so far remdesivir is the only effective and safe antiviral monotherapy for COVID-19 based on evidences from RCT, and combined therapy with IFN-β1b, lopinavir-ritonavir and ribavirin was effective and safe, and weak evidences support efficacy of combined therapy with sofosbuvir and daclatasvir. Five RCTs demonstrated that neither chloroquine nor hydroxychloroquine was effective in treatment of COVID-19, and higher doses showed poor safety. The strengths of the RCTs in design should be applied and the limitations should be taken as lessons for pediatric clinical trials. COVID-19) Dashboard. https:// covid19.who.int Antiviral treatment of SARS: can we draw any conclusions? COVID-19) Update: FDA issues emergency use authorization for potential COVID-19 Treatment s Principles and Practice of Infectious Diseases Clinical practice guidelines: A primer on development and dissemination National Comprehensive Cancer Network Guidelines: Who makes them? What are they? Why are they important? Oseltamivir compared with the Chinese traditional therapy maxingshigan-yinqiaosan in the treatment of H1N1 influenza: a randomized trial Advances in the possible treatment of COVID-19: A review Multicenter initial guidance on use of antivirals for children with COVID-19/SARS-CoV-2 Fundamentals of Clinical Trials Remdesivir in adults with severe COVID-19: a randomised, double-blind, placebo-controlled, multicentre trial Triple combination of interferon beta-1b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with COVID-19: an open-label, randomised, phase 2 trial Remdesivir for severe acute respiratory syndrome coronavirus 2 causing COVID-19: an evaluation of the evidence A randomized, controlled trial of Ebola virus disease therapeutics Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro Remdesivir for the treatment of Covid-19-Preliminary report Remdesivir for the treatment of Covid-19-Final report A comparative study on the time to achieve negative nucleic acid testing and hospital stays between danoprevir and lopinavir/ritonavir in the treatment of patients with COVID-19 Challenges and opportunities in developing respiratory syncytial virus therapeutics Remdesivir in adults with severe COVID-19: a randomised, double-blind, placebo-controlled, multicentre trial Effect of remdesivir vs standard care on clinical status at 11 days in patients with moderate COVID-19: A randomized clinical trial Remdesivir for severe covid-19: a clinical practice guideline Repurposed antiviral drugs for COVID-19 -interim WHO SOLIDARITY trial results Natural history of COVID-19 and current knowledge on treatment therapeutic options COVID-19 length of hospital stay: a systematic review and data synthesis A trial of lopinavir-ritonavir in adults hospitalized with severe Covid-19 Arbidol monotherapy is superior to lopinavir/ritonavir in treating COVID-19 Arbidol: A potential antiviral drug for the treatment of SARS-CoV-2 by blocking trimerization of the spike glycoprotein The anti-influenza virus drug, arbidol is an efficient inhibitor of SARS-CoV-2 in vitro Arbidol/ IFN-α2b therapy for patients with corona virus disease 2019: a retrospective multicenter cohort study Potential of arbidol for post-exposure prophylaxis of COVID-19 transmission: A preliminary report of a retrospective cohort study Effectiveness of arbidol for COVID-19 prevention in health professionals Therapeutic use of chloroquine and hydroxychloroquine in COVID-19 and other viral infections: A narrative review Targeting endosomal acidification by chloroquine analogs as apromising strategy for the treatment of emerging viral diseases Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro Effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection: A randomized clinical trial Hydroxychloroquine with or without azithromycin in mild-to-moderate Covid-19 Hydroxychloroquine in patients with mainly mild to moderate coronavirus disease 2019: open label, randomised controlled trial A randomized trial of hydroxychloroquine as postexposure prophylaxis for Covid-19 A pilot study of hydroxychloroquine in treatment of patients with moderate COVID-19 Novel coronavirus treatment with ribavirin: Groundwork for an evaluation concerning COVID-19 Pharmacologic treatments for coronavirus disease 2019 (COVID-19): A review Type 1 interferons as a potential treatment against COVID-19 A randomized clinical trial of the efficacy and safety of interferon β-1a in treatment of severe COVID-19 Interferon-α2b treatment for COVID-19 The immunomodulatory effects of macrolides-A systematic review of the underlying mechanisms Azithromycin attenuates airway inflammation in a mouse model of viral bronchiolitis Azithromycin in addition to standard of care versus standard of care alone in the treatment of patients admitted to the hospital with severe COVID-19 in Brazil (COALITION II): A randomised clinical trial Safety and effectiveness of azithromycin in patients with COVID-19: An open-label randomised trial Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open-label non-randomized clinical trial No evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe COVID-19 infection Clinical trials on drug repositioning for COVID-19 treatment Class A G proteincoupled receptor antagonist famotidine as a therapeutic alternative against SARS-CoV2: An in silico analysis Repurposing chlorpromazine to treat COVID-19: The reCoVery study Sofosbuvir and daclatasvir compared with standard of care in the treatment of patients admitted to hospital with moderate or severe coronavirus infection (COVID-19): a randomized controlled trial Evaluation of the efficacy of sofosbuvir plus daclatasvir in combination with ribavirin for hospitalized COVID-19 patients with moderate disease compared with standard care: a single-centre, randomized controlled trial Arbidol combined with LPV/r versus LPV/r alone against corona virus disease 2019: A retrospective cohort study Progresses in clinical studies on antiviral therapies for COVID-19-Experience and lessons in design of clinical trials None of the authors has any conflict of interest concerning this manuscript.